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    • 专利摘要:
    The present invention relates to a process for the elimination of histamine. The present invention is useful in biotechnology and refers to an isolated nucleic acid molecule that, comprised in a microorganism, allows the elimination of histamine and imidazoleacetic acid (ImAA) from different media, such as food, as well as the use of some mutants which accumulate well said ImAA, either alanine or aspartic acid. The microorganisms of the present invention are capable of transforming Hin into ImAA thanks to the gene pool called hin1 and/or are capable of transforming ImAA into aspartic acid or into salts thereof, thanks to the gene pool called hin2 as well as the hinK gene, and/or are capable of transforming histamine into fumaric acid or salts thereof thanks to the Hin1, Hin2, Hink and Hin3 gene cluster. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2684421A2
    申请号:ES201830155
    申请日:2018-02-21
    公开日:2018-10-02
    发明作者:José Luis GÓMEZ BOTRÁN；Manuel DE LA TORRE GARCÍA；Elías Rodríguez Olivera；José María Luengo Rodríguez
    申请人:Bioges Starters S A；BIOGES STARTERS SA；
    IPC主号:
    专利说明:

    Histamine removal procedure Field of the Invention
    The present invention belongs to the field of Biotechnology and refers to the use of
    5 microorganisms (wild or recombinant) that contain the hin genes to eliminate or reduce the amounts of histamine (Hin) and imidazoleacetic acid (ImAA) in those media or foods that contain them. These genes encode the enzymes that catalyze the different reactions of a catabolic pathway that carries out the transformation of histamine and its ImAA catabolic derivative, into aspartic acid (or some of its salts). 10 Background Biogenic amines
    Amines are chemical compounds derived from ammonia that result from the replacement of the hydrogens of that molecule by alkyl radicals. As one, two or three hydrogens are substituted, the amines will be primary, secondary or tertiary. When they are originated as a result of the activity of living organisms and have biological activity (they perform important functions in cells) they are called biogenic or biogenic amines. Depending on the number of amino groups present in the molecule we can differentiate monoamines, diamines and polyamines. Aliphatic monoamines are widespread in nature where putrescine diamine is also abundant, while
    20 that spermidine and spermine polyamides are produced by animals, plants and most bacteria (1).
    Aromatic amines, caused by decarboxylation of amino acids, are the most common amines in food (histamine, 2-phenylethylamine, tyramine, etc.) and also have great importance as transmitters within the central nervous system (dopamine,
    25 norepinephrine, epinephrine, serotonin, etc.).
    We can make a distinction between endogenous biogenic amines, which are those that are synthesized in different tissues of higher organisms (such as adrenaline produced in the adrenal medulla or histamine in mast cells) and exogenous biogenic amines, which are ingested in the diet. These exogenous biogenic amines may be present in foods of plant origin (fruits and vegetables), or they may appear in foods as a result of microbial activity during processing (curing of meats and cheeses) or during storage thereof .


    Because they can cause harmful effects in both man and animals, they are considered toxic substances.
    The most important biogenic amines that can be found in food are histamine, putrescine, cadaverine, tyramine, tryptamine, phenylethylamine, spermine and spermidine; and the foods that contain them can be very varied (fish, meat, eggs, cheeses, fermented drinks, etc.) (2).
    Fortunately, organisms have different natural detoxification systems (monoamine oxidase -MAO- or diaminooxidase -DAO-) that allow them to eliminate biogenic amines, avoiding the harmful effects caused by these compounds. However, there may be cases in which these systems do not work properly, or that are inhibited by the action of certain drugs, so the presence of biogenic amines in food can pose a serious health problem.
    For all these reasons, it is very interesting to select microorganisms that, when used in food processing processes (cured, fermentations, etc.), do not accumulate biogenic amines, or that do so in concentrations that are not dangerous to health. Genetic Engineering and Metabolic Engineering could contribute to obtaining this type of strains, ensuring, in addition, that another series of properties and characteristics that are necessary to maintain the standards of identity and quality of food are preserved. Biogenic amines as neurotransmitters.
    It has been known for decades that the catecholaminergic transmission is mediated by biogenic amines, including catecholamines (dopamine, norepinephrine and adrenaline) derived from the amino acid tyrosine; indolamine serotonin, synthesized from tryptophan; and histamine, produced from the amino acid histidine. Catecholamines
    Under the term catecholamines, all those biogenic amines derived from tyrosine that contain a catechol group and an amino group in its molecule are included. The first step in the synthesis of catecholamines is catalyzed by the enzyme tyrosine hydroxylase through a reaction that requires oxygen as a substrate and tetrahydrobiopterin as a cofactor, and allows dihydroxyphenylalanine (DOPA) as a final product (Figure 1). Therefore, the


    Tyrosine hydroxylase rate will be the limiting factor for the synthesis of the three catecholaminergic neurotransmitter amines (dopamine, norepinephrine and adrenaline).
    Dopamine is produced by decarboxylation of L-DOPA. This reaction is carried out by the enzyme DOPA decarboxylase. The area of the brain where it is found in greater abundance is in the corpus estriatum, playing an essential role in the coordination of body movements (3). In patients suffering from Parkinson's disease, for example, degeneration of dopaminergic neurons has been observed, which will lead to the characteristic motor dysfunction associated with this disease (4).
    Norepinephrine, also called norepinephrine, requires for its synthesis, from dopamine, the action of dopamine-�-hydroxylase. This catecholamine is mainly produced in neurons of the sympathetic ganglia and its action is related to sleep, wakefulness, attention and behavior.
    Adrenaline, also called epinephrine, is present in the brain at lower levels than the other two catecholamines. The enzyme that synthesizes adrenaline, phenylethanolamine-N-methyltransferase, is located only in the secretory neurons of this catecholamine.
    The most important enzymes in the catabolism of catecholamines are monoamine oxidase (MAO) and catechol O-methyltransferase (COMT) (5). These enzymes are found respectively in the mitochondria and cytoplasm of both neuronal and glial cells. Inhibitors of these enzymes are used clinically as antidepressants (6). Histamine
    This biogenic neurotransmitter amine is produced by decarboxylation of histidine due to the action of histidine decarboxylase (Figure 2A). Its metabolism involves both histidine methyltransferase and MAO. The highest concentration of this neurotransmitter is found in the neurons of the hypothalamus and its action is related to the alert and attention processes. Histamine is also released by macrophages in response to allergic reactions or tissue damage. Serotonin
    This indolamine, also called 5-hydroxytryptamine, is synthesized in neurons from tryptophan ingested with food after being hydroxylated to 5-hydroxytryptophan by


    a reaction catalyzed by the enzyme tryptophan-5-hydroxylase. Subsequently, 5-hydroxytryptophan is decarboxylated by means of the action of a 5-hydroxytryptophan decarboxylase to give rise to serotonin (Figure 2B). The main enzyme responsible for its degradation is MAO, as in the other biogenic amines. Serotonin is involved in the regulation of sleep and wakefulness.
    In addition to neurotransmitter monoamines, there are other biogenic amines that have a similar molecular structure and act as neuromodulators or "false neurotransmitters." These endogenous amines, also called "trace" amines or microamines, are found in small amounts in the central nervous system and their study is becoming important in recent years. "Trace" amines
    With the term "trace" amines or microamines, reference is made to a family of endogenous amines structurally and metabolically related to dopamine, norepinephrine and serotonin (7-8). This group includes p- and m-octopamine, p- and m-tyramine, tryptamine and �-phenylethylamine (Figure 3). All these molecules are heterogeneously distributed in the brain of mammals in very low concentrations (0.1-100 ng / g of tissue) (9), but they play an important role in coordinating the synaptic response mediated by neurotransmitter biogenic amines.
    Two specific receptors of these "trace" amines have been characterized which cannot be activated by neurotransmitter monoamines. These receptors are called TA1 and TA2, belong to the family of receptors associated with G proteins (GPCRs) and are located in the pre-and post-synaptic plasma membrane of receptor neurons. They are activated by tryptamine, p-tyramine and by �-phenylethylamine, as well as by amphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and other types of hallucinogenic drugs. (10) This discovery aroused great interest in these compounds which, given their physiological relevance, are called "endogenous amphetamines" (11-12).
    In addition to these recently discovered aspects, the co-transmitter function played by these “trace” amines in the neurotransmission systems mediated by dopamine, norepinephrine or serotonin mediated (8) has been known for years. The structural similarity of these "trace" amines with neurotransmitter monoamines, will allow them to act as substitutes or "false neurotransmitters" in the dopamine and norepinephrine systems. In addition, due to their functional similarity they are being used in the treatment of hepatic encephalopathy (13) or in Parkinson's disease (14).


    Finally, “trace” amines can serve as neuromodulators in the central nervous system, but before explaining this activity, a clear distinction between neurotransmitter and neuromodulator should be made (15).
    It is called a neurotransmitter, the molecule released by a neuron to the synaptic channel in response to an electrical activity and which will subsequently specifically bind to its post-synaptic receptors, causing the induction of a change in the excitability of the post-synaptic cell and thus, allow the passage of information.
    A neuromodulator is also a molecule released by a neuron, but in this case it is not capable of causing a change in the excitability of the post-synaptic cell membrane by itself, since it needs the presence of a neurotransmitter. The release of a neuromodulator acts by modifying the action (increasing or decreasing it) of a coexisting neurotransmitter.
    Therefore, it is not surprising that “trace” amines have been implicated in most neuropsychiatric disorders associated with dysfunctions in the catecholamine and indolamines systems, since these compounds modulate the signaling processes that occur in the post terminals. -and pre-synaptics of these systems. It is believed that alterations in the function of these "trace amines" are involved in the etiology of a wide variety of neuropathological disorders, including hallucinations, schizophrenia, depression, anxiety states, hyperactivity, bipolar disorder, etc. (11, 16-18). Presence of biogenic amines in food.
    Biogenic amines, in addition to being present in the central nervous system fulfilling neurotransmitter and neuromodulatory functions, are present in fermented foods and beverages, where they are generated by decarboxylation of their precursor amino acids. Their accumulation (especially that of aromatic biogenic amines) can make the intake of these foods harmful to health. The main biogenic amines that can cause toxicity when they accumulate in food, are reflected in Figure 4.
    Amines and polyamines (PAs) are only found naturally in plant-based foods, since these molecules are found in plants and in their fruits, forming part of their cell walls or acting as a defensive system against attack of pathogens or predators (19). Due to their chemical nature these


    Compounds participate in numerous basic cellular processes, as well as in different events related to growth, development and response of plants to certain stress conditions. PAs, in addition to being essential for plant growth, under appropriate conditions, can exercise specific morphogenesis control functions (20).
    The predominant amine will depend on the type of fruit or plant considered; Thus, for example, in fruits such as lemon, tangerine and strawberry, putrescine predominates, while in raspberry and mushrooms, the predominant amine is tyramine (21). It has also been proven that between different varieties of the same fruit there may be a great variation in the levels of amines (22).
    However, the biogenic amines present in many foods can also have an exogenous origin, being generated by decarboxylation of the precursor amino acids. Thus, they appear in a wide variety of foods, whether they are unfermented (fish, dairy products, meat, etc.) or those that have undergone some type of fermentation during their production (wine, beer, cheese, etc.) Its accumulation is an aspect to be taken into account due to the toxicological problems that its ingestion can generate.
    There are factors that will limit the accumulation of biogenic amines (especially those that are due to microbial activity) in food. Thus, for example, the availability of substrate, the pH of the medium, the concentration of salts and the temperature will also have a great influence on the production of amines. Pyridoxal phosphate is a factor required for the decarboxylation of amino acids in most bacteria and, therefore, their presence or absence will be decisive for the synthesis of biogenic amines. Presence of amines in unfermented foods
    The presence of biogenic amines in unfermented foods is an indicator of the presence of unwanted microbial activity, and, therefore, the level of amines present can be used as an indicator of food spoilage by microorganisms. Normally, the amount of histamine, putrescine and cadaverine increases during food spoilage, while spermine and spermidine levels decrease. Due to this characteristic, the Biogenic Amines Index (IAB), defined by Karmas (23) and expressed in mg / Kg, has been used to calculate the quality grade of a food. Currently the detection and quantification of biogenic amines


    It is performed using HPLC techniques, as will be described later in this work.
    IAB = [histamine] + [utrescine] + [cadaverine] / 1 + [spermine] + [spermidine]
    Fish or meat with an IBA value below 1 are considered top quality, 5 while values around 10 indicate a poor microbiological quality of the product.
    Among the unfermented foods that accumulate endogenous amines, fish and meat stand out, which are characterized by accumulating large concentrations of histamine and tyramine, respectively, during storage, even if it is not prolonged. Moreover, it has been shown that the accumulation of amines as a consequence
    10 of the microbial activity cannot be avoided with vacuum packaging of the product (24). The only effective measure to prevent the accumulation of biogenic amines is the storage of products at low temperatures (24). Presence of amines in fermented foods
    During the fermented food preparation processes, the product is usually
    15 incubated for days, weeks and even months, until reaching the necessary degree of fermentation or maturation, so that a greater proliferation of microorganisms and, therefore, a greater presence of biogenic amines in these products can be expected. In addition, the development of these foods requires the participation of microorganisms that modify the properties of the original raw material, so that the
    The elimination of these microorganisms would distort the quality, properties and characteristics of the products. This is what happens with foods as popular as cheese, sausages, sauerkraut or wine (25).
    The most important amine that accumulates in cheese during ripening is tyramine (26) and, to a lesser extent, phenylethylamine. During this process, casein is
    25 slowly degraded by proteolytic enzymes, thereby increasing the content of free amino acids that may be susceptible to serve as substrate for specific bacterial decarboxylases, to give rise to the formation of CO2 and an amine.
    On the other hand, the amine that accumulates mostly in sausages is histamine (21), but in this case its accumulation will depend on the manufacturing process, the type of meat used, its proportion and the quality of it, as well as of the ripening time. In the case of sausages, the amount of accumulated amines in the final product can be greatly reduced through the use of starter cultures


    (starters) that contain the microorganisms suitable for carrying out the required fermentation, but that do not produce these undesirable amines. This measure, which has meant a great advance in the regularization of fermentation processes, is not always effective, since the endogenous microbial flora (present in the original raw materials) may already be capable of producing biogenic amines by itself.
    A group of important products regarding the accumulation of biogenic amines are fermented beverages. Such is the case of beer and especially wine. The presence of amines in these drinks is responsible for the characteristic headache that is experienced after abusive consumption (26).
    In the wine there are mainly histamine, tyramine and putrescine, in very variable amounts depending on the type of wine. The concentration of these amines is low during alcoholic fermentation and increases during malolactic fermentation. This explains why red wines have higher concentrations of these amines compared to white wines, since the latter do not suffer from malolactic fermentation. After this fermentation, the wine is usually sulphited to eliminate undesirable populations of bacteria and yeasts from that moment, but even so, the concentration of biogenic amines continues to evolve and can reach up to 50 mg / l during aging (27 ). Although there is no definite regulation regarding the concentration of biogenic amines in wine, there are countries that have established import limits (Canada and Switzerland 10 mg / l, Netherlands 5 mg / l). This is due to the fact that the presence of amines in wine entails more risk than in other foods, since when alcohol interacts with them, the mechanisms of detoxification of the organism will be affected and the possibilities of poisoning due to the intake of amines
    The levels of biogenic amines in alcoholic beverages made by fermenting with yeasts are generally lower than those found in beverages in whose production lactic acid fermentation takes place (except yogurt), but even so they can contain considerable amounts of putrescine, cadaverine , histamine and tyramine (2).
    In summary, the main conclusions that can be drawn about the presence of biogenic amines in food are the following:
    - Most foods are susceptible to deterioration by the action of microorganisms capable of producing biogenic amines.


    - High concentrations of certain amines in food can be harmful to health
    - Great importance should be given to the evaluation of the amines content of foods, as well as the presence of other agents that enhance their effect, such as other amines, alcohol or certain drugs.
    - High concentrations of biogenic amines in food can and should be avoided, through good manufacturing and storage practices (control of hygiene, contamination, temperature, etc.)
    - In the production of foods that require acid lactic fermentation, starter cultures of microorganisms that are amino acid negative decarboxylase should be used. In this sense, it would be very useful to prepare starter cultures that have in their composition microorganisms that not only do not produce these amines, but are capable of degrading them. Toxicology of biogenic amines.
    As indicated previously, histamine, tyramine, tryptamine and �phenylethylamine are biologically active amines that can cause important physiological effects in humans, both psychoactive (neuromodulatory) and vasoactive. The consumption of foods with a high content of biogenic amines can cause a large number of pharmacological effects (Table 1) that characterize certain diseases, such as for example histamine poisoning or the "cheese reaction" caused by tyramine intake. In addition, amines are currently being studied as precursors of carcinogenic compounds (21).
    Among all the biogenic amines present in food, it should be noted for their high toxicity (consequence of the greater number of physiological effects they cause), histamine and tyramine.
    Histamine is a very biologically active amine because it performs many actions within the body. Although mast cells and blood basophils contain large amounts of histamine, it is stored in characteristic granules and will not be released unless special reactions occur (allergic reaction). Histamine can stimulate the heart rhythm and this effect results in the release of adrenaline and norepinephrine by the adrenal glands, excitation of the uterine musculature and respiratory tract, stimulation of both motor and sensory neurons and


    control of gastric secretion (28). Therefore, it is not surprising that histamine poisoning manifests skin symptoms such as hives and the appearance of edema or rashes, as well as gastrointestinal symptoms, such as nausea, vomiting and diarrhea. Other symptoms such as hypotension, headache or palpitations may also occur (29).
    Table 1. Biogenic amines present in food and its effects on the organism.
    Biogenic amine Pharmacological effects
    Histamine Adrenaline and norepinephrine release Stimulation of uterine, intestinal and respiratory tract muscles.
    Putrescina tyramine and cadaverine Stimulation of sensory and motor neurons. Increase in blood pressure. Control of gastric secretion. Peripheral vasoconstriction Increased heart and respiratory rate. Stimulation of lacrimation and salivation. Norepinephrine release Migraine. Hypotension Bradycardia Enhancement of the effect of other amines.
    �-phenylethylamine Norepinephrine release Increase in blood pressure. Migraine.
    Tryptamine Increase in blood pressure.
    Histamine is a very biologically active amine since it performs many actions
    10 within the organism. Although mast cells and blood basophils contain large amounts of histamine, it is stored in characteristic granules and will not be released unless special reactions occur (allergic reaction). Histamine can stimulate the heart rhythm and this effect results in the release of adrenaline and norepinephrine by the adrenal glands, muscle excitation
    15 uterine and respiratory tract, stimulation of both motor and sensory neurons and control of gastric secretion (28). Therefore, it is not surprising that in poisoning


    With histamine, skin symptoms such as hives and the appearance of edema or rashes, as well as gastrointestinal symptoms, such as nausea, vomiting and diarrhea. Other symptoms such as hypotension, headache or palpitations may also occur (29).
    Despite the toxic nature caused by an excess of histamine, the presence of this amine in food does not have to be dangerous. There are many foods that contain small amounts of histamine and, therefore, will be easily tolerated by the body thanks to the existence of efficient detoxification systems in the digestive tract, which will metabolize both ingested histamine and formed histamine by the intestinal flora itself. This detoxification system is composed of two different enzymes: diamino oxidase and histidine-N-methyltransferase. These two enzymes are responsible for converting histamine into products without biological activity, and their efficiency is high when there is a normal consumption of amines in the diet. However, these mechanisms are less effective if large amounts of amines are ingested, or in the presence of other amines that potentiate the toxic effect caused by histamine.
    The presence of tyramine induces the release of norepinephrine from the sympathetic nervous system, which will cause an increase in blood pressure through peripheral vasoconstriction and an increase in heart rate. It can also cause pupil dilation, increased salivation, breathing and blood sugar levels (30).
    Regarding the presence of tyramine in food, it is worth noting only its own toxicity, but also its harmful effects in the presence of monoamine oxidase (MAO) inhibitors, which can cause critical hypertension states (31-33). The MAO is responsible for the oxidative deamination of the amines derived from food, and they constitute the best endogenous defensive system against these toxic compounds, allowing their degradation before they pass into the bloodstream. The use of MAO inhibitor drugs during the treatment of certain mental illnesses (depression, schizophrenia, etc.) will cause the inhibition of this natural detoxification system and, as a consequence of this inhibition, the accumulation of tyramine in the blood, which will generate critical states of hypertension in patients. The first food that was associated with this process was cheese, so this increase in blood pressure is known as a "cheese reaction" and can cause severe headaches, brain hemorrhages or heart failure (1).


    In addition to being able to act as toxic agents, the involvement of amines in the synthesis of derivatives that could act as mutagenic agents is currently being studied. By adding nitrates to foods as preservatives, and by reacting these with the amines present in these foods, N-nitrosamines are generated, which are carcinogenic compounds and constitute a serious risk to human health. Some examples of these processes are the reaction between tyramine and nitrites that will cause 3diazothiramine, a compound that induces the appearance of cancer in the oral cavity of rats (30), and the reaction between tyramine and nitrates that when it occurs in acidic conditions gives place a mutagenic compound identified as 4- (2-aminoethyl) -6-diazo-2,4-cyclohexadienone (34). Production of biogenic amines in food.
    As we have already indicated, most of the amines present in food are generated by decarboxylation of their corresponding precursor amino acids, through the action of specific enzymes (amino acid decarboxylases) produced by the microorganisms present in those foods. These decarboxylases are present in a large number of species belonging to different bacterial genera.
    For the formation of biogenic amines in food, the following requirements are needed: a) availability of free amino acids (generally caused by proteolytic action); b) presence of microorganisms possessing decarboxylase enzyme (positive decarboxylase); and c) that the appropriate physicochemical conditions exist that allow both bacterial growth and synthesis and decarboxylase activity. The positive decarboxylase microorganisms may be part of the endogenous flora of the food, or be introduced by contamination during the processing or storage processes. In the case of food and beverages that undergo fermentation processes, the introduction of starter cultures can affect the production of biogenic amines interacting, directly or indirectly, with both the endogenous flora and the contaminating flora (2).
    Most decarboxylases maintain their activity even after the pasteurization process. This fact, together with the fact that most of the amines are thermostable, implies not only that the amount of amines already formed in the food will not be eliminated with the pasteurization process, but that it will even increase during storage.
    Decarboxylation of amino acids.


    In the decarboxylation of amino acids the elimination of the �-carboxyl group of the amino acid in question occurs to give rise to CO2 and the corresponding amine. This reaction is catalyzed by bacterial decarboxylases that are specific for each amino acid. Thus, for example, ornithine can be degraded to putrescine and lysine to cadaverine by the action of ornithine decarboxylase (35) and lysine decarboxylase (36) respectively. In the same way and provided that the appropriate conditions exist, histidine, tyrosine, tryptophan and phenylalanine will be decarboxylated to histamine, tyramine, tryptamine and �-phenylethylamine by the action of histidine decarboxylase (37), tryptophan decarboxylase ( 38), tyrosine decarboxylase (39) and phenylalanine decarboxylase
    (40) respectively. There is also an aromatic L-amino acid decarboxylase (AADC) that catalyzes the irreversible decarboxylation reaction of L-DOPA to dopamine, but which has a lower substrate specificity than the previous ones, since it is capable of carrying out the decarboxylation of both Tyrosine, such as phenylalanine, 5-hydroxytryptophan and tryptophan (41).
    Amino acid decarboxylases have been widely studied in recent years (42). Most of them use pidoxal-5’-phosphate or pyruvate as coenzyme, and are dependent on vitamin B6. Currently there are a large number of sequences corresponding to amino acid decarboxyls that are collected in the different databases. Their comparative analysis has allowed them to be classified into four groups (Table 2) and identify functionally important regions within those sequences.
    Two mechanisms of action for the decarboxylation of amino acids have been proposed, one is based on a pyridoxal phosphate-dependent reaction, and another requires a pyruvate molecule as a cofactor (43).
    In pyridoxal-5-phosphate-dependent decarboxylation reactions, a Schiff base is formed due to the reaction of the aldehyde group of the pyridoxal with one of the amino groups belonging to a lysine located in the active center of the enzyme (internal aldimine) . The carbonyl group of pyridoxal-5-phosphate reacts easily with amino acids to form a new Schiff base (external aldimine) that functions as an intermediate, and allows these to be subsequently decarboxylated to give rise to the corresponding amine and the pyridoxal molecule original phosphate
    In decarboxylation reactions not dependent on pyridoxal-5-phosphate, a pyruvate molecule (44) is involved. In this case it will be the pyruvil group that covalently binds to the amino group of the amino acid forming a Schiff base allowing


    which are subsequently decarboxylated by a reaction very similar to the decarboxylation reaction dependent on pyridoxal-5-phosphate.
    The decarboxylation of amino acids has an important energy function for bacteria in those nutrient-poor environments, since, being a reaction
    5 endothermic, it constitutes a system of generation of ATP while leading to the synthesis of amines. On the other hand, the formation of amines will cause an increase in the pH of the medium, favoring bacterial growth. For all these reasons, the decarboxylation of amino acids can be considered as a very advantageous mechanism that allows the adaptation to a large number of microorganisms.
    10 Table 2. P-pyridoxal-P-dependent amino acid decarboxylases with their known sequences (42).
    Enzyme Access numbers and sources
    Group I
    Glycine decarboxylase (EC 1.4.4.2) Group II P23378, human; P15505, chicken; P26969, Pisum sativum
    Glutamate decarboxylase (EC 4.1.1.15) M84024, Escherichia coli (GAD-a); M84025, Escherichia coli (GADp); P20228, Drosophila melanogaster; mouse "; JH0423, rat (GAD65); P18088, rat (GAD67); P14748, cat; M74826, human (GAD65); M81883, human (GAD67)
    Histidine decarboxylase (EC 4.1.1.22) P28577, Enterobacter aerogenes; P28578, Klebsiella planticola; P05034, Morganella morganii; X70644, Drosophila melanogaster; P23738, mouse; P16453, rat; P19113, human.
    Tyrosine decarboxylase (EC 4.1.1.25) M96070, Petroselinum crispurn '
    Decarboxylase of aromatic amino acids (EC 4.1.1.28) S19796, Caenorhabditis elegans; P05031, Drosophila melanogaster; P14173, rat; P22781; Guinea pig, P80041, P27718 pig, bovine; P20711, human
    Tryptophan decarboxylase (EC 4.1.1.17) P17770, Catharanthus roseus
    Group III
    Ornithine decarboxylase (EC 4.1.1.18) P21169, Escherichia coli; P24169, Escherichia coli (inducible)


    Lysine decarboxylase (EC 4.1.1.19) P05033, Hafiia alvei; P26934, Bacillus subtilis; P23892, Escherichia coli
    Arginine decarboxylase (EC 4.1.1.17) P28629, Escherichia coli
    Group IV Ornithine decarboxylase (EC 4.1.1.19) P28629, Escherichia coli (biodegradative) P07805, Trypanosoma brucei; P27116, Leishmania donovani; P27121, Neurospora crassa; P08432, Saccharomyces cerevisiae; P27120, Xenopus laevis; P27118, chicken; P00860, mouse; P27119, Mus pahari; P09057, rat; P14019, hamster; P27117, bovine; P11926, human.
    Arginine decarboxylase (EC 4.1.1.20) P21170, Escherichia coli; P22220, Avena sativa Microorganisms producing biogenic amines.
    Enzymes with decarboxylating activity have been found in a large number of bacteria, including species of enterobacteria, pseudomonadids, 5 enterococci and lactobacilli, among others (44-49).
    There are several enterobacteria with decarboxylastic activity (mainly related to the production of cadaverine and putrescine). Studies carried out in vitro with Enterobacter cloacae and with different species of Serratia, as well as in Citrobacter freundii and Enterobacter aerogenes, have revealed the ability of these microorganisms to form large amounts of putrescine and cadeverin (46). Other enterobacteria are characterized instead by being large producers of histamine; such is the case of Klebsiella oxytoca (47), Escherichia coli (48) or Morganella morganii (46). Although these enterobacteria are found in very low proportion in the final products, poor storage practices, or uncontrolled fermentation during
    15 the elaboration, can cause an important proliferation of the same.
    There are other microorganisms that produce biogenic amines of different nature. Such is the case of Pseudomonas putrefaciens, Aeromonas hydrophila and Plesiomonas shigelloides, microorganisms that are usually found in fish in poor condition (21).
    20 Acid-lactic bacteria are used profusely in processes intended to obtain various foods (sausages, wine, cheeses, etc.) by fermentation, and although not


    They can be considered toxic or pathogenic species, many of them are capable of producing biogenic amines. Thus, for example, some strains of Lactococcus and Leuconostoc produce appreciable amounts of tyramine and histamine (39) and strains of lactobacilli belonging to the Lactobacillus buchneri, L. alimentaius, L. plantarum, L. curvatus, L. farciminis, L. bavaricus, L. homohiochii, L. reuteri and L. sakei, are large producers of amines, especially tyramine (49-51). Many of these lactic bacteria are used in the manufacture of cheeses, and are included in the starter cultures used by the dairy industry. Such is the case of Lactococcus lactis subsp. lactis, Streptococcus faecium, S. mitis, Lactobacillus helveticus, L. casei, L. acidophilus and L. arabinose, all identified as histamine producers (26).
    When studying the accumulation of biogenic amines in meat, it has been observed that species such as Carnobacterium. divergens, C. piscicola and C. gallinarum are responsible for the presence of high concentrations of tyramine in it (52).
    Other studies have shown that Enterococcus faecalis is responsible for the accumulation of biogenic amines (�-phenylethylamine among others) in fermented foods (53).
    The production of biogenic amines by fungi and yeasts in fermented foods has also been revealed. This is the case of Debaryomyces and Candida, two yeasts isolated from fermented meat, which have a greater histidine decarboxylase activity even than that observed in acid-lactic bacteria (51). Analytical methods applied to the assessment of the capacity of amines production by microorganisms: Detection and quantification.
    Due to the toxic effects that the amines present in food can cause, it has been necessary to design methods and techniques that allow the detection of amines producing capacities in the microorganisms used in the processes of manufacturing food products, as well as quantifying the presence of Amines in these foods. Methods used to detect microorganisms with aminobiogenic capacity.
    Several biochemical methods have been developed that allow the detection of strains producing histamine and tyramine from fermented meats and cheeses (46, 54-57). These methods are based on an assay that involves the use of a solid medium containing the amino acid precursor of the amine to be investigated and a pH indicator. Since the formation of amines from amino acids implies an elevation of the pH of the medium, if the


    Study strain has the capacity to form amines, this will be reflected by a color change of the pH indicator present in the medium.
    There are also molecular detection methods that are based on the design of specific primers for the sequences of the genes that code for the decarboxylases responsible for the formation of amines. So far, three methods of gene detection involved in the production of amines have been developed:
    Detection system of the hdc gene that encodes a histidine decarboxylase (55). For the design of the primers, the nucleotide sequences of the Lactobacillus sp30A, Clostridium perfringens hdcA gene and the histidine decarboxylase amino acid sequences of these two microorganisms were compared with those of Lactobacillus buchneri and Micrococcus. The analysis of the different sequences revealed the existence of a high degree of identity between the hdc genes of the different lactic bacteria, which allowed designing specific primers for the detection of this gene.
    Detection system of the tdc gene encoding a tyrosine decarboxylase (56). For the design of the primers, the nucleotide sequences of the tdc gene of Enterococcus faecalis, Carnobacterium divergens and Lactobacillus brevis were compared.
    Odc detection system encoding an ornithine decarboxylase (57). The nucleotide sequence of the ornithine decarboxylase gene in an Oenococcus oeni strain was used for the design of the primers. Analytical methods used to detect amines in food.
    So far, several methodologies have been described that allow us to detect and quantify the presence of amines in food. Although the most commonly used method is high performance liquid chromatography (HPLC), the first valuation of amines was performed by thin layer chromatography and aimed at determining histamine in cat food (58). Currently, many specific methods of detection and quantification of amines in many foods have been described, all based on HPLC techniques (59-64). In recent years, commercial ELISA methods have appeared for the analysis of histamine in wines and other foods (65).
    Some countries have set maximum values for the presence of amines in certain foods. Thus, for example, Switzerland has established a maximum concentration of 10 mg / L of histamine in wine as tolerable values for human health, while other countries such as Germany, Belgium and France recommend lower maximum values (2 mg / L, 5 -6


    mg / L and 8 mg / L), respectively. No maximum limits have been imposed on the rest of biogenic amines, although given the importance of the effects of histamine in the organism, the prompt appearance of recommendations or regulations on maximum permitted values for this amine is very likely. Degradation of biogenic amines.
    The presence of amines in the environment is increasing considerably in recent years as a result of industrial activity. Some of these amines, especially methylated ones, are very volatile and are involved in the formation of nitric oxide, an important gas causing the “greenhouse effect” (66-67). On the other hand, other amines that are present in a wide variety of foods can cause, as explained above, toxic effects in the body.
    The identification of the pathways and enzymes involved in the metabolism of these amines can be of great help for the conversion of these toxic compounds into other less harmful compounds and thus avoid their damaging effect on organisms and the environment.
    Many microorganisms can oxidize primary amines by generating products that can be used as a source of carbon and energy, as a source of nitrogen, or as both (68) and that are no longer toxic.
    R-CH2NH2 + H2O
    RIGHT + 2 [H] + + NH3
    This mechanism of oxidation of primary amines is a widely distributed process in nature, since it has been identified in both eukaryotic and prokaryotic organisms and is catalyzed by different enzymes, including quinoproteins amino oxidases and quinoproteins or amino dehydrogenases quininoproteins ( 69). Both amino oxidases and amino dehydrogenases catalyze the conversion of amines into their corresponding aldehydes. The difference is that while amino oxidases (present in both eukaryotes and prokaryotes) produce toxic peroxides, amino dehydrogenases (present exclusively in bacteria) produce reduced equivalents that directly transfer the e-to the respiratory chain (70-71).
    As for the location of these enzymes; it seems that they have a periplasmic location in the G- microorganisms; some are soluble and others are attached to the outer face of the cytoplasmic membrane. However, in G + microorganisms these enzymes are found in the cytoplasm or attached to the cytoplasmic membrane by their face


    internal On the other hand, in certain yeasts, it has been found that its location is exclusively peroxisomal (69).
    As mentioned above, the enzymes responsible for the oxidative deamination of the amines use different cofactors that are reduced after the oxidation of the substrate and that participate in the transfer of e to one or two exogenous acceptors, such as cytochrome c, cupredoxins (azurine or amocyanine) or molecular oxygen. Depending on who is a natural e-acceptor, the natural distinction can be made between amino oxidases and amino dehydrogenases.
    Most of the enzymes involved in the oxidation of the different amines to their corresponding aldehydes are oxidoreductases that are characterized by using cofactors other than NAD (P) + or FAD + (although there are exceptions of flavoproteins amino oxidases that use FAD as cofactor and that will be explained later). These enzymes are called quinoproteins, because they use cofactors that have a quinone group (72) such as TPQ (topaquinone), TTQ (tryptophan tryptopilquinone), LTQ (lysine tyrosylquinone) or CTQ (tryptopilquinone cysteine). These cofactors are characterized in that each of them is formed from one or two amino acids present in the enzyme itself after undergoing a post-transcriptional chemical modification (73-75).
    Depending on the identity of the cofactor used, we can differentiate several types of enzymes that catalyze oxidative deamination reactions of amines: amino oxidases, amino dehydrogenases and quinoproteins amino dehydrogenases Quinoproteins amino oxidases.
    These enzymes are also called copper dependent amino oxidases (Q-AmO). As for the structure, they are generally homodimers formed by two identical subunits of the same size. They are characterized in that in each subunit they present a TPQ molecule, an enzymatic cofactor that is formed by post-transcriptional modification from one of the tryptophan residues existing in the enzyme. They also have a copper molecule, Cu (II) that is coordinated with three histidine residues and two water molecules (76-77). Cu (II) is necessary, both for the biosynthesis of the enzyme cofactor, and for enzymatic catalysis (77).
    These enzymes are characterized because they are the only ones that are widely distributed in both bacteria and higher organisms and catalyze the oxidation of a large number of primary amines. Thus, a Q-AmO has been characterized in Klebsiella oxytoca and


    which is related to the degradation of PhEtNH2 and tyramine (69). When E. coli (78)
    o Klebsiella aerogenes (K. pneumoniae) grown in the presence of tyramine has also been observed to express a Q-AmO similar to that of Klebsiella oxytoca (69). Likewise, other Q-AmO have been identified, in G + bacteria and in yeasts. For example, in Arthrobacter globiformis a Q-AmO containing copper and using as a substrate PhEtNH2 (76, 79) has been characterized, and in the yeast Hansenula polymorpha a methylamine oxidase implicated in the degradation of methylamine (77) has been identified.
    The catalytic reaction carried out by these amino oxidases quinoproteins, takes place through a ping-pong transamination mechanism that can be divided into two hemirreactions or stages, depending on the oxidation state of the cofactor (76, 79).
    I. Reductive hemirreaction: Oxidative deamination of the substrate.
    Ia. The TPQ cofactor participates in this stage by forming a covalent bond with the substrate (through the C5 carbonyl group), resulting in the formation of a Schiff base with the substrate.
    Ib. Schiff's base is then deprotonated by an aspartate residue near the active site and at the same time the cofactor is reduced. In this step another Schiff base is formed, but now it is the product that forms the Schiff base.
    Ic. Subsequently, hydrolysis of this Schiff base occurs, releasing the aldehyde and leaving the aminoresorcinol form of the reduced TPQ.
    The stereospecificity of proton abstraction from the C1 position of the substrate has been studied in different enzymes, both bacterial and plant and animal. It has been found that the specificity varies depending on the nature of the enzyme and the substrates used. Thus, for example, in the case of dopamine, sometimes the pro-R proton and sometimes the pro-S are abstracted, depending on whether the reaction takes place in plants or animals (78).
    II. Oxidative hemirreaction: reduction of molecular oxygen.
    In this stage the re-oxidation of the reduced TPQ (TPQred) and the release of ammonium occurs. The participation of Cu (II) in this stage was pointed out after the discovery of the Cu (I) / semiquinone topa (TPQsq) form. Because the transfer of e-from TPQred to Cu (II) to form Cu (I) / TPQseq is very fast, and because Cu (I) reacts easily with O2, the Cu (I) / state TPQseq has been proposed as a kinetically intermediary


    competent with Cu (I) that oxidizes directly to O2. Thus, a balance is established between the form Cu (II) / TPQred and Cu (I) / TPQsq for the intramolecular transfer of e (76). Subsequently, the aldehyde generated after deamination of the substrate is oxidized to the corresponding acid. 5 Quinohemoproteins amino dehydrogenases.
    They are heterotrimeric enzymes consisting of three subunits (���) that use CTQ as a cofactor, which is formed from a cysteine residue present in the small subunit (�). The possibility that a fourth protein, whose function is still unknown, could intervene facilitating the formation of the cofactor is not ruled out.
    10 These enzymes have, in addition to the quinone group, two heme c groups, linked to one of the enzyme subunits, which act as active redox groups (69, 79). In this case, the e-released during the oxidation process are ultimately transferred to a cytochrome oxidase present in the transport chain, via the cytochrome c550, as seen in Paracoccus denitrificans; or via the azurine as in P. putida (81-82).
    15 To this group belongs the quinohemoprotein amino dehydrogenase (QH-AmDH) of Pseudomonas putida U, responsible for the oxidative deamination of 2-phenylethylamine (83) and that of Paracoccus denitrificans. The latter bacterium has two enzymes with amino dehydrogenase activity in its periplasm. A methylamine dehydrogenase (MADH) that recognizes methylamine and a QH-AmDH that recognizes aliphatic primary amines and
    20 aromatic, although it seems to show greater activity with n-butylamine and benzylamine (82, 84-85). Another QH-AmDH within this group has been purified from P. putida IFO 15633 and ATCC 12633 (70, 89-81).
    The four genes encoding the QH-AmDH of P. putida and Paracoccus denitrificans have been identified and sequenced. ORF1 encodes the subunit �, which has two
    25 heme groups that act as redox groups during oxidation; ORF2 encodes a protein, whose function is unknown, but which plays an essential role in the oxidation of these compounds; ORF3 encodes the small subunit (to which the cofactor is attached); and ORF4 encodes the subunit �. These genes are transcribed together, so they constitute a single operon (70).
    30 The mechanism of reactions catalyzed by this group of enzymes is as follows (8182) (Figure 5):


    I. The reaction is initiated by the nucleophilic attack of the nitrogen of the C6 amino group belonging to the carbonyl group of the CTQ cofactor, resulting in the formation of a carbinolamine intermediate (a).
    II. Carbinolamine loses a water molecule and an imine (b) is formed.
    5 III. Next, an active site residue (probably Asp33�) abstractsa proton of the C� of the amine to form the carbanionic intermediate (c). To theat the same time the CTQ is reduced after capturing a molecule of H2O, givingplace of intermediary d.
    IV. The product resulting from this oxidation, the aldehyde (e), is finally
    10 released by hydrolysis of the new imine bond that had formed between the C� and the amino group.
    It seems, therefore, that there is an intramolecular transfer of e-from the complex generated by the reduced substrate-CTQ to heme I, from here to heme II, and from there an intermolecular transfer to the exogenous acceptor occurs. This oxidative reaction will have
    15 place through two sequential reactions until re-oxidation of the cofactor and heme groups occurs.
    It has been proven that P. putida QH-AmDH can be inhibited by pnitrophenylhydrazine. This compound binds to the active site of the enzyme, located between the subunit � and �, of the same m as the substrate would. This union causes some
    20 important changes in the CTQ cofactor and also conformational changes in the side chains of the residues belonging to the amino acids of the active site, thus causing the modification of the active center and, thereby, the loss of enzymatic activity (81). Quinoproteins amino dehydrogenases.
    25 These enzymes (Q-AmDH) use as a cofactor the TTQ that is formed from two tryptophan residues present in the small subunit of the enzyme. They have a structure �2�2, where the cofactor is attached to the subunits � (84).
    This group includes methylamine dehydrogenase (MADH) ofMethylobacterium extorquens AM1 or Paracoccus denitrificans (66) and the amino
    30 aromatic amino acid dehydrogenase (ADHD) described in Alcaligenes faecalis and that


    It seems to be involved in the catabolism of different primary amines such as PhEtNH2, tyramine and tryptamine (67).
    These enzymes usually use blue copper proteins of type I (cupredoxins) as an e-acceptor. Thus, MADH use amycinin and AADH azurine (84, 86).
    5 Regarding their genetic organization, nine genes are identified in Alcaligenes faecalis that are transcribed in the same direction and are apparently involved in the degradation of amines (ORF1, aauBEDA, ORF2, ORF3, ORF4 and hemE). The aauA and aauB genes encode the small and large subunits of the AADH, respectively, and are homologous to the mauA and mauB genes encoding MADH. The genes AauE and AauD are
    10 homologues of mauE and mauD and, apparently, encode proteins that perform the same function (transport and folding of the small subunit in the periplasm, respectively). The homologous genes of mauF, mauG, mauL, mauM and mauN, which participate in the biosynthesis of the TTQ cofactor, are not found in the aau cluster. However, other ORFs, such as ORF2, that encode a cytochrome have been identified.
    Type C monoheme, and ORF1, ORF3, and ORF4, which have not yet been assigned any function, but which appear to be essential for the degradation of certain amines, since their disruption is lethal in this bacterium (66 ).
    The catalytic mechanism described for ADHD and MADH is similar to that proposed for QH-AmDH, although these enzymes lack both heme groups.
    In the reactions catalyzed by the Q-AmDH, the transfer of 2e-from the reduced TTQ cofactor to the external e-acceptor is produced by two consecutive reductions. The e-acceptor is azurine in the case of ADAs and amycinin in the case of MADH. Both physiological acceptors mediate the transfer of e-up to different types of soluble cytochrome c. In the case of ADHD, the reaction is favored
    25 when the ionic strength is high, while the activity of the MADH decreases when the ionic strength increases (86).
    In both cases, after the release of the product, a quinol form of the TTQ is generated. For example, in the reduction of TTQ by methylamine, formaldehyde is released but the amino group remains attached to the cofactor. Therefore, the reduction of the substrate implies the
    Formation of an aminoquinol form of TTQ in which one of the oxygens of the carbonyl group is replaced by the amino group derived from the substrate. The release of the amino group occurs after the second transfer of e-allowing the regeneration of the quinone (86).

    Tyramine deamination.
    The deamination of tyramine is carried out primarily by amino oxidases. These enzymes are widely distributed in all living organisms and allow the conversion, by oxidation, of tyramine and other primary amines into their corresponding aldehydes by releasing a molecule of hydrogen peroxide (hydrogen peroxide). Due to the high reactivity of these products (aldehyde and hydrogen peroxide), they must be rapidly transformed, by reactions mediated by a NAD-dependent phenylacetaldehyde dehydrogenase and by a cytoplasmic localization catalase respectively.
    R-CH2NH2 + O2 + H2O
    RCHO + NH3 + H2O2
    The amino oxidases (AOs) that carry out this tyramine deamination reaction can be classified into two subgroups: Flavoproteins amino oxidases (EC 1.4.3.4), which use FAD as a cofactor, and quinoproteins amino oxidases (EC 1.4. 3.6) that, as explained above, are characterized by containing Cu (II) and TPQ as cofactor.
    Among the amino oxidases flavoproteins, it is worth mentioning the monoamine oxidases (MAOs) found in countless organisms, and such as the tyramine oxidase of Klebsiella aerogenes (87), that of Salmonella typhimurium (88) or that of Sarcina lutea (89) , are capable of oxidizing tyramine, dopamine and norepinephrine. These enzymes are induced by the presence of tyramine and their genes are subjected to catabolic repression in the presence of glucose. But MAO is also present in higher organisms, and in them two isoenzymes have been described, MAOA and MAOB, this classification was made based on the specific inhibition of MAOA by the antidepressant clogiline and MAOB by deprenil (90) . Both isoforms are mitochondrial enzymes that play an important role in the inactivation of biogenic amines, such as adrenaline, norepinephrine, serotonin, dopamine and various "trace amines" (including tyramine).
    These two isoforms of the MAO are distributed very heterogeneously in the different tissues of the human body, for example, in the liver the MAOB form is predominant, while in the mucosa of the duodenum the majority isoform is the MAOA, being this isoenzyme the responsible for deamination of the amines introduced with the diet. For this reason critical hypertension processes will be caused in those patients who consume foods rich in amines and continue treatments with MAOA inhibitors. This is one of the reasons why MAO inhibitor drug treatments are currently aimed at inhibiting the activity of the MAOB isoform (91).


    But tyramine deamination is not always catalyzed by amino oxidases flavoproteins. In many organisms, amino oxidases quinoproteins have been identified, (whose mechanism of action has already been explained above), responsible for tyramine deamination, such as Klebsiella oxytoca (69), E. coli (78) and Euphorbia characias (92). In the latter case the enzyme is a soluble homodimer that contains in the active center a Cu (II) ion and TPQ as cofactor.
    In contrast, other G-bacteria deaminate tyramine through the activity of amino dehydrogenase enzymes. This is the case of Alcaligenes faecalis and Pseudomonas aeruginosa (69) that have a periplasmic amine dehydrogenase that uses TTQ as a cofactor.
    Finally, it is worth mentioning the existence of another tyramine deamination system that is mediated by a peroxidase (EC 1.11.1.7) and that in the presence of H2O2 catalyzes, as a previous step to the oxidation of tyramine, the formation of an intermediate dimer (dithyramine). This peroxidase requires the presence of H2O2 to carry out its action and it has been proven that this enzyme works cooperatively with amino oxidases, the latter acting as donors of H2O2. Subsequently, the intermediate generated will undergo oxidation mediated by the action of the corresponding (flavoprotein or quinoprotein) amino oxidase in the two amino groups, resulting in the formation of a di-phydroxyphenylacetaldehyde. (91-93). It could also happen that this peroxidase used aldehyde and H2O2 as substrates obtained as products of tyramine deamination by amino oxidases, giving rise to di-p-hydroxyphenylacetaldehyde without prior formation of dithyramine (Figure 6). Microbial degradation of tyramine.
    As indicated above, tyramine can trigger toxic effects in patients treated with MAO inhibitors, or in healthy people when their dietary intake is very high. Therefore, knowledge of the pathways responsible for the degradation of this compound by a microbial agent can have important applications in the field of public health. On the other hand, the transfer of the genes responsible for this pathway could provide the ability to degrade tyramine to organisms used in the food industry, thereby reducing the concentration of tyramine in foods (cheeses, wines, etc.). Finally, the fact that tyramine is an important “trace” amine with function in the central nervous system and that it has structural similarity with important neurotransmitters (for example, dopamine), makes the study of the metabolic pathway responsible for degradation of this compound may have interesting


    Pharmacological applications, and can be used, for example, for the treatment of certain neurological diseases.
    In certain microorganisms such as Klebsiella aerogenes (87), Micrococcus luteus (68), Salmonella typhimurium (88) and E. coli (63) the presence of enzymes with oxydase tyramine activity that are capable of oxidizing tyramine to 4-hydroxyphenylacetaldehyde has been described. In addition, several of the genes and proteins responsible for the degradation of this compound have already been characterized (63). Thus, for example, in E. coli K12, the degradation of tyramine and other related aromatic amines (2-phenylethylamine, dopamine) requires the sequential action of a monoamine oxidase (member of the family of amino oxidases quinoproteins) encoded by the maoA gene, and a phenylacetaldehyde dehydrogenase, a product of the padA gene, which transforms the phenylacetaldehyde generated by the MAOA protein into 4-OH-PhAc, which will later be degraded by a specific route (95). Upstream of the maoA gene is the maoB gene that is transcribed in the same direction and encodes a transcriptional regulator belonging to the AraC family. The product of the maoB gene activates the expression of maoA, but not that of the padA gene, which is transcribed in the opposite direction. This indicates that, although these genes are physically close to the chromosome and are part of the same catabolic pathway, they do not constitute an operon (95). All microorganisms in which tyramine degradation has been studied use the same enzymes described in E. coli. Histamine catabolism in P. putida U.
    Pseudomonas putida U (Spanish Culture Collection Type CECT 4848) is a bacterial strain capable of growing in numerous culture media with defined chemical composition (minimum media or MM), containing as carbon sources different molecules that are hardly degradable, some of which they can even be harmful or toxic to prokaryotes and eukaryotes (96-99). This metabolic versatility has made this bacterium the subject of numerous biochemical and genetic studies, which has led to the clarification of some of the catabolic pathways responsible for the degradation of these molecules. Thus, the routes involved in the degradation of phenylacetic acid and of various structurally related compounds have been identified (n-phenylalkanoic acids, phenylacetaldehyde, 2-phenylethanol, etc.) (83,97-99). Additionally, it has been proven that P. putida U is capable of efficiently degrading other aromatic compounds such as the amino acids phenylalanine, tyrosine, and 3-hydroxyphenylacetic acid, using a pathway, characterized by our research group, which involves the conversion of all those compounds in


    homogetic which is subsequently degraded to fumaric acid and acetoacetic acid (100-102).
    This bacterium is also capable of growing in MM containing aliphatic (propyl, butyl, pentyl, hexyl, octal and nonylamines) and aromatic (2-phenylethylamine, tyramine and dopamine) as the only carbon sources. The routes responsible for the degradation of aliphatic amines, tyramine 2-phenylethylamine and dopamine have been identified by our group (83, 102) while the route responsible for the histamine assimilation in this bacterium was unknown until the date. In fact, the few researchers who have addressed the microbial degradation of histamine in bacteria (usually in those belonging to the genus Pseudomonas) have only described some of the possible degradative stages and the genes encoding those enzymes have not been identified or sequenced. We have reliably demonstrated that P. putida U degrades histamine through a new catabolic pathway whose enzymes are encoded by the hin genes as we will describe later. The data included in this patent increases our knowledge about the degradation of biogenic amines (103115) and describes the use of biotechnological tools useful for the elimination of histamine from different media. Description of the invention
    In a first aspect, the present invention relates to an isolated nucleic acid molecule comprising sequences with at least 60% identity with the sequences represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 , SEQ ID NO: 10 and SEQ ID NO: 12. This isolated nucleic acid molecule comprising sequences with at least 60% identity with these five sequences is the common inventive concept of the present invention, since these sequences allow the Histamine elimination and all of them are present in all aspects of the invention. These five sequences are those corresponding to the hin C, hin A, hinD, hinE and hinB genes, respectively.
    For greater ease in understanding the present invention, the correspondence between nucleotide sequences and genes is indicated below:
    - hin C: SEQ ID NO: 4
    - hinA: SEQ ID NO: 6
    - hinD: SEQ ID NO: 8
    - hinE: SEQID NO: 10
    - hinB: SEQ ID NO: 12


    In the same way, the correspondence between amino acid sequences and proteins is indicated below:
    - HinC: SEQ ID NO: 5
    - HinA: SEQ ID NO: 7
    - HinD: SEQ ID NO: 9
    - HinE: SEQ ID NO: 11
    - HinB: SEQ ID NO: 13
    The term "% identity" is understood as the percentage of coincidence in nucleotides when it is a nucleotide sequence, or in amino acids, when it is an amino acid sequence. The percent identity is related to the ability of a nucleic acid molecule to mate with another sequence under strict hybridization conditions (the conditions under which complementary nucleic acid chains hybridize, which are easily identifiable by one skilled in the art in each specific case, but that can be summarized as conditions of high temperatures -65 º C, for example - and low concentration of salts).
    When it is indicated that the sequences have at least 60% identity, in the present invention it is understood that they have at least 60% identity, preferably at least 70% identity, more preferably at least 80% identity, even more preferably at least 90% identity, and in the most preferred situation, the sequences are identical with the sequences represented in the sequence listing. The histamine elimination route described by the present invention was not known to date. Although 100% identical sequences are preferred to the indicated sequences, as Table 3 shows, other sequences with identities of at least 60% of other microorganisms are equally useful for histamine removal.
    Table 3 below shows that the identity percentages of the amino acid sequences of the HinA, HinB, HinC, HinD, HinE, HinF, HinG, HinH, HinI, HinJ and HinK proteins, in different microorganisms of the Pseudomonas genus, are so low. about 24% The Protein_id column indicates the protein reference in the NCBI database (https://www.ncbi.nlm.nih.gov/protein). Due to the nature of the genetic code, where most amino acids can be encoded by more than one codon, the percentages of nucleotide sequence identity of the hinA, hinB, hinC, hinD, hinE, hinF, hinG, hinH, hinI genes , hinJ and hinK, in different microorganisms of the genus Pseudomonas, could be even lower than the percentages of identity of the amino acid sequences shown in table 3.


    Table 3a. Identity percentages of amino acid sequences encoded by the hinA genes in different microorganisms of the Pseudomonas genus. 9999999/99 / I9I!
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    5 {a {hÇíí h b.w / {{{{w ítt! Íí! Í! Ííí! Í! ! íí !!! í !!
    ttttttttttt ttttttttttttttt Çb5 {í ítt! í !! í! ííí í !! !!!!!!!!! b. {Ç {{ítt !! í! íí! íí íí! ! ííí !! í !! bWù {{ítt! íí !!! íí! í !! !! í !!!!!!
    ttttttttttt ttttttttttttt [aD {h {íw: 3B í !! ¡ííí !! íí! ttttttttttt tttt [aD {higw {{9wí !! íí íí! ! ííí !! í !! ttttttttttt tttttttttt. {{hh {{5b !!! í! íí! ! ííí !!!!!
    t! a / {hwíw ítt !!!! í! í !! ! í! !! í !!! íí! ttttttttttt tttttttttt. {{{í {ítt !! íííí! í! í !! ! Here !!!!!! ttttttttttt tttttttttttttt And [{h ítt! íí !!!!! í í !! ! ííí !!!!!


    ttttttttttt ttttttttttt [aD {{{{í í {{ítt! í !! íííí! íí! ! Here !!!!!!. {{hh {ítt! í !! íííí! íí! ! Here !!!!!!ttttttttttt tttttttttttt. {{{{h ítt !!! í! ííí! íí! ! Here !!!!!!wD /. {{{ítt !!! í! ííí! íí! ! Here !!!!!!ttttttttttt ttttttttttt! Ü {{Çh ítt! í !! íí !! í íí! ! ííí !!!!!! Ü {whÇí: 3B íí! ! ííí !!!!!Çw {: 3B íí! ! ííí !!!!!bùÇÇh ítt! í !!! í! í! íí! ! ííí !!!!!b / La. {{h {w ítt !! í! íííí! ! í! !! íí! íí!{{{Ç {: 3B íí! ! ííí !!!!![.Üa {{{ítt !! í! Í! Í! Í í !! ! Here !!!!!!th {w ítt !!! í! í! íí í !! ! Here !!!!!!! íÇ {ítt !!! í! í! íí í !! ! Here !!!!!!ttttttttttt ttttttttttt hÇ {í ítt! í! í! íííí í !! ! ííí !! í !!
    awò {{DÇÇí {ítt !! íí !! í! í í !! ! ííí !! í !! ttttttttttt ttttttttttttttttt YChÇh h b.w / {{Ç {hÇ ítt !! í! íííí! í !! ! ííí !! í !! ttttttttttt ttttttttt W / a {hh {ítt! ííí! íí! í! í! !! íí! íí! ttttttttttt ttttttttttttt b.w / {{{íw ítt! í! í! ííí! íí! ! ííí !!!!!
    ttttttttttt ttttttttttttt [aD {h {hh: 3B í !! ! ííí !!! í!
    {WÇ9D {: 3B íí! ! ííí !!! í! ttttttttttt tttttttttt b.w / {Ç {Çwh: 3B í !! !!!!!!! í! ttttttttttt tttttttt [bÜD9DÇÇ {: 3B íí! ! ííí !!! í!
    ttttttttttt ttt í / {{hw ítt !!! í! í! íí í !! ! Here !!!!!! Dahw: 3B! ! ííí !!!!! Üíw: 3B í !! !!!!!!! í!
    Table 3b Identity percentages of amino acid sequences encoded by the hinB genes in different microorganisms of the Pseudomonas genus.
    9999999 / 99 /I9I.
    tttttttttt LttttttLt
    !!!!
    ttttttttttt tttttt ORItt.¡íí !! íí !!
    {{{{{{{ ítt! íí !! íííííí! ¡ííí! íí !!
    {{{ ítt !!! íí !!!!í !! ! í! í! íí !!
    {{{ ítt !! íííí! ííí !! ! í! í! íí !!
    b.w / {w {{w ítt !!! í !! í! ííí! ¡ííí! íí !!
    D.D { ítt !! ííííííííí! ! í! í! íí !!
    .Lw5D { ítt !!! íí !!!!í !! ! í! í! íí !!
    YÇ {wwÇ bttí !!í !! ! í! í! íí !!
    C{ ítt !!! ííí! ííí !! ! í !!! íí !!
    í {{í ítt !! íí !! ííííí! !!! íííí!


    í {íh9h {h ítt !! í! í !! í! íí! ¡íí !! íí !! . {D {ítt !!! ííííí! í !! ! í !!! íí !! b5 {ítt !!! ííí! íí íí! ! í! í! íí !! !! h ítt! ííí! í !!! íí! !!! íííí!
    b.w / {Ç {ÇwÇ h 5 {a
    : 3B
    ttttttttttt tttttttt {{{wh í !! ¡íí! íí !! ttttttttttt ttttttttttt [wh ítt !!! íí !! íí íí! !!!!! ttttttttttt ttttttttt {. {Çhh ítt !! íííí! íí í !! ! í! í! íí !!
    {. {{Ç {ítt !! íííí! Íí í !! ! í! í! íí !!
    ttttttttttt tttttttt {W {Ç ítt! ííí !! ííí íí! !!!!! D9W {ítt! Íí! Íí! Íí íí! !!! í! íí !! í {{DÇ {.D {Ç ítt! í! í! ííí! íí! !!! í! íí !! 5 {a {hwíh ítt! Íí! Íí! Íí íí! !!! í! íí !!
    ttttttttttt ttttttttt b.w / {{{{{h 5 {a {hÇÇw: 3B íí! ! íííí! ttttttttttt ttttt a9WÇh {: 3B íí! ! í !! íí !! ttttttttttt ttttttttttt 5 {a {{ÇÇ {: 3B íí! ! í !! íí !! ttttttttttt tttttttttttttt b.w / {Ç {{íí: 3B íí! ! íí! í !!!
    ttttttttttt tttttttttt í / {{íh ítt! í! í !!! íí íí! ! í! í! í !!! ttttttttttt ttttttttttttttt b5Çh: 3B í !! ¡íí! íí !! 5 {a {hÇíí h b.w / {{{{w: 3B í !! !!!! í !!
    ttttttttttt ttttttttttttttt Çb5 {í ítt !! íííí! íí í !! ! í! í! íí !! b. {Ç {{: 3B íí! !!!!! bWù {{: 3B íí! ! í! í! íí !! ttttttttttt ttttttttttttt [aD {h {íw: 3B !!! !!! íííí! ttttttttttt tttt [aD {hig {: 3B íí! !!! í! íí !! ttttttttttt tttttttttt. {{hh {ítt! ííí !!!! í í !! ! í !!! ííí! t! a / {hwíw: 3B !!! !!! í! í! í! ttttttttttt tttttttttt. {{{í {: 3B íí! ! í !!! í! í! ttttttttttt tttttttttttttt And [{h: 3B íí! !!! í! íí !! ttttttttttt ttttttttttt [aD {{{{í í {{: 3B íí! ! í! í! ííí! . {{hh {: 3B íí! ! í! í! ííí! ttttttttttt tttttttttttt. {{{{h: 3B íí! ! í! í! í! í! wD /. {{{: 3B íí! ! í! í! í! í! ttttttttttt ttttttttttt! Ü {{Çh: 3B íí! ! í !!! ííí! ! Ü {whÇí: 3B íí! ! í !!! í! í! Çw {ítt! Íí !!!! íí íí! ! í! í! ííí! bùÇÇh: 3B íí! ! í! í! ííí! b / La. {{h {w: 3B íí! ! í !! í! í! {{{Ç {: 3B íí! ! í! í! ííí! [.Üa {{{: 3B í !! ! í! í! ííí!
    th {w ítt! íííííííí íí! ! í! í! ííí!


    ! íÇ { ítt !!! í! í! í!íí! ! í! í! ííí!
    ttttttttttt ttttttttttt hÇ {íítt! í! íí! í! ííí! ! íí! í !!!
    awò {{DÇÇí { ítt! í! íí! í! ííí! ! íí! í !!!
    ttttttttttt ttttttttttttttttt YChÇh h b.w / {{Ç {hÇítt !!!! í! ííííí! ! íí! í! í!
    ttttttttttt ttttttttt W / a {hh {ítt! ííí! íí! ííí! ! í !! í !!!
    ttttttttttt ttttttttttttt b.w / {{{íwítt! í! íííííí! í! !!!!!
    ttttttttttt ttttttttttttt [aD {h {hh: 3B! Í! !!! í! í! í!
    {WÇ9D { : 3B! Í! !!! í! í! í!
    ttttttttttt tttttttttt b.w / {Ç {Çwh: 3B
    ttttttttttt tttttttt [bÜD9DÇÇ {: 3B! Í! !!! í! í! í!
    ttttttttttt ttt í / {{hw: 3B! ! í! í! ííí!
    Dahw : 3B! !!!!!
    Üíw : 3B í !! ! íí! í !!!
    Table 3c Identity percentages of amino acid sequences encoded by hinC genes in different microorganisms of the genus Pseudomonas. 9999999/99 / I9I /
    tttttttttt LttttttLt !!!! ttttttttttt tttttt Ü Itt / !!!!!!!!! !!!! {{{{{{ítt! íí !! ííí! !!!!!!!!! {{{ítt !! í! ííííí íí! !! {{{ítt !! íííí! íí íí! !! b.w / {w {{w ítt !!! í !! í !! íí! !! D.D {ítt !! íííííí! íí! !! í !!!!!! .Lw5D {ítt !!! ííí! Í! íí! !! í !!!!!! YÇ {wwÇ bttí !! ííí íí! !! C {ítt !! ííí! Í !! íí! !! í {{í ítt !! íí !! íí! í !! !! í {íh9h {h ítt !! í! í !! í! íí! !! . {D {ítt !! ííí! Í !! íí! !!
    b5 {ítt !! ííí! í !! íí! !! !! h ítt! ííí! í !! í í !! !! í !!!!!! b.w / {Ç {ÇwÇ h 5 {a
    ttttttttttt tttttttt {{{wh ítt !! í! ííí! í íí! ¡ííí !! íí!
    ttttttttttt ttttttttttt [wh ítt !!! íí !! í! íí! !! í !!!!!!
    ttttttttttt ttttttttt {. {Çhh ítt !! íííí! íí íí! !! {. {{Ç {ítt !! íííí! Íí íí! !!
    ttttttttttt tttttttt {W {Ç ítt! ííí !! ííí íí! !! í !!!! í! D9W {ítt !! íí! Íííí íí! !! í !!! íí! í {{DÇ {.D {Ç ítt !! íí! íííí íí! !! í !!! íí!


    5 {a {hwíh ítt! Íí! Íí! Í! íí! !! í !!! íí! ttttttttttt ttttttttt b.w / {{{{{h 5 {a {hÇÇw: 3B íí! ! ííí !!! í! ttttttttttt ttttt a9WÇh {: 3B íí! ! í! í !! í !! ttttttttttt ttttttttttt 5 {a {{ÇÇ {: 3B í !! ! ííí !!!!! ttttttttttt tttttttttttttt b.w / {Ç {{íí: 3B í !! ! í !!!!! í! ttttttttttt tttttttttt í / {{íh ítt! íí! íííí! íí! !!!!!!!!! ttttttttttt ttttttttttttttt b5Çh ítt! íí !!!!!! íí! ! íí !!!! í!
    5 {a {hÇíí h b.w / {{{{w ítt! Íí! Íí !!! íí! ! íí !!!! í! ttttttttttt ttttttttttttttt Çb5 {í ítt !! íííí! íí íí! !!
    b. {Ç {{ítt !!! íí !! íí íí! !!
    bWù {{: 3B íí! !! ttttttttttt ttttttttttttt [aD {h {íw: 3B í !! !!! í !!!!! ttttttttttt tttt [aD {higw {{9 {!!! íí íí! !! í !!!! í! ttttttttttt tttttttttt. {{hh {{5b !!! í! íí! !!!!!!!!!
    t! a / {hwíw ítt !!!! í !!!! í !! ! í !!!!! í! ttttttttttt tttttttttt. {{{í {ítt !! íííí! í! í !! !!!!!!!!! ttttttttttt tttttttttttttt And [{h ítt! íí !!!! íí íí! !! í !!! íí! ttttttttttt ttttttttttt [aD {{{{í í {{ítt! í !! ííííí íí! !!! í !!!!!
    . {{hh {: 3B íí! !!! í !!!!! ttttttttttt tttttttttttt. {{{{h ítt !! ííí! íí! í !! !!! í !!!!!
    wD /. {{{: 3B í !! !!! í !!!!!
    ttttttttttt ttttttttttt! Ü {{Çh ítt! í !! íí !! í í !! !!! í !!!!! ! Ü {whÇí: 3B í !! !!! í !!!!! Çw {ítt! Íí !!!! íí í !! !!! í !!!!! bùÇÇh ítt !! í! í! íí! íí! !!! í !!!!! b / La. {{h {w: 3B íí! ¡ííí !! íí! {{{Ç {: 3B í !! !!!!!!!!! [.Üa {{{: 3B í !! !!! í !!!!! th {w: 3B í !! !!! í !!!!! ! íÇ {: 3B í !! !!! í !!!!!
    ttttttttttt ttttttttttt hÇ {í: 3B íí! ¡íí !!! íí!
    awò {{DÇÇí {ítt !! íí !! ííí í !! ¡ííí !! íí! ttttttttttt ttttttttttttttttt YChÇh h b.w / {{Ç {hÇ ítt !! í! ííííí í !! ! ííí !!!!! ttttttttttt ttttttttt W / a {hh {ítt! ííí !!! í! íí! ! í !!!! í !! ttttttttttt ttttttttttttt b.w / {{{íw ítt !! í! ííííí í !! ¡íí !!! íí!
    ttttttttttt ttttttttttttt [aD {h {hh: 3B íí! ¡íí !!! íí!
    {WÇ9D {: 3B íí! ¡ííí !! íí! ttttttttttt tttttttttt b.w / {Ç {Çwh: 3B ttttttttttt tttttttt [bÜD9DÇÇ {: 3B í !! ! ííí !!!!! ttttttttttt ttt í / {{hw: 3B í !! !!! í !!!!!
    Dahw: 3B í !! !! í !!!!!!


    Üíw: 3B í !! ¡ííí !! íí!
    3d table. Identity percentages of amino acid sequences encoded by the hinD genes in different microorganisms of the Pseudomonas genus. 9999999/99 / I9I5
    tttttttttt LttttttLt !!!! ttttttttttt tttttt Ü Itt5 !! íí !! íí! {{{{{{ítt! íí !! íííí íí! !! íí! íí! {{{ítt !!! íí !! íí íí! !! í !!! íí! {{{ítt !! íííí !!! íí! !! íí !! í !! b.w / {w {{w ítt !!! í !! í !! íí! !! íí! íí! D.D {ítt !! íííííí! íí! !! í !!! í !! .Lw5D {ítt !!! íí !! íí íí! !! í !!! íí! YÇ {wwÇ bttí !! ííí íí! !! íí! íí! C {ítt !! í! Ííí! Í í !! !! í !!! íí! í {{í ítt !! íí !! ííí í !! !!! í !! í !! í {íh9h {h: 3B íí! !! íí! íí! . {D {ítt !! ííí! Í! Í í !! !! íí! íí!
    b5 {ítt !!! ííí! íí í !! !! íí! íí! !! h ítt! íííííí! í íí! !! íí! íí! b.w / {Ç {ÇwÇ h 5 {a
    ttttttttttt tttttttt {{{wh ítt! í! í! ííí! í !! !!! í !! í !!
    ttttttttttt ttttttttttt [wh ítt !!! íí !! íí íí! !!! í !! í !!
    ttttttttttt ttttttttt {. {Çhh ítt! í !! í! íí! íí! !! í !!! í !! {. {{Ç {ítt! Í !! í! Íí! íí! !! í !!! í !!
    ttttttttttt tttttttt {W {Ç ítt! ííí !! íí! í !! ! ííí !! í !! D9W {ítt !! í !! í! Íí í !! !!!!!! í !! í {{DÇ {.D {Ç ítt! í! í! íííí í !! !!! í !! í !! 5 {a {hwíh ítt! Íí! Íí! Í! í !! !!! í !! í !!
    ttttttttttt ttttttttt b.w / {{{{{h 5 {a {hÇÇw ítt! íí! ííí! í í !! ! í! í !! íí!
    ttttttttttt ttttt a9WÇh {ítt !! ííííííí í !! ! ííí !! í !!
    ttttttttttt ttttttttttt 5 {a {{ÇÇ {: 3B í !! ! ííí !! í !!
    ttttttttttt tttttttttttttt b.w / {Ç {{íí: 3B íí! ¡íí !!! íí!
    ttttttttttt tttttttttt í / {{íh: 3B íí! !! íí !! í !!
    ttttttttttt ttttttttttttttt b5Çh: 3B í !! ! í! í !! íí! 5 {a {hÇíí h b.w / {{{{w: 3B í !! ! í! í !! íí!
    ttttttttttt ttttttttttttttt Çb5 {í ítt !! íííí !!! íí! !! íí !! í !! b. {Ç {{: 3B í !! !!! í !! í !! bWù {{: 3B íí! !! íí !! í !!
    ttttttttttt ttttttttttttt [aD {h {íw: 3B íí! !!!!!! í !!


    ttttttttttt tttt [aD {hw {: 3B í !! !!!!!! í !! ttttttttttt tttttttttt. {{hh {: 3B í !! ! ííí !! í !!
    t! a / {hwíw: 3B í !! ! í !!!! íí! ttttttttttt tttttttttt. {{{í {: 3B í !! !!!!!! í !! ttttttttttt tttttttttttttt Y [{h: 3B í !! ! ííí !! í !! ttttttttttt ttttttttttt [aD {{{{í í {{: 3B í !! ! ííí !! í !!
    . {{hh {: 3B í !! ! ííí !! í !! ttttttttttt tttttttttttt. {{{{h: 3B íí! !!!!!! í !!
    wD /. {{{: 3B íí! !!! í !! í !! ttttttttttt ttttttttttt! Ü {{Çh: 3B íí! !!! í !! í !!
    ! Ü {whÇí: 3B í !! !!!!!! í !!
    Çw {: 3B íí! !!! í !! í !!
    bùÇÇh: 3B í !! !!!!!! í !!
    b / La. {{h {w: 3B íí! ! íí !!! í !!
    {{{Ç {: 3B í !! ! ííí !! í !!
    [.Üa {{{: 3B í !! !!!!!! í !!
    th {w: 3B í !! !!!!!! í !!
    ! íÇ {: 3B í !! !!!!!! í !!
    ttttttttttt ttttttttttt hÇ {í ítt! í! í! íí !! íí! !!! í !! í !!
    awò {{DÇÇí {ítt !! íí !! ííí íí! !!! í !! í !! ttttttttttt ttttttttttttttttt YChÇh h b.w / {{Ç {hÇ ítt !! í! ííííí íí! !!!!!! í !! ttttttttttt ttttttttt W / a {hh {ítt! ííí! í! íí íí! ! í! í !! íí! ttttttttttt ttttttttttttt b.w / {{{íw ítt! í! ííííí! íí! ! ííí !! í !!
    ttttttttttt ttttttttttttt [aD {h {hh: 3B íí! ! íí !!! í !!
    {WÇ9D {: 3B íí! ! ííí !! í !! ttttttttttt tttttttttt b.w / {Ç {Çwh: 3B ttttttttttt tttttttt [bÜD9DÇÇ {: 3B íí! ! íí !!! í !! ttttttttttt ttt í / {{hw: 3B í !! !!!!!! í !!
    Dahw: 3B í !! !!! í !! í !!
    Üíw: 3B í !! ! ííí !! í !!
    Table 3e Identity percentages of amino acid sequences encoded by the hinE genes in different microorganisms of the Pseudomonas genus.
    9999999 / 99 /I9I9
    tttttttttt LttttttLt
    ttttttttttt tttttt ORItt9!!!! ¡íí !! íí !!
    {{{{{{{ ítt! íí !! íííííí! ! í! í! íí !!
    {{{ ítt !!! íí !!! íí !! !! íí! íí !!
    {{{ ítt !! íííí! í!í !! !! íí! íí !!
    36


    b.w / {w {{w ítt !!! í !! í !! íí! ¡íí !! íí !! D.D {ítt !! íííííí! íí! ! í !!! íí !! .Lw5D {ítt !!! íí !!! í í !! !! íí! íí !! YÇ {wwÇ bttí !! ííí í !! !! íí! íí !! C {ítt !! í! Ííí !! í !! !! íí! íí !! í {{í ítt !! íí! íííí í !! ! íí !! í! í!
    í {íh9h {h: 3B íí! ! í !!! íí !! . {D {ítt !! ííí! Í !! í !! !! íí! íí !! b5 {ítt !!! íííí !! í !! !! íí! íí !! !! h ítt! íííííí !! íí! ! ííí! í! í!
    b.w / {Ç {ÇwÇ h 5 {a ttttttttttt tttttttt {{{wh ítt !! í! ííí !! í !! ! í! í! í! í! ttttttttttt ttttttttttt [wh ítt !!! íííí !! í !! ! í !!! í! í! ttttttttttt ttttttttt {. {Çhh ítt !! íííí! í! í !! !! íí! íí !! {. {{Ç {ítt !! íííí! Í! í !! !! íí! íí !! ttttttttttt tttttttt {W {Ç: 3B í !! ! í! í! í! í! D9W {: 3B íí! ! ííí! í! í! í {{DÇ {.D {Ç: 3B íí! ! ííí! í! í! 5 {a {hwíh: 3B íí! ! íí !! í! í! ttttttttttt ttttttttt b.w / {{{{{h 5 {a {hÇÇw: 3B íí! ! íí !! í! í! ttttttttttt ttttt a9WÇh {: 3B í !! ! ííí! í! í! ttttttttttt ttttttttttt 5 {a {{ÇÇ {: 3B íí! ! íí !! í! í! ttttttttttt tttttttttttttt b.w / {Ç {{íí: 3B í !! ¡ííí! íí !! ttttttttttt tttttttttt í / {{íh: 3B í !! ! íí !! í! í! ttttttttttt ttttttttttttttt b5Çh: 3B íí! !!! íííí! 5 {a {hÇíí h b.w / {{{{w: 3B í !! ! í !!! í! í! ttttttttttt ttttttttttttttt Çb5 {í ítt !! íííí! í! í !! !! íí! íí !! b. {Ç {{: 3B íí! ! ííí! í! í! bWù {{: 3B í !! !! í !! ííí! ttttttttttt ttttttttttttt [aD {h {íw: 3B íí! ! ííí! í! í! ttttttttttt tttt [aD {hw {: 3B í !! ! í! í! í! í! ttttttttttt tttttttttt. {{hh {: 3B í !! ! íí !! í! í! t! a / {hwíw: 3B íí! ! ííí! í! í! ttttttttttt tttttttttt. {{{í {: 3B íí! ! íí !! í! í! ttttttttttt tttttttttttttt And [{h: 3B íí! ! ííí! í! í! ttttttttttt ttttttttttt [aD {{{{í í {{: 3B íí! ! ííí! í! í! . {{hh {: 3B íí! ! ííí! í! í! ttttttttttt tttttttttttt. {{{{h: 3B íí! ! ííí! í! í! wD /. {{{: 3B íí! ! íí !! í! í! ttttttttttt ttttttttttt! Ü {{Çh: 3B í !! ! ííí! í! í! ! Ü {whÇí: 3B íí! ! ííí! í! í!


    Çw { : 3Bíí! ! íí !! í! í!
    bùÇÇh : 3B! í! ! í! í! í! í!
    b / La. {{h {w : 3Bí !! ! ííí! í! í!
    {{ {C{ : 3Bí !! ! ííí! í! í!
    [.Üa {{{ : 3Bí !! ! ííí! í! í!
    th {w : 3Bíí! ! íí !! í! í!
    ! íÇ { : 3Bíí! ! íí !! í! í!
    ttttttttttt ttttttttttt hÇ {íítt! í! íí! íí!íí! ! ííí! í! í!
    awò {{DÇÇí { ítt !! íí! ííííí !! ! ííí! í! í!
    ttttttttttt ttttttttttttttttt YChÇh h b.w / {{Ç {hÇítt !!!! í! í !!íí! ¡ííí! íí !!
    ttttttttttt ttttttttt W / a {hh {6) (íí! ! ííí! í !!! tLtttL /
    ttttttttttt ttttttttttttt b.w / {{{íwítt! í! ííí! í!íí! ! íí !! í! í!
    ttttttttttt ttttttttttttt [aD {h {hh: 3Bíí! !!!!! í! í!
    {WÇ9D { : 3Bíí! !!!!! í! í!
    ttttttttttt tttttttttt b.w / {Ç {Çwh: 3B   
    ttttttttttt tttttttt [bÜD9DÇÇ {: 3Bí !! ! íí !! í! í!
    ttttttttttt ttt í / {{hw: 3Bíí! ! íí !! í! í!
    Dahw : 3Bí !! ! í !!! í! í!
    Üíw : 3Bí !! ! ííí! í! í!
    Table 3f. Identity percentages of the amino acid sequences encoded by the hinF genes in different microorganisms of the Pseudomonas genus. 9999999/99 / I9IC
    tttttttttt LttttttLt !!!! ttttttttttt tttttt Ü IttC !! íí !! íí! {{{{{{ítt! íí !! í! íí íí! !! í !!! íí! {{{ítt !!! íí !! í! í !! !! í !!! íí! {{{ítt !! ííííí! í íí! !!! ííí! b.w / {w {{w ítt !!!! ííííí íí! !! íí! íí! D.D {ítt !! ííí! Íí! íí! !! í !!! íí! .Lw5D {ítt !!! íí !! í! í !! !! í !!! íí! YÇ {wwÇ 13B í !! !! í !!! íí! C {ítt !! í! Íí! Í! í !! !! í !!! íí! í {{í ítt !! íí !! í! í í !! !!! ííí! í {íh9h {h ítt !! í! í! íí! íí! !! íí! íí! . {D {ítt !! ííí! Í !! í !! !! í !!! íí! b5 {ítt !!! ííí! í! íí! !!! ííí! !! h ítt! ííííí! í! íí! !!! ííí! ttttttttttt tttttttt b.w / {Ç {ÇwÇ h 5 {a ítt !! í! íí! íí í !! ! í! í !! íí!


    {{{wh ttttttttttt ttttttttttt [wh ítt !!! ííííí! íí! !!!!!! ttttttttttt ttttttttt {. {Çhh ítt !! ííííí! í íí! !!! ííí! {. {{Ç {ítt !! ííííí! Í íí! !!! ííí!
    ttttttttttt tttttttt {W {Ç: 3B íí! !!! ííí!D9W {: 3B íí! !!! ííí!í {{DÇ {.D {Ç: 3B íí! !!!!!!5 {a {hwíh: 3B íí! !!! ííí!ttttttttttt ttttttttt b.w / {{{{{h 5 {a {hÇÇw: 3B íí! !!!!!!ttttttttttt ttttt a9WÇh {: 3B í !! ! í !!!! íí!ttttttttttt ttttttttttt 5 {a {{ÇÇ {: 3B í !! ¡ííí !! íí!ttttttttttt tttttttttttttt b.w / {Ç {{íí: 3B í !! ! í !!!! íí!ttttttttttt tttttttttt í / {{íh: 3B íí! !!! ííí!ttttttttttt ttttttttttttttt b5Çh: 3B í !! ¡íí !!! íí!5 {a {hÇíí h b.w / {{{{w: 3B í !! ¡íí !!! íí!ttttttttttt ttttttttttttttt Çb5 {í ítt !! ííííí! í íí! !!! ííí!b. {Ç {{: 3B í !! !!!!!!bWù {{: 3B í !! !! í !!! íí!ttttttttttt ttttttttttttt [aD {h {íw: 3B í !! !!!!!!!!!ttttttttttt tttt [aD {hig {: 3B íí! !!! ííí!ttttttttttt tttttttttt. {{hh {: 3B íí! ! íí !!!! í!t! a / {hwíw: 3B! í! ! íí !!!! í!ttttttttttt tttttttttt. {{{í {: 3B íí! ! ííí !! í !!ttttttttttt tttttttttttttt And [{h: 3B íí! ! ííí !! í !!ttttttttttt ttttttttttt [aD {{{{í í {{: 3B íí! ! ííí !!! í!. {{hh {: 3B íí! ! ííí !!! í!ttttttttttt tttttttttttt. {{{{h: 3B íí! ! Here !!!!!!wD /. {{{: 3B íí! ! Here !!!!!!ttttttttttt ttttttttttt! Ü {{Çh: 3B íí! ! ííí !!! í!! Ü {whÇí: 3B íí! ! ííí !!! í!Çw {: 3B íí! ! íí !!!! í!bùÇÇh: 3B íí! ! ííí !!! í!b / La. {{h {w: 3B íí! ¡íí !!! íí!{{{Ç {: 3B íí! ! ííí !!! í![.Üa {{{: 3B íí! ! ííí !!! í!th {w: 3B íí! ! ííí !!! í!! íÇ {: 3B íí! ! ííí !!! í!ttttttttttt ttttttttttt hÇ {í ítt! í! í !! íí! í !! ¡ííí !! íí!awò {{DÇÇí {ítt! í! í !! ííí í !! ¡ííí !! íí!ttttttttttt ttttttttttttttttt YChÇh h b.w / {{Ç {hÇ ítt !! í !! í !! í í !! ! í !!!! í !!


    ttttttttttt ttttttttt W / a {hh {ítt! íí! ííí! í íí! ¡íí !!! íí! ttttttttttt ttttttttttttt b.w / {{{íw ítt! í! í! ííí! íí! ! í !!!! íí! ttttttttttt ttttttttttttt [aD {h {hh: 3B í !! ! í !!!! í !! {WÇ9D {: 3B í !! ! í !!!! í !! ttttttttttt tttttttttt b.w / {Ç {Çwh: 3B ttttttttttt tttttttt [bÜD9DÇÇ {: 3B íí! ¡íí !!! íí! ttttttttttt ttt í / {{hw: 3B íí! ! ííí !!! í! Dahw: 3B í !! !!! ííí! Üíw: 3B íí! ! ííí !!! í!
    3g table. Identity percentages of the amino acid sequences encoded by the hinG genes in different microorganisms of the Pseudomonas genus.
    9999999 / 99 /I9ID
    tttttttttt LttttttLt
    !!!!
    ttttttttttt tttttt ORIttD¡ííí! ííí!
    {{{{{{{ ítt! íí !! í! íííí! ¡íí !! ííí!
    {{{ ítt !!! íí! í! ííí! ! í !!! íí !!
    {{{ ítt !! ííííí! ííí! ! í !!! ííí!
    b.w / {w {{w ítt !!!! ííí !!íí! ¡íí !! ííí!
    D.D { ítt !! ííí! íí!í !! ! í! í! ííí!
    .Lw5D { ítt !!! íí! í! ííí! ! í !!! íí !!
    YÇ {wwÇ 13Bíí! ! í !!! íí !!
    C{ ítt !! í! íí! íííí! ! í !!! íí !!
    í {{í ítt !! íí !! í! íí !! ¡íí !! ííí!
    í {íh9h {h ítt !! í! í! ííííí! ¡ííí! ííí!
    . {D { ítt !! ííí! í! ííí! ! í !!! íí !!
    b5 { ítt !!! ííí !! ííí! ! í !!! íí !!
    !! h ítt! ííííí! íííí! ¡íí !! ííí!
    b.w / {Ç {ÇwÇ h 5 {a
    ttttttttttt tttttttt {{{whítt !! í! ííííííí! ! í! í! íí !!
    ttttttttttt ttttttttttt [whítt !!! ííííí!íí! ¡ííí! ííí!
    ttttttttttt ttttttttt {. {Çhhítt! í !! íííííí !! ! í! í! ííí!
    {.{{C{ ítt! í !! íííííí !! ! í! í! ííí!
    ttttttttttt tttttttt {W {Çítt! íí! ííííííí! ! í! í! íí !!
    D9W { ítt !! í !! ííí!íí! ¡ííí! ííí!
    í {{DÇ {.D {Ç ítt! í! í! íí !!íí! ¡ííí! ííí!
    5 {a {hwíh ítt! íí! ííí !!í !! ¡ííí! ííí!
    ttttttttttt ttttttttt b.w / {{{{{h 5 {a {hÇÇw ítt! íí! ííííííí! ¡ííí! ííí!
    ttttttttttt ttttt a9WÇh {ítt !! ííí! í! ííí! ! í! í! ííí!
    ttttttttttt ttttttttttt 5 {a {{ÇÇ {: 3B í !! ¡íí !! ííí!


    ttttttttttt tttttttttttttt b.w / {Ç {{íí: 3B! í! ¡ííí! ííí! ttttttttttt tttttttttt í / {{íh ítt! íí! íí! íí í !! ! í !!! ííí! ttttttttttt ttttttttttttttt b5Çh: 3B í !! ¡íí !! ííí!
    5 {a {hÇíí h b.w / {{{{w: 3B íí! ¡ííí! íí !! ttttttttttt ttttttttttttttt Çb5 {í ítt! í !! ííííí í !! ! í! í! ííí!
    b. {Ç {{ítt !!! íí! ííí íí! ¡íí !! ííí!
    bWù {{ítt !! ííííí! í íí! ! í !!! ííí!
    ttttttttttt ttttttttttttt [aD {h {íw: 3B í !! ! í !!! ííí! ttttttttttt tttt [aD {higw {{9víí! í! íí! ! í! í! íí !! ttttttttttt tttttttttt. {{hh {ítt! íííííííí í !! ! í! í! íí !!
    t! a / {hwíw ítt !!!! ííííí !!! ¡ííí! íí !! ttttttttttt tttttttttt. {{{í {ítt !! íííí! íí í !! ! í !!! ííí! ttttttttttt tttttttttttttt And [{h ítt! íí !!!!!! íí! ¡ííí! ííí! ttttttttttt ttttttttttt [aD {{{{í í {{ítt! í !! ííí! í í !! ! í! í! íí !!
    . {{hh {ítt! í !! ííí! í í !! ! í! í! íí !! ttttttttttt tttttttttttt. {{{{h ítt !! ííí! ííí íí! ¡ííí! íí !!
    wD /. {{{ítt !!! í! íííí íí! ¡ííí! íí !! ttttttttttt ttttttttttt! Ü {{Çh: 3B! í! ! í! í! ííí!
    ! Ü {whÇí: 3B í !! ! í! í! ííí!
    Çw {: 3B! Í! ! í! í! ííí!
    bùÇÇh: 3B í !! ¡íí !! ííí!
    b / La. {{h {w ítt !! ííííí! í í !! ! í! í! íí !!
    {{{Ç {: 3B í !! ! í !!! ííí!
    [.Üa {{{: 3B í !! ! í! í! ííí!
    th {w: 3B! í! ! í !!! ííí!
    ! íÇ {: 3B í !! ! í! í! ííí!
    ttttttttttt ttttttttttt hÇ {í ítt! í! í! íí! í í !! ¡ííí! ííí! awò {{DÇÇí {ítt !! íí !! ííí íí! ! í! í! ííí!
    ttttttttttt ttttttttttttttttt YChÇh h b.w / {{Ç {hÇ ítt !! í !! í !!! íí! ! í! í! íí !! ttttttttttt ttttttttt W / a {hh {: 3B íí! ! í! í! ííí! ttttttttttt ttttttttttttt b.w / {{{íw ítt! í! í! ííí! í !! ¡ííí! íí !! ttttttttttt ttttttttttttt [aD {h {hh ítt! í! íí! íí! í !! ¡íí !! íí !!
    {WÇ9D {ítt !!! íí! Í! Í í !! ¡ííí! íí !! ttttttttttt tttttttttt b.w / {Ç {Çwh ítt! í! í !!! íí íí! ¡ííí! ííí! ttttttttttt tttttttt [bÜD9DÇÇ {ítt !!! íí! í !! í !! ! íí !! í! í! ttttttttttt ttt í / {{hw ítt! íí !! íííí! í! ! í !!! ííí!
    Dahw ítt !! í! Í! Í! Í íí! ¡ííí! ííí! Üíw: 3B íí! ¡íí !! íí !!
    3h table. Identity percentages of amino acid sequences encoded by the hinH genes in different microorganisms of the Pseudomonas genus.

    9999999/99 / I9II
    tttttttttt Lttttttttv !!!! ttttttttttt tttttt Ü IttI! ííí! ííí! {{{{{{{ítt! íí !! í! í! íí! ¡ííí! ííí! {{{ítt !! í! íí! íí íí! ! í! í! ííí! {{{ítt !! ííííí !! í !! ! í! í! ííí! !!!! b.w / {w {{w ítt !!!! ííííí! ííí! ííí! D.D {ítt !! ííí! Ííí íí! ¡íí !! ííí! .Lw5D {ítt !!! íí! Í! Í íí! ¡íí !! ííí! YÇ {wwÇ 13B íí! ! í! í! ííí! C {ítt !! í! Íí! Íí íí! ! í! í! ííí! í {{í: 3B íí! ! í !!! ííí! í {íh9h {h ítt !! í! í! ííí íí! ¡ííí! ííí! . {D {ítt !! ííí! Í! Í íí! ! í! í! ííí!
    b5 {ítt !!! íí !! íí íí! ! í! í! ííí! !! h ítt! ííííí! íí í !! ! í! í! ííí! b.w / {Ç {ÇwÇ h 5 {a
    : 3B ttttttttttt tttttttt {{wh wh! ! í !!! ííí!
    ttttttttttt ttttttttttt [wh ítt !!! íííííí í !! ! í! í! ííí!
    ttttttttttt ttttttttt {. {Çhh ítt! í !! ííííí íí! ! í !!! ííí! {. {{Ç {ítt! Í !! ííííí íí! ! í !!! ííí!
    ttttttttttt tttttttt {W {Ç ítt! ííí! íííí íí! ! í! í! ííí! D9W {ítt! Íí! Ííííí í !! ! í! í! ííí! í {{DÇ {.D {Ç ítt !! ííííí !! í !! ! í !!! ííí! 5 {a {hwíh ítt! Íí! Ííííí í !! ! í! í! ííí!
    ttttttttttt ttttttttt b.w / {{{{{h 5 {a {hÇÇw ítt! íí! íííí! íí! ! í! í! ííí!
    ttttttttttt ttttt a9WÇh {ítt !! ííí! í !! í !! ¡¡íí! ííí!
    ttttttttttt ttttttttttt 5 {a {{ÇÇ {: 3B í !! ¡¡íí! ííí!
    ttttttttttt tttttttttttttt b.w / {Ç {{íí: 3B !!! !!! íííí!
    ttttttttttt tttttttttt í / {{íh ítt! íí! íí! í! íí! ! í! í! ííí!
    ttttttttttt ttttttttttttttt b5Çh: 3B íí! !! í !! ííí! 5 {a {hÇíí h b.w / {{{{w: 3B íí! ¡¡íí! ííí!
    ttttttttttt ttttttttttttttt Çb5 {í ítt! í !! ííííí íí! ! í !!! ííí! b. {Ç {{ítt !!! íí! ííí í !! ! í !!! ííí! bWù {{ítt !! ííííí !! í !! ! í! í! ííí!
    ttttttttttt ttttttttttttt [aD {h {íw: 3B íí! ! í! í! ííí!
    ttttttttttt tttt [aD {higw {{9víí! í! íí! ! í !!! ííí!
    ttttttttttt tttttttttt. {{hh {ítt! íííííííí í !! !!!!! t! a / {hwíw: 3B! í! ¡¡íí! ííí! ttttttttttt tttttttttt. {{{í {ítt !! íííí! í! ! í! ¡¡íí! ííí! ttttttttttt tttttttttttttt And [{h ítt! íí !!!!! í íí! !!! íííí! ttttttttttt ttttttttttt [aD {{{{í í {{: 3B í !! ¡¡íí! ííí!


    . {{hh {: 3B í !! ¡¡íí! ííí!ttttttttttt tttttttttttt. {{{{h ítt !! ííí! íí! í !! ¡¡íí! ííí!wD /. {{{ítt !! ííí! íí! í !! ¡¡íí! ííí!ttttttttttt ttttttttttt! Ü {{Çh: 3B í !! ¡¡íí! ííí!! Ü {whÇí: 3B í !! ¡¡íí! ííí!Çw {: 3B í !! ¡¡íí! ííí!bùÇÇh: 3B í !! ¡¡íí! ííí!b / La. {{h {w: 3B! í! !! í !! ííí!{{{Ç {: 3B í !! ¡¡íí! ííí![.Üa {{{: 3B! Í! !! í !! ííí!th {w: 3B í !! ¡¡íí! ííí!! íÇ {: 3B í !! ¡¡íí! ííí!ttttttttttt ttttttttttt hÇ {í ítt! í! í! íí !! íí! !! í !! ííí!
    awò {{DÇÇí {ítt !! íí !! í !! íí! !! í !! ííí! ttttttttttt ttttttttttttttttt YChÇh h b.w / {{Ç {hÇ ítt !! í !! í !! í íí! ¡¡íí! ííí! ttttttttttt ttttttttt W / a {hh {ítt! íí! ííí !! í !! ¡¡íí! ííí! ttttttttttt ttttttttttttt b.w / {{{íw ítt! í! í! íííí íí! ¡¡íí! ííí!
    ttttttttttt ttttttttttttt [aD {h {hh: 3B í !! ¡¡íí! ííí!
    {WÇ9D {: 3B í !! !! í !! ííí! ttttttttttt tttttttttt b.w / {Ç {Çwh: 3B í !! !! í !! ííí! ttttttttttt tttttttt [bÜD9DÇÇ {: 3B íí! ¡¡íí! ííí! ttttttttttt ttt í / {{hw: 3B í !! ¡¡íí! ííí!
    Dahw ítt !! í! Í! Í! Í í !! ! í !!! ííí! Üíw: 3B í !! ¡¡íí! ííí!
    Table 3i. Identity percentages of the amino acid sequences encoded by the hinI genes in different microorganisms of the Pseudomonas genus.
    9999999 / 99 /I9IL
    tttttttttt LttttttLt
    !!!!
    ttttttttttt tttttt ORIttL!! í !!! í !!
    {{{{{{{ ítt! í! í! íí! ííí! !! íí !! í !!
    {{{ ítt !! ííííííííí! !!! í !! í !!
    {{{ ítt !! íí !!! íííí! !!!!!! í !!
    !!!!
    b.w / {w {{w ítt !!!!! í! íí!! í !!! í !!
    D.D { ítt !!!!!! í! ííí! !!! í !! í !!
    .Lw5D { ítt! íííí! í !!íí! !!! í !! í !!
    YÇ {wwÇ 13Bíí! !!! í !! í !!
    C{ ítt !! ííííííííí! !!! í !! í !!


    í {{í ítt !! í !!! íí! íí! !!!!!! í !! !!!! í {íh9h {h ítt !!!!! í! íí !! í !!! í !!
    . {D {ítt! Íííí! Í !! íí! !!! í !! í !! b5 {ítt !! ííííííí íí! !!! í !! í !! !! h ítt! íííí !! íí íí! !!! í !! í !!
    b.w / {Ç {ÇwÇ h 5 {a ttttttttttt tttttttt {{{wh ítt !! í! í !!!! í !! !! íí !! í !! ttttttttttt ttttttttttt [wh ítt !!!! ííííí íí! !!!!!! í !! ttttttttttt ttttttttt {. {Çhh ítt !! íí !!! íí íí! !!!!!! í !! {. {{Ç {ítt !! íí !!! íí íí! !!!!!! í !! ttttttttttt tttttttt {W {Ç ítt! í! í !! í! í íí! !! íí !! í !! D9W {ítt! Íí! Ííííí íí! !!!!!! í !! í {{DÇ {.D {Ç ítt! í! í! íí! í íí! !!!!!! í !! 5 {a {hwíh ítt! Íí! Ííííí íí! !!!!!! í !! ttttttttttt ttttttttt b.w / {{{{{h 5 {a {hÇÇw ítt! íí! íííí! íí! !!!!!! í !! íí!
    : 3B ttttttttttt ttttt a9WÇh {!!! í !! íí!
    ttttttttttt ttttttttttt 5 {a {{ÇÇ {ítt! ííí! íí !! íí! !!!!!!ttttttttttt tttttttttttttt b.w / {Ç {{íí: 3B íí! !!! ííí!ttttttttttt tttttttttt í / {{íh ítt! ííí! ííí! íí! !! íí !! í !!ttttttttttt ttttttttttttttt b5Çh: 3B íí! !!!!!! í !!5 {a {hÇíí h b.w / {{{{w: 3B íí! !!!!!! í !!ttttttttttt ttttttttttttttt Çb5 {í ítt !! ííí !!! í íí! !!! í !! í !!b. {Ç {{ítt !!! íí! íí! íí! !! í !!! í !!bWù {{ítt !! íí !!! íí íí! !!!!!! í !!ttttttttttt ttttttttttttt [aD {h {íw: 3B íí! !!!!!! í !!ttttttttttt tttt [aD {hig {: 3B íí! !! íí !! í !!ttttttttttt tttttttttt. {{hh {: 3B íí! !! íí !! í !!t! a / {hwíw: 3B íí! !! íí !! í !!ttttttttttt tttttttttt. {{{í {: 3B íí! !! íí !! í !!ttttttttttt tttttttttttttt And [{h ítt! íí !! íííí íí! !! í !!! í !!ttttttttttt ttttttttttt [aD {{{{í í {{: 3B íí! !! íí !! í !!. {{hh {: 3B íí! !! íí !! í !!ttttttttttt tttttttttttt. {{{{h: 3B íí! !! íí !! í !!wD /. {{{: 3B íí! !! íí !! í !!ttttttttttt ttttttttttt! Ü {{Çh: 3B íí! !!!!!! í !!! Ü {whÇí: 3B íí! !! íí !! í !!Çw {: 3B íí! !!!!!! í !!bùÇÇh: 3B íí! !! íí !! í !!b / La. {{h {w: 3B íí! !!!!!! í !!


    {{{Ç {: 3B íí! !!!!!! í !! [.Üa {{{: 3B íí! !!!!!! í !! th {w: 3B íí! !!!!!! í !! ! íÇ {: 3B íí! !!!!!! í !!
    ttttttttttt ttttttttttt hÇ {í ítt! í! íííííí í !! !! íí !! í !!
    awò {{DÇÇí {ítt !! íí! í! íí í !! !! íí !! í !! ttttttttttt ttttttttttttttttt YChÇh h b.w / {{Ç {hÇ ítt !! í! ííí! í íí! !! í !!! í !! ttttttttttt ttttttttt W / a {hh {ítt! ííí !! ííí íí! !! íí! íí! ttttttttttt ttttttttttttt b.w / {{{íw ítt !! í! íí! íí íí! !! í !!! í !!
    ttttttttttt ttttttttttttt [aD {h {hh: 3B í !! !! í !!! í !!
    {WÇ9D {: 3B í !! !! í !!! í !! ttttttttttt tttttttttt b.w / {Ç {Çwh: 3B í !! !! íí !! í !! ttttttttttt tttttttt [bÜD9DÇÇ {: 3B í !! !! íí !! í !! ttttttttttt ttt í / {{hw: 3B íí! !!!!!! í !!
    Dahw ítt !! ííííííí íí! !! í !!! í !! Üíw: 3B íí! !! íí !! í !!
    Table 3j. Identity percentages of amino acid sequences encoded by the hinJ genes in different microorganisms of the Pseudomonas genus.
    9999999 / 99 /I9IW
    tttttttttt LttttttLt
    !!!!
    ttttttttttt tttttt ORIttW! í! í! í! í!
    {{{{{{{ ítt !! í! í! í !!íí! ! í! í! í! í!
    {{{ ítt !! í! íí! íííí! ! ííí! í !!!
    {{{ ítt! íí !!!!!!íí! ! ííí! í !!!
    !!!!
    b.w / {w {{w ítt !!!!! íí !!! í! í! í! í!
    D.D { ítt !!!!!!!íí! ! í !!! í! í!
    .Lw5D { ítt !! í! í !! íííí! ! ííí! í !!!
    YÇ {wwÇ bttí! í! íííí! ! ííí! í! í!
    C{ ítt !! í! í !! íííí! ! ííí! í !!!
    í {{í ítt !! íí! í! ííí !! ¡íí !! ííí!
    í {íh9h {h ítt !! í! ííí! ííí! ! í !!! í! í!
    . {D { ítt !! ííííí !!íí! ! ííí! í !!!
    b5 { ítt !! í! í !! íííí! ! ííí! í !!!
    !! h ítt! íííí! ííííí! ¡íí !! ííí!
    b.w / {Ç {ÇwÇ h 5 {a
    ttttttttttt tttttttt {{{whítt !! í! í !!!!íí! ¡íí !! ííí!
    ttttttttttt ttttttttttt [wh: 3B! ! íí !! í !!!
    ttttttttttt ttttttttt {. {Çhhítt! íí !!!!!!íí! ! ííí! í !!!


    {. {{Ç {ítt! Íí !!!!!! íí! ! ííí! í !!!ttttttttttt tttttttt {W {Ç ítt! íí! ííí !! íí! ! íí !! í !!!D9W {ítt! Íí! Ííííí íí! ! íí !! í !!!í {{DÇ {.D {Ç ítt !! íííí! í! íí! ! ííí! í !!!5 {a {hwíh ítt! Íí! Ííííí íí! ! íí !! í !!!ttttttttttt ttttttttt b.w / {{{{{h 5 {a {hÇÇw ítt! íííííí! í í !! ! í !!! í! í!ttttttttttt ttttt a9WÇh {: 3B í !! ¡íí !! íí !!ttttttttttt ttttttttttt 5 {a {{ÇÇ {ítt! ííí! íí !! íí! ¡ííí! íí !!ttttttttttt tttttttttttttt b.w / {Ç {{íí: 3B í !! ! í! í! ííí!ttttttttttt tttttttttt í / {{íh ítt! ííí! íííí íí! ! í! í! í! í!ttttttttttt ttttttttttttttt b5Çh ítt! íí !!!! íí íí! ¡ííí! ííí!5 {a {hÇíí h b.w / {{{{w ítt! Íí! Íí! Í! í !! ! ííí! í !!!ttttttttttt ttttttttttttttt Çb5 {í ítt! íí !!!!!! íí! ! ííí! í !!!b. {Ç {{ítt !!! íí! íí! íí! ! íí !! í !!!bWù {{ítt! íí !!!!!! íí! ! ííí! í !!!ttttttttttt ttttttttttttt [aD {h {íw: 3B íí! ! ííí! í! í!ttttttttttt tttt [aD {hig {: 3B íí! ! íí !! í !!!ttttttttttt tttttttttt. {{hh {: 3B í !! ¡íí !! ííí!t! a / {hwíw: 3B í !! ! í! í! ííí!ttttttttttt tttttttttt. {{{í {: 3B íí! ! í! í! í! í!ttttttttttt tttttttttttttt And [{h ítt! íí !! íííí íí! ¡íí !! íí !!ttttttttttt ttttttttttt [aD {{{{í í {{: 3B í !! ! í! í! ííí!. {{hh {: 3B í !! ! í! í! ííí!ttttttttttt tttttttttttt. {{{{h: 3B íí! ! í! í! í! í!wD /. {{{: 3B íí! ! í! í! í! í!ttttttttttt ttttttttttt! Ü {{Çh: 3B íí! ! í! í! í !!!! Ü {whÇí: 3B íí! ! íí !! í !!!Çw {: 3B í !! ¡íí !! ííí!bùÇÇh: 3B í !! ! í! í! ííí!b / La. {{h {w ítt !! íí !! í! í í !! ! í! í! ííí!{{{Ç {: 3B í !! ! í! í! ííí![.Üa {{{: 3B íí! ! í! í! í! í!th {w ítt !! íí! í! íí í !! ¡íí !! ííí!! íÇ {ítt !!! íí! íí! í !! ! í! í! ííí!ttttttttttt ttttttttttt hÇ {í ítt! í! íí! íí! íí! ! í !!! ííí!awò {{DÇÇí {ítt! í! íí! ííí íí! ! í !!! ííí!ttttttttttt ttttttttttttttttt YChÇh h b.w / {{Ç {hÇ ítt !! í! ííí! í í !! ¡ííí! ííí!ttttttttttt ttttttttt W / a {hh {ítt! ííí !! ííí í !! ! í! í! íí !!ttttttttttt ttttttttttttt b.w / {{{íw ítt! í! í !! í! í í !! ! í !!! í !!!ttttttttttt ttttttttttttt [aD {h {hh: 3B íí! ! í !!! íí !!{WÇ9D {: 3B íí! ! í! í! íí !!ttttttttttt tttttttttt b.w / {Ç {Çwh: 3B í !! ! í !!! íí !!


    ttttttttttt tttttttt [bÜD9DÇÇ {: 3B íí! ! í !!! íí !!ttttttttttt ttt í / {{hw ítt !! íí! í! íí í !! ¡íí !! ííí!Dahw ítt !! í! Ííííí íí! ! íí !! í !!!Üíw: 3B íí! ! í !!! ííí!
    3k table. Identity percentages of amino acid sequences encoded by the hinK genes in different microorganisms of the Pseudomonas genus.
    9999999 / 99 /I9IY
    tttttttttt LttttttLt
    !!!!
    ttttttttttt tttttt ORIttY! Í !!! í !!!
    !!!!
    {{{{{{{ ítt !!! í !! í !! ! í !!! í !!!
    {{{ ítt !!! íí !! í! íí! ! ííí! í !!!
    {{{ ítt !! íííí! í! íí! ! ííí! í !!!
    !!!!
    b.w / {w {{w ítt !!! í !! í !! ! í !!! í !!!
    D.D { ítt !!!!!! ííí íí! ! íí !! í !!!
    .Lw5D { ítt !!! íí !! í! íí! ! ííí! í !!!
    YÇ {wwÇ bttí !! í! í íí! ! ííí! í !!!
    C{ ítt !! ííí! í! í íí! ! ííí! í !!!
    í {{í ítt !! íí !! í !! íí! ! íí !! í !!!
    í {íh9h {h ítt !! í! íí! íí í !! !!! íííí!
    . {D { ítt !! ííí! í! í íí! ! íí !! í !!!
    b5 { ítt !!! íííí! í íí! ! ííí! í !!!
    !! h ítt! íííí! ííí íí! ! ííí! í !!!
    b.w / {Ç {ÇwÇ h 5 {a
    ttttttttttt tttttttt {{{whítt !! í! íííí! íí! ! í !!! ííí!
    ttttttttttt ttttttttttt [whítt !!! íííííí íí! ! ííí! í !!!
    ttttttttttt ttttttttt {. {Çhhítt !! íííí! í! íí! ! ííí! í !!!
    {.{{C{ ítt !! íííí! í! íí! ! ííí! í !!!
    ttttttttttt tttttttt {W {Ç: 3B! ! ííí! í !!!
    D9W { : 3B í !! ! íí !! í !!!
    í {{DÇ {.D {Ç : 3B í !! ! íí !! í !!!
    5 {a {hwíh : 3B í !! ! íí !! í !!!
    ttttttttttt ttttttttt b.w / {{{{{h 5 {a {hÇÇw: 3B! ! ííí! í !!!
    ttttttttttt ttttt a9WÇh {: 3B! ! í !!! í !!!
    ttttttttttt ttttttttttt 5 {a {{ÇÇ {: 3B! ! íí !! í !!!
    ttttttttttt tttttttttttttt b.w / {Ç {{íí: 3B! !!! íííí!
    ttttttttttt tttttttttt í / {{íhítt! íí! íííí! í !! ! ííí! í !!!
    ttttttttttt ttttttttttttttt b5Çh: 3B í !! ¡ííí! ííí!
    5 {a {hÇíí h b.w / {{{{w : 3B! ¡íí !! ííí!
    47

    ttttttttttt ttttttttttttttt Çb5 {í ítt !! íííí! í! íí! ! ííí! í !!!
    b. {Ç {{ítt !!! íí! ííí íí! ! ííí! í !!!
    bWù {{: 3B íí! ! ííí! í !!! ttttttttttt ttttttttttttt [aD {h {íw: 3B í !! ! ííí! í !!! ttttttttttt tttt [aD {higw {{9wíí !! íí! ! ííí! í !!! ttttttttttt tttttttttt. {{hh {{5b !! ííí íí! ! íí !! í !!!
    t! a / {hwíw: 3B íí! !!! íííí! ttttttttttt tttttttttt. {{{í {ítt !! íííí! í! íí! ! ííí! í !!! ttttttttttt tttttttttttttt And [{h ítt! íí !!!! í! íí! ! íí !! í !!! ttttttttttt ttttttttttt [aD {{{{í í {{ítt! í !! ííí! í í !! ! íí !! í !!!
    . {{hh {: 3B í !! ! íí !! í !!! ttttttttttt tttttttttttt. {{{{h ítt !! ííí! í! í í !! ! ííí! í !!!
    wD /. {{{: 3B í !! ! ííí! í !!! ttttttttttt ttttttttttt! Ü {{Çh ítt! í !! ííí !! íí! ! íí !! í !!!
    ! Ü {whÇí: 3B íí! ! íí !! í !!!
    Çw {: 3B íí! ! íí !! í !!!
    bùÇÇh ítt !!!! í! ííí í !! ! ííí! í !!!
    b / La. {{h {w ítt! í! í! í! íí í !! ¡ííí! ííí!
    {{{Ç {: 3B íí! ! íí !! í !!!
    [.Üa {{{: 3B íí! ! ííí! í !!!
    th {w: 3B í !! ! ííí! í !!!
    ! íÇ {: 3B í !! ! ííí! í !!!
    ttttttttttt ttttttttttt hÇ {í: 3B íí! ! í! í! ííí!
    awò {{DÇÇí {: 3B íí! ! í! í! ííí! ttttttttttt ttttttttttttttttt YChÇh h b.w / {{Ç {hÇ: 3B íí! ! í! í! ííí! ttttttttttt ttttttttt W / a {hh {: 3B í !! ! í !!! í !!! ttttttttttt ttttttttttttt b.w / {{{íw: 3B í !! ! í !!! ííí! ttttttttttt ttttttttttttt [aD {h {hh: 3B íí! ! í !!! ííí!
    {WÇ9D {: 3B íí! ! í !!! ííí! ttttttttttt tttttttttt b.w / {Ç {Çwh: 3B íí! ! í! í! ííí! ttttttttttt tttttttt [bÜD9DÇÇ {: 3B íí! ! íí !! í !!! ttttttttttt ttt í / {{hw: 3B í !! ! ííí! í !!!
    Dahw: 3B! ! ííí! í !!!
    Üíw: 3B í !! ¡ííí! ííí!
    Thus, in a preferred embodiment of the molecule of the first aspect, the sequences are the sequences represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12. In another preferred embodiment, the sequence of the molecule of the first aspect is the sequence represented by SEQ ID NO: 1.


    Therefore, in a preferred embodiment of the molecule of the first aspect, the sequences are coding sequences for the proteins represented by SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13.
    In the present invention, "coding sequence" means any nucleic acid sequence that encodes, according to the genetic code, the proteins represented in the sequence listing, or any nucleic acid sequence that encodes proteins with the same function as the proteins represented. in the sequence listing.
    In a preferred embodiment of the nucleic acid molecule of the first aspect, said nucleic acid molecule further comprises: a sequence with at least 60% identity with the sequence represented by SEQ ID NO: 14; a coding sequence for the protein represented by SEQ ID NO: 15; or the sequence represented by SEQ ID NO:
    14.
    In a preferred embodiment of the nucleic acid molecule of the first aspect, said nucleic acid molecule further comprises: a sequence with at least 60% identity with the sequence represented by SEQ ID NO: 16; a coding sequence for the protein represented by SEQ ID NO: 17; or the sequence represented by SEQ ID NO:
    16.
    In a preferred embodiment of the nucleic acid molecule of the first aspect, said nucleic acid molecule further comprises: a sequence with at least 60% identity with the sequence represented by SEQ ID NO: 18; a coding sequence for the protein represented by SEQ ID NO: 19; or the sequence represented by SEQ ID NO:
    18.
    In a preferred embodiment of the nucleic acid molecule of the first aspect, said nucleic acid molecule comprises the sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2.
    In a preferred embodiment of the nucleic acid molecule of the first aspect, said nucleic acid molecule further comprises: a sequence with at least 60% identity with the sequence represented by SEQ ID NO: 24; a coding sequence for the protein represented by SEQ ID NO: 25; or the sequence represented by SEQ ID NO:
    24.
    In a preferred embodiment of the nucleic acid molecule of the first aspect, said nucleic acid molecule further comprises: a sequence with at least 60% of


    identity with the sequence represented by SEQ ID NO: 20; a coding sequence for the protein represented by SEQ ID NO: 21; or the sequence represented by SEQ ID NO:
    twenty.
    In a preferred embodiment of the nucleic acid molecule of the first aspect, said nucleic acid molecule further comprises: a sequence with at least 60% identity with the sequence represented by SEQ ID NO: 22; a coding sequence for the protein represented by SEQ ID NO: 23; or the sequence represented by SEQ ID NO:
    22
    In a second aspect, the present invention relates to a microorganism capable of degrading histamine or a methylated derivative thereof, characterized in that it comprises the nucleic acid molecule of the first aspect, where said molecule has been artificially introduced into said microorganism.
    In a third aspect, the present invention relates to a microorganism capable of degrading the imidazolacetic acid characterized in that it comprises the nucleic acid molecule of the first aspect, wherein said molecule has been artificially introduced into said microorganism.
    In a fourth aspect, the present invention relates to a method for the elimination of histamine or a methylated derivative thereof from a medium, characterized in that in said medium a microorganism comprising the nucleic acid molecule of the first aspect is cultured. .
    As used herein, the term "medium" can be any medium on which the microorganisms of the invention can be grown, including a food
    or beverage intended for consumption by humans or animals, a starting raw material for obtaining said food or drink or an intermediate product in obtaining the food or drink. As used herein, the term "microorganism" includes prokaryotic and eukaryotic microbial species of the Archaea, Bacteria and Eucarya domains, the latter including yeast and filamentous fungi, protozoa, algae or higher Protista. The terms "microbial cells" and "microbes" are used interchangeably with the term microorganism.
    In a preferred embodiment of the process for the removal of histamine or a methylated derivative thereof from the fourth aspect, the microorganism is the strain Pseudomonas putida U CECT4848. Preferably, the medium from which the


    Histamine or the methylated derivative thereof is a fermented food that is treated with a starter culture of the microorganism.
    In a preferred embodiment of the process for the removal of histamine or a methylated derivative thereof from the fourth aspect, the histamine or the methylated derivative thereof is transformed into imidazoleacetic acid and where the activity of the monooxygenase enzyme, which transforms the Imidazolacetic acid in 4-hydroxy-imidazol-5-acetic acid or its analogue, 4-imidazolon-5-acetic acid, is prevented by a mutation in the sequence represented by SEQ ID NO: 14. Preferably, said mutation is introduced by use of the transposon Tn5.
    In a preferred embodiment of the process for the removal of histamine or a methylated derivative thereof from the fourth aspect, the histamine or the methylated derivative thereof is transformed into alanine and where the activity of the enzyme D-amino acid dehydrogenase, which transforms alanine into pyruvate, is prevented by a mutation in the dadA gene; or where the activity of the enzyme racemase, which transforms L-alanine into Dalanin, is prevented by a mutation in the dadX gene. Preferably, said mutation is introduced by the use of the Tn5 transposon.
    In a preferred embodiment of the process for the removal of histamine or a methylated derivative thereof from the fourth aspect, the histamine or the methylated derivative thereof is transformed into aspartic acid and where the activity of the enzyme aspartate ammonium lyase, which transforms aspartate into fumarate, is prevented by a mutation in the sequence represented by SEQ ID NO: 20 or by a mutation in the sequence represented by SEQ ID NO: 22. Preferably, said mutation is introduced by the use of the transposon Tn5.
    In a fifth aspect, the present invention relates to a process for the elimination of histamine or a methylated derivative thereof and also of the imidazoleacetic acid of a medium, characterized in that in said medium a microorganism comprising the molecule of nucleic acid of the first aspect, when said molecule comprises, in addition to sequences with at least 60% identity with the sequences represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 AND SEQ ID NO: 12, at least one sequence with at least 60% identity with the sequence represented by SEQ ID NO: 14. In preferred embodiments, the first aspect nucleic acid molecule further comprises a sequence with at least one 60% identity with the sequence represented by SEQ ID NO: 18, a sequence with at least 60% identity with the sequence represented by SEQ ID NO: 16, a


    sequence with at least 60% identity with the sequence represented by SEQ ID NO: 24, a sequence with at least 60% identity with the sequence represented by SEQ ID NO: 20 and / or a sequence with at least 60 % identity with the sequence represented by SEQ ID NO: 22. In a preferred embodiment, the nucleic acid molecule of the first aspect comprises at least one sequence with at least 60% identity with the sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2. The fifth aspect of the invention relates to a process for the elimination of imidazolacetic acid derived from histamine (or a methylated derivative thereof), where the microorganism that is grown has at least the gene Pseudomonas putida U CECT4848 hinF, or any sequence encoding a protein with the same function as the HinF enzyme, which transforms the imidazolacetic acid into a hydroxylated derivative.
    In a preferred embodiment of the process for the removal of histamine or a methylated derivative thereof and imidazolacetic acid of the fifth aspect, the microorganism is the strain Pseudomonas putida U CECT4848. Preferably, the medium from which histamine or a methylated derivative thereof and imidazoleacetic acid is removed is a fermented food that is treated with a microorganism starter culture.
    In a sixth aspect, the present invention relates to a process for obtaining imidazoleacetic acid comprising culturing a microorganism comprising the nucleic acid molecule of the first aspect of the invention comprising the nucleotide genes or sequences with at least 60% identity. with the hinC, hinA, hinD, hinE and hinB genes, or with the hin1 cluster, which allow the transformation of histamine or a methylated derivative thereof into imidazoleacetic acid. Preferably, in addition, the activity of the enzyme monooxygenase, which converts the imidazolacetic acid into 4-hydroxy-imidazol5-acetic acid or its analogue, 4-imidazolon-5-acetic acid, is prevented by a mutation in the sequence represented by SEQ ID NO: 14. In another preferred embodiment of the sixth aspect procedure, the expression and / or function of the HinK protein is prevented by a mutation in the sequence represented by SEQ ID NO: 24. In both cases, preferably the mutation is introduced by using the transposon Tn5.
    In a preferred embodiment of the sixth aspect procedure, the microorganism is the strain Pseudomonas putida U CECT4848.
    In a seventh aspect, the present invention relates to a process for obtaining alanine which comprises culturing a microorganism comprising the nucleic acid molecule of the first aspect of the invention comprising the genes or sequences.


    nucleotides with at least 60% identity with the hinC, hinA, hinD, hinE and hinB genes, or with the hin1 cluster. Preferably, in addition, the activity of the D-amino acid dehydrogenase, which transforms the alanine into pyruvate, is prevented by a mutation in the gene that gives. Preferably, said mutation is introduced by the use of the Tn5 transposon. Preferably, the microorganism is the strain Pseudomonas putida U CECT4848.
    In an eighth aspect, the present invention relates to a process for obtaining aspartic acid comprising culturing a microorganism comprising the nucleic acid molecule of the first aspect of the invention comprising the nucleotide genes or sequences with at least 60% identity. with the hinC, hinA, hinD, hinE and hinB genes, or with the hin1 grouping. Preferably, in addition, the activity of the enzyme aspartate ammonium lyase, which converts aspartate into fumarate, is prevented by a mutation in the sequence represented by SEQ ID NO: 20 or by a mutation in the sequence represented by SEQ ID NO: 22. Preferably , said mutation is introduced through the use of the Tn5 transposon. In a preferred embodiment, the microorganism is the strain Pseudomonas putida U CECT4848.
    In a ninth aspect, the present invention relates to a method for removing L-histidine from a medium, characterized in that in said medium a microorganism is formed which comprises the nucleic acid molecule of the first aspect and additionally comprises a nucleic acid molecule. which codes for an enzyme with histidine decarboxylase activity. Preferably, the medium from which L-histidine is removed is a fermented food that is treated with a microorganism starter culture.
    The present invention relates to the description of an alternative method for the degradation of histamine (Hin) and imidazoleacetic acid (ImAA) in samples containing them. It is based on the discovery of a new catabolic route (Fig. 7 and Fig. 8), identified by the authors of the invention in Pseudomonas putida U CECT4848, capable of degrading these two molecules to fumaric acid. The complete pathway involves the intervention of: (i) the enzymes encoded in the clusters, hin1 (HinEABCD), hin2 (HinGHF), hin3 (HinIJ) and in the hinK (HinK) gene, some of which are essential for degradation of histamine; and (ii) enzymes encoded in the dadXAR and coxBA-C genes, which are only required when histamine is the only carbon source (Fig. 7).
    Enzymes encoded in the genes belonging to the hin1 cluster are responsible for the transformation of histamine into imidazoleacetic acid. The HinA protein is a transporter belonging to the family of transporters called ABC that is involved in the


    taking histamine from the culture medium inside the bacteria. HinC is a histamine deaminase or histamine pyruvate aminotransferase that uses histamine and pyruvic acid as substrates and generates imidazolacetaldehyde (ImAdh) and L-alanine as products. This enzyme removes the amino group from histamine and transfers it to pyruvic acid (or pyruvate) to generate ImAdh and L-Ala. HinD and HinE are aldehyde dehydrogenases that can catalyze the oxidation of ImAdh to ImAA and that use NAD + as coenzymes. Finally, HinB, the other protein belonging to this cluster, is a regulator belonging to the LuxR family, which acts as a transcriptional activator of the other genes.
    Enzymes encoded in the hin2 cluster (HinGHF) sequentially catalyze the transformation of ImAA into a hydroxylated derivative (HinF); the opening of the imidazole ring, generating the N-formyl-Aspartic acid (FA), prior formation of N2-formylisoasparragine (FiAsn) and, finally, the hydrolysis of FA to generate L-Aspartic (HinG). The HinH and HinG enzymes could act forming a complex that, from the hydroxylated derivative of ImAA, generated L-aspartic. The compound indicated as 4-imidazolon-5-acetic acid is a structure equivalent to hydroxylated ImAA (ketoenolic equilibrium) (Fig. 7).
    Additionally, we have shown that an affected mutant in the hinK gene, which encodes a regulator belonging to the LysR family (HinK), located outside the hin1, hin2 and hin3 clusters (Fig. 8), cannot catabolize either histamine or Imidazolacetic acid (ImAA). This protein (HinK) activates the transcription of the hinGHF genes so that, when it is mutated, and whenever there is another carbon source in the medium that supports bacterial growth, histamine is transformed into ImAA, a compound that, when not continue to degrade, accumulates as such in the broth.
    The aspartic acid generated by the action of HinF, HinH and HinG enzymes, to be transformed into fumaric acid requires the participation of two additional proteins (HinI and HinJ) encoded in the hinI and hinJ genes belonging to what we have called cluster hin3. HinI corresponds to an enzyme with aspartate ammonium lyase activity that deaminates aspartic acid generating fumaic acid (Fig. 7), which is the final catabolite of the route and that connects this pathway with the general metabolism of the bacteria when incorporated into the cycle of the tricarboxylic acids or Krebs cycle. The HinJ protein is a transcriptional regulator of the LysR family. A mutation in this gene prevents the degradation of aspartic acid, which indicates that it is an activator that enhances the transcription of the hinI gene (the one that encodes aspartate ammonium lyase, HinI). Mutations in either of these two genes will lead to the accumulation of aspartic acid (or aspartate) from histamine or


    from imidazolacetic, provided that a source of carbon is added to the culture medium that allows the growth of that bacterium.
    The mission of the DadXAR system is twofold. On the one hand, it allows regenerating the pyruvate required as an amino group acceptor in the transamination reaction. In addition, it participates in an electron transfer system, in which the final stage is catalyzed by a cytochrome C oxidase complex of type aa3 (CoxBA-C), and which is responsible for the genesis of the energy required to transport the histamine The DadX protein, a racemase, catalyzes the transformation of L-alanine generated by histamine-pyruvate aminotransferase (HinC) into the D-Alanine isomer (Fig. 7). Subsequently, this amino acid is deaminated by an FAD-dependent D-amino acid dehydrogenase (DadA) that catalyzes the deamination of the D-Ala, generating the pyruvate required to deaminate another histamine and ammonia molecule. During this oxidative deamination process, the FAD linked to DadA is reduced to FADH2 and, this, is coupled to an electronic transport chain in which, as we have indicated, a cytochrome C oxidase complex type aa3 (CoxBA-C) participates composed of three CoxA, CoxB and CoxC subunits and an assembly protein (here indicated as Cox-) (Fig. 7). In this way, the energy needed to transport histamine is generated when it is being used as the only carbon source. The fact that a mutation in the dadA gene prevents growth in histamine and L-Ala indicates that this pathway is the only one existing in P. putida U to degrade both compounds. For this reason, the mutation of the dadA gene does not allow the genesis of pyruvate from D-Alanine, so, provided that this mutant is grown in a medium in which there is histamine and pyruvate or a source of pyruvate (acid 4- Hydroxy-phenylacetic -4-OH-AFA-or other) alanine will accumulate in the culture broth (Fig. 9). The same happens when the dadX gene that encodes a racemase (DadX) is mutated.
    Both the catabolic route itself, and the enzymes that catalyze the reactions that are part of it and, in particular, those catalyzed by the HinA, HinB, HinC, HinD, HinE proteins; HinH, HinG, HinK and HinJ are new and their nature was not obvious from the prior art since, as previously mentioned, the route responsible for the assimilation of histamine in Pseudomonas putida U CECT4848 and in other bacteria it was unknown until the date. In addition, in other cases in which some of the enzymes had been described, their participation had not been related to this route (cases of DadXAR, CoxBA-C and HinI), or the amino acid sequences of those proteins and that of the nucleotides. corresponding to the genes that encode them were completely unknown (case of HinK).


    The discovery of this route further demonstrates that the degradation of histamine in P. putida is not performed by the enzymes described in other living things. That is why, both the histamine and ImAA degradation process that leads to fumarate (through the action of the successive enzymes of the route), as well as the amino acid sequence 5 of the different enzymes that catalyze said reactions, and the coding sequences of these proteins (enzymes and regulators), are part of the present invention. Also part of the invention are the methods described for the expression of these genes in different microorganisms using vectors that allow synthesizing said enzymes (or enzymes with a high degree of identity thereto, which are capable of catalyzing the
    10 same reactions), as well as the recombinant microorganisms that have been transformed with said vectors.
    Given the great interest that the application of the process of the invention may have in foods, to decrease its histamine content, additional alternative aspects of the invention are the use of microorganisms capable of synthesizing at least the first, a subset or all of the enzymes necessary to complete the complete catabolic route described, to decrease the content of histamine in foods that contain it or, even, the use of said microorganisms to act on raw materials to be transformed, in order to be converted into food or beverages ready for human or animal consumption and that either contain naturally histamine, or that are likely to generate it during the process of transforming said raw materials into final foods. This is true both for the microorganism that, as disclosed in the present application, naturally contains all the genes necessary to synthesize the enzymes that catalyze the reactions of the complete pathway (Pseudomonas putida U CECT4848), as well as for possible recombinant microorganisms
    25 that have been transformed with some vector that allows the expression of at least one of the genes coding for the enzymes that catalyze the reactions of the catabolic process.
    The hin1, hin2 and hin3 clusters also contain other open reading frames that encode polypeptides that are involved in the regulation of the described catabolic pathway. Additionally, as we have indicated before, the hinK gene also encodes another regulator (HinK). Said polypeptides, the sequences that encode them, the embodiments of the procedure that are carried out while the regulatory polypeptides encoded in such clusters are present, are also included within the scope of the invention, as well as the possible expression vectors that include those coding sequences. and the
    35 microorganisms transformed with said vectors.


    Thus, the present invention provides both the alternative method described for the degradation of histamine and / or ImAA in any sample, as well as the sequences of the proteins capable of catalyzing and regulating the process of degradation of histamine and ImAA to fumaric acid, as well as the nucleotide sequences encoding said proteins. Expression vectors comprising sequences encoding at least one of said proteins and the microorganisms transformed therewith also constitute aspects of the invention, as well as the use of Pseudomonas putida U or any of said recombinant microorganisms to decrease the content of histamine or ImAA in a sample of a food or beverage intended for human or animal use or in some intermediate product of the transformation of raw materials into the final food or beverage, including the raw materials themselves.
    Thus, one aspect of the invention relates to nucleic acid molecules (cluster hin1, cluster hin2 and cluster hin3) (SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 from which they can synthesized proteins capable of catalyzing and / or regulating the process of the invention, or encoding proteins with a high degree of identity with them (defined or by the percentage of coincidence in the nucleotides (at least 60%), or by the ability to mate under strict hybridization conditions (the conditions under which complementary nucleic acid chains hybridize, which are easily identifiable by one skilled in the art in each specific case, but which can be summarized as elevated temperature conditions (65 ° C , for example) and low concentration of salts, as long as they are capable of carrying out the same activity.
    Specifically, in the cluster hin1, they refer to the nucleic acid molecules from which the enzyme that catalyzes the essential stage of the degradation process, histamin-aminotransferase (histamine deaminase or histamine pyruvate aminotransferase) (HinC), can be synthesized. or proteins homologous to it that have the same activity. Therefore, a first aspect of the present invention relates to an isolated nucleic acid molecule encoding a protein or protein complex capable of acting as histamine deaminase, selected from the group consisting of:
    a) a nucleic acid molecule comprising a sequence that is identical, at least 60%, to the sequence represented by SEQ ID No: 4 (corresponding to the DNA encoding HinC);
    b) a nucleic acid molecule comprising a sequence that hybridizes under strict conditions with the sequence of a);


    c) a nucleic acid molecule comprising a sequence encoding a polypeptide whose amino acid sequence is identical, at least 60%, to the sequence represented by SEQ ID No: 5 (HinC amino acid sequence);
    d) a nucleic acid molecule comprising a sequence encoding a variant
    Natural allelic of a polypeptide comprising the amino acid sequence represented by SEQ ID No: 5, where the hybridized nucleic acid molecule, under strict conditions, with a DNA sequence comprising the sequence mentioned in a), or sequences complementary to the same.
    In a preferred embodiment of the invention, the nucleic acid molecule comprises a
    10 sequence in which the percentage of identity in alternative a) or in alternative c) is 100%.
    In a further preferred embodiment, the isolated nucleic acid molecule of the first aspect of the invention also comprises a nucleic acid fragment from which the protein that acts as a histamine transporter, HinA, can be synthesized.
    15 (belonging to the ABC type transporter family) or a homologous protein to it that performs the same function. Thus, in said embodiment, the isolated nucleic acid molecule of the first aspect further comprises a sequence encoding a protein capable of acting as a transporter, selected from the group consisting of:
    a) a nucleic acid sequence that is identical, at least 60% to the sequence 20 represented by SEQ ID No: 6 (HinA coding DNA);
    b) a nucleic acid sequence comprising a sequence that hybridizes under strict conditions with the sequence of a);
    c) a nucleic acid sequence comprising a sequence encoding a polypeptide whose amino acid sequence is at least 60% identical to the sequence
    25 represented by SEQ ID No: 7 (HinA amino acid sequence);
    d) a nucleic acid sequence comprising a sequence encoding a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEQ ID No: 7, where the hybrid nucleic acid molecule, under strict conditions, with a sequence of DNA comprising the sequence mentioned in a), or a
    30 sequence complementary to it.


    Again, it is especially preferred that the nucleic acid molecule corresponds to that of alternative a) or c), in which the percent identity is 100%.
    In a further preferred embodiment, the isolated nucleic acid molecule also comprises two nucleic acid fragments from which either enzyme that catalyzes the next stage of the degradation process, Imidazolacetaldehyde (ImAdh) dehydrogenases (HinD) can be synthesized. and HinE) or a homologous protein that performs the same function. Both enzymes (HinD and HinE) can catalyze the oxidation of ImAdh to imidazoleacetic acid (ImAA). Thus, in said embodiment, the isolated nucleic acid molecule additionally comprises a sequence encoding a protein capable of acting as Imidazolacetaldehyde dehydrogenase, selected from the group consisting of:
    a) a nucleic acid sequence that is identical, at least 60% to the sequence represented by SEQ ID No: 8 (HinD coding DNA) and a nucleic acid sequence that is identical, at least 60% a the sequence represented by SEQ ID No: 10 (HinE encoding DNA);
    b) a nucleic acid sequence comprising a sequence that hybridizes under strict conditions with the sequences of a);
    c) a nucleic acid sequence comprising a sequence encoding a polypeptide whose amino acid sequence is identical, at least 60%, to the sequence represented by SEQ ID No: 9 (HinD amino acid sequence) and a sequence of nucleic acid comprising a sequence encoding a polypeptide whose amino acid sequence is identical, at least 60%, to the sequence represented by SEQ ID No: 11 (HinE amino acid sequence);
    d) a nucleic acid sequence comprising a sequence encoding a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEQ ID No: 9, where the hybrid nucleic acid molecule, under strict conditions, with a sequence of DNA comprising the sequence mentioned in a), or a sequence complementary to it. A nucleic acid sequence comprising a sequence encoding a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEQ ID No: 11, where the hybrid nucleic acid molecule, under strict conditions, with a DNA sequence that it comprises the sequence mentioned in a), or a sequence complementary to it.


    Again, it is especially preferred that the nucleic acid molecule corresponds to that of alternative a) or c), in which the percent identity is 100%.
    Another additional embodiment, complementary to the above, is one in which the nucleic acid molecule also comprises the polypeptide coding sequence (HinB) that acts as a regulator belonging to the LuxR family and that activates the transcription of the hinA and hinC genes . Thus, in said embodiment, the isolated nucleic acid molecule additionally comprises a sequence encoding a protein (HinB) capable of acting as a regulator of the sequence of reactions catalyzed by HinA and HinC, selected from the group consisting of:
    a) a nucleic acid sequence that is identical, at least 90%, to the sequence represented by SEQ ID No: 12 (HinB coding DNA);
    b) a nucleic acid sequence comprising a sequence that hybridizes under strict conditions with the sequence of a);
    c) a nucleic acid sequence comprising a sequence encoding a polypeptide whose amino acid sequence is at least 90% identical to the sequence represented by SEQ ID No: 13 (HinB amino acid sequence);
    d) a nucleic acid sequence comprising a sequence encoding a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEQ ID No: 13, where the hybrid nucleic acid molecule, under strict conditions, with a sequence of DNA comprising the sequence mentioned in a, or a sequence complementary thereto.
    Again, it is especially preferred that the nucleic acid molecule corresponds to that of alternative a) or c), in which the percent identity is 100%.
    In another possible embodiment, the nucleic acid molecule corresponding to the isolated hin2 cluster comprises the sequence corresponding to the hinF gene encoding a monooxygenase (HinF), selected from the group consisting of:
    a) a nucleic acid sequence that is identical, at least 60%, to the sequence represented by SEQ ID No: 14 (HinF coding DNA);
    b) a nucleic acid sequence comprising a sequence that hybridizes under strict conditions with the sequence of a);


    c) a nucleic acid sequence comprising a sequence encoding a polypeptide whose amino acid sequence is at least 60% identical to the sequence represented by SEQ ID No: 15 (HinF amino acid sequence);
    d) a nucleic acid sequence comprising a sequence encoding a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEQ ID No: 15, where the hybrid nucleic acid molecule, under strict conditions, with a sequence of DNA comprising the sequence mentioned in a), or a sequence complementary to it.
    Again, it is especially preferred that the nucleic acid molecule corresponds to that of alternative a) or c), in which the percent identity is 100%.
    In addition to the above combinations, the isolated nucleic acid molecule may also comprise the sequence encoding any of the remaining polypeptides identified in the hin2 cluster: HinG, HinH, or combinations thereof. Thus, another possible embodiment is one in which the isolated nucleic acid molecule additionally comprises a sequence encoding a protein, selected from the group consisting of:
    a) a nucleic acid sequence that is identical, at least 60%, to the sequence represented by SEQ ID No: 16 (HinG encoding DNA) and / or a nucleic acid sequence that is identical, at least in one 60%, to the sequence represented by SEQ ID No: 18 (HinH coding DNA)
    b) a nucleic acid sequence comprising a sequence that hybridizes under strict conditions with at least one of the sequences of a);
    c) a nucleic acid sequence comprising a sequence encoding a polypeptide whose amino acid sequence is at least 60% identical to the sequence represented by SEQ ID No: 17 (HinG amino acid sequence) and / or a sequence encoding a polypeptide whose amino acid sequence is identical, at least 60%, to the sequence represented by SEQ ID No: 19 (HinH amino acid sequence);
    d) a nucleic acid sequence comprising a sequence encoding a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEQ ID No: 17 and / or a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEQ ID No: 19, where the hybridized nucleic acid molecule, under strict conditions, with a DNA sequence comprising the


    less one of the sequences mentioned in a), or a sequence complementary to it.
    Again, it is especially preferred that the nucleic acid molecule corresponds to that of alternative a) or c), in which the percent identity is 100%.
    Another additional embodiment, complementary to the previous ones, is that in which the nucleic acid molecule also comprises the coding sequence (hinK) of the polypeptide (HinK) that acts as a regulator belonging to the LysR family and that activates the transcription of the hinF, hinG and hinH genes. Thus, in said embodiment, the isolated nucleic acid molecule additionally comprises a sequence encoding a protein.
    10 (HinK) capable of acting as a regulator of the sequence of reactions catalyzed by HinF, HinG and HinH, selected from the group consisting of:
    a) a nucleic acid sequence that is identical, at least 60%, to the sequence represented by SEQ ID No: 24 (HinK coding DNA);
    b) a nucleic acid sequence comprising a sequence that hybridizes under strict conditions with the sequence of a);
    c) a nucleic acid sequence comprising a sequence encoding a polypeptide whose amino acid sequence is at least 60% identical to the sequence represented by SEQ ID No: 25 (HinK amino acid sequence);
    d) a nucleic acid sequence comprising a sequence encoding a variant
    Natural allelic of a polypeptide comprising the amino acid sequence represented by SEQ ID No: 25, where the hybrid nucleic acid molecule, under strict conditions, with a DNA sequence comprising the sequence mentioned in a, or a sequence complementary to the same.
    In addition to the above combinations, the isolated nucleic acid molecule can
    25 also comprise the sequence coding for any of polypeptides identified in the hin3 cluster: HinI, HinJ, or combinations thereof. Thus, another possible embodiment is one in which the isolated nucleic acid molecule additionally comprises a sequence encoding a protein, selected from the group consisting of:
    a) a nucleic acid sequence that is identical, at least 60%, to the sequence 30 represented by SEQ ID No: 20 (HinI encoding DNA) and / or an acid sequence


    nucleic that is identical, at least 60%, to the sequence represented by SEQ ID No: 22 (HinJ coding DNA);
    b) a nucleic acid sequence comprising a sequence that hybridizes under strict conditions with at least one of the sequences of a);
    5 c) a nucleic acid sequence comprising a sequence encoding a polypeptide whose amino acid sequence is identical, at least 60%, to the sequence represented by SEQ ID No: 21 (HinI amino acid sequence) and / or a sequence encoding a polypeptide whose amino acid sequence is identical, at least 60%, to the sequence represented by SEQ ID No: 23 (HinJ amino acid sequence);
    D) a nucleic acid sequence comprising a sequence encoding a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEQ ID No: 21 and / or a natural allelic variant of a polypeptide comprising the amino acid sequence represented by SEQ ID No: 23, where the hybrid nucleic acid molecule, under strict conditions, with a DNA sequence comprising the
    15 minus one of the sequences mentioned in a), or a sequence complementary to it.
    Again, it is especially preferred that the nucleic acid molecule corresponds to that of alternative a) or c), in which the percent identity is 100%.
    So that the sequence of reactions catalyzed by the enzymes of the hin1, hin2 clusters
    20 and hin3 is carried out in its entirety, it is particularly preferred that the isolated nucleic acid molecule comprises coding sequences of all polypeptides that appear to be encoded in those gene clusters. Thus, in a particularly preferred embodiment of this aspect of the invention, the isolated nucleic acid molecules (hin1, hin2 and hin3) (SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3) comprise the sequences
    25 represented by SEQ ID No: 4 (DNA hinC), SEQ ID No: 6 (DNA hinA), SEO ID No: 8 (DNA hinD), SEQ ID No: 10 (DNA hinE), SEQ ID No: 12 (DNA hinB), SEQ ID No: 14 (DNA hinF), SEQ ID No: 16 (DNA hinG), SEQ ID No: 18 (DNA hinH), SEQ ID No: 24 (DNA hinK), SEQ ID No: 20 (DNA HinI) and SEQ ID No: 22 (HinJ DNA) and corresponding respectively to sequences encoding polypeptides whose amino acid sequence is identical, at least
    30 in 60%, to the sequence represented by SEQ ID No: 5 (HinC protein), SEQ ID No: 7 (HinA protein), SEQ ID No: 9 (HinD protein), SEQ ID No: 11 (HinE protein) , SEQ ID No: 13 (HinB protein), SEQ ID No: 15 (HinF protein), SEQ ID No: 17 (HinG protein), SEQ ID


    No: 19 (HinH protein), SEQ ID No: 25 (HinK protein), SEQ ID No: 21 (HinI protein) and SEQ ID No: 23 (HinJ protein).
    One possibility would be for the nucleic acid molecule to comprise the sequence represented by the sequences of the clusters hin1, hin2, hin3 and hinK of fused Pseudomonas U CECT4848 (SEQ ID No: 1 + SEQ ID No: 2 + SEQ ID No: 3 + SEQ ID No: 24). That is, in a preferred embodiment, the nucleic acid molecule would comprise not only the open-frame reading sequences identified in the hin1-3 and hinK clusters, and encoding all the enzymes and regulators required to transform histamine and ImAA into fumaric acid. . Thus, in a particularly preferred embodiment of the nucleic acid molecule of the invention, it would comprise the sequences encoding identical or function-like proteins and sequence homologous to those of the hin1-3 clusters. This is, at least 10 sequences (hinABCDEFGHIJK), of which each of them encodes a protein capable of catalyzing or regulating a stage of the transformation of histamine into fumaric acid through the sequence of reactions described in Fig. 7.
    The inclusion in any one of the nucleic acid molecules described in the invention of a coding sequence of a histidine decarboxylase (hdc) would allow, when expressing the proteins encoded therein, decarboxylation of histidine present in the medium could occur to give rise to to histamine, which could then be degraded thanks to the other enzymes encoded also in the nucleic acid molecules corresponding to hin1, hin2, hin3 and hinK (Fig. 10). Therefore, the case in which the isolated nucleic acid molecule further comprises a sequence encoding a histidine decarboxylase (Hdc) is preferred. Particularly preferred is the case in which the histidine decarboxylase is the Enterobacter aerogenes Hdc (CECT684) (GenBank accession number: M62745.1 sequence comprised in the ENTHDCA locus, 1327bp, the amino acid sequence of said enzyme is represented by SEQ ID No: 26 and its coding sequence, by SEQ ID NO: 27).
    Another aspect of the invention is related to an expression vector comprising at least the sequence corresponding to hin1, that of hin2 + hinK or the hin1 + hin2 + hinK described above. In a preferred embodiment, said vector is a plasmid. Particularly preferred is the case in which said plasmid comprises a nucleic acid fragment comprising the coding sequences comprised in the hin1, hin2 and the HinK clusters of Pseudomonas putida U CECT4848. A particular embodiment refers to the case where said nucleic acid fragment is inserted into plasmid pK18 :: mob, pJQ200SK or pMC, which are plasmids capable of remaining as a form


    Autonomous replicative in some bacterial species (pK18 :: mob, pJQ200SK and pMC) and with integrative capacity in others (pK18 :: mob, pJQ200SK) (99,102).
    Another preferred embodiment corresponds to the case in which each of these plasmids contains a nucleic acid fragment comprising the coding sequences present in the cluster hin1 of Pseudomonas putida, another those of hin2, and another those of hinK. Even more preferred is the case in which the cluster hin1 is inserted into plasmid pK18 :: mob, hin2 in the pJQ200SK and the hink in the pMC.
    In another additional embodiment the plasmid pK18 :: mob the plasmid pJQ200SK or the plasmid pMC further comprise a sequence encoding a histidine decarboxylase (Hdc). The case in which the histidine decarboxylase is the histidine decarboxylase of Enterobacter aerogenes is preferred.
    In a further aspect of the invention, a host organism transformed with any one of the expression vectors described above is described. A preferred case is one in which the host organism is a bacterium, this being preferably a bacterium capable of transforming a sugar into lactic acid (acid lactic acid bacteria). These microorganisms are important because the formation of lactic acid in different raw materials results in denaturation and gelation of proteins, initiating their transformation into food derivatives such as cheese, yogurt, sausages, etc. they act as initiators of the transformation of many raw materials and, in some cases (yogurt), are responsible for their culmination; in others, such as cheese, particularly cured cheese varieties, favor the action of other microorganisms, molds in many cases, which are responsible, for example, for the appearance of characteristic slabid substances. Since in the process of transforming raw materials such as milk or meat into cheese or sausages, there are often amounts of histamine that could make it advisable to reduce the present content of said amine, to have lactic bacteria that, in addition to performing their function in the process of food transformation, have the enzymes to catalyze the catabolic route disclosed in the present application, which allows the degradation of histamine, would facilitate the reduction of the final levels of this biogenic amines present in the final food intended for human consumption or animal
    A possible embodiment of this aspect of the invention contemplates the case in which the expression vector is inserted into the host genome. In another alternative embodiment, said expression vector remains as an autonomous replicative form.


    Another aspect of the invention, of great importance, relates to methods that employ the polypeptide sequences described above, to decrease the histamine and imidazoleacetic content in any sample, preferably for food and beverages for human or animal consumption, raw materials from which they originate or the intermediate products of transformation of said raw materials into food or final beverages. The procedure contemplates all the steps of histamine and imidazoleacetic transformation of the catabolic route discovered by the inventors. Within the scope of the invention, all variants thereof comprising the first stage, the transformation of histamine into imidazolacetaldehyde or imidazoleacetic acid, as well as the transformation of the latter into aspartic and / or smoking acids or in any of its components are considered included you go out.
    Thus, one aspect of the invention relates to a process for decreasing the content in a histamine sample (Fig. 7) comprising a step in which the compound of Formula I is transformed into the compounds of Formula II (ImAdh) first and in Formula III (ImAA) later. The transformation of the compound of Formula I into a compound of Formula II is catalyzed by a histamine pyruvate aminotransferase (histamin deaminase) encoded by the nucleic acid molecule of SEQ ID No: 4. or by an aminotransferase whose amino acid sequence is at least 60% identical to the polypeptide sequence represented by SEQ ID No: 5. The second stage, which catalyzes the oxidation of the imidazolacetaldehyde to imidazoleacetic acid is carried out by either of the two dehydrogenase aldehydes whose amino acid sequences are at least 60% identical to a polypeptide sequence represented by SEQ ID No: 9 and SEQ ID No: 11), respectively. In a third stage, the imidazolacetic (ImAA) is hydroxylated to 4-hydroxy-ImAA (see Fig. 7) by the action of a monooxygenase (HinF) whose amino acid sequence is at least 60% identical to the polypeptide sequence represented by SEQ ID No: 15. Subsequently, this cyclic compound or its structural analog, 4-imidazolon-5acetic acid, are substrates of the HinF enzyme, whose amino acid sequence is at least 60% identical to the polypeptide sequence represented by SEQ ID No: 15 giving rise to N2-formylisoasparragine (FiAsn) (Fig. 7). This compound is transformed into N-formyl aspartic acid (FA) by the action of an amidohydrolase (HinH) whose amino acid sequence is at least 60% identical to a polypeptide sequence represented by SEQ ID No: 19. FA is transformed into L-Aspartic acid, CO2 and NH3 · by the HinG enzyme, whose amino acid sequence is at least 60% identical to a polypeptide sequence represented by SEQ ID No: 17.


    Both enzymes, HinG and HinH could act in complex form transforming N2formilisoasparragina (FiAsn) in L-Aspartico. Finally, L-aspartic acid is transformed into fumaric acid by an enzyme with asparto-amonioliase (HinI) activity whose amino acid sequence is at least 60% identical to a polypeptide sequence represented by SEQ ID No: 21.
    As mentioned, embodiments of the process in which the histamine compound is transformed into fumaric acid or any of its salts are preferred, following a reaction sequence analogous to that catalyzed by the enzymes of the hinse, hin2 and hin3 clusters of Pseudomonas putida U. In said embodiment, the process comprises the following steps:
    a) transform histamine into Imidazolacetaldehyde (ImAdh);
    b) transform ImAdh into imidazoacetic acid (ImAA);
    c) transforming the ImAA into a hydroxylated derivative (4-hydroxy-imidazol-5-acetic acid);
    d) transforming 4-hydroxy-imidazol-5-acetic acid (or its structural analogue 4imidazolon-5-acetic acid into N2-formylisoasparragine (FiAsn);
    e) transform N2-formylisoasparragine (FiAsn) into N-formyl aspartic acid (FA) or any of its salts;
    f) Transform N-formyl aspartic acid (FA) into aspartic acid or any of its salts.
    g) transform aspartic acid into fumaric acid or any of its salts.
    In a preferred embodiment, the enzymes used in the respective stages are enzymes with an activity analogous to those of the hin1, hin2 and hin3 clusters and with a high degree of identity with them (at least 60%), that is:
    a) a histamine pyruvate aminotransferase (histamine deaminase) whose sequence is at least 60% identical to the polypeptide sequence represented by SEQ ID No: 5,
    b) an aldehyde dehydrogenase whose sequence is at least 60% identical to the polypeptide sequence represented by SEQ ID No: 9 or to SEQ ID No: 11,
    c) a monoxygenase whose sequence is at least 60% identical to the polypeptide sequence represented by SEQ ID No: 15,


    d) an amidohydrolase whose amino acid sequence is at least 60% identical to the polypeptide sequence represented by SEQ ID No: 19,
    e) an amidase whose amino acid sequence is at least 60% identical to the polypeptide sequence represented by SEQ ID No: 17,
    5 f) an aspartate amonioliase whose sequence is at least 60% identical to the polypeptide sequence represented by SEQ ID No: 21.
    Even more preferred is the case in which the aforementioned identity percentages are 100%. As previously mentioned, in a possible further embodiment of the process of the invention the compound of Formula I (histamine), the process comprises a step
    10 in which the compound of Formula I is the result of a histidine decarboxylation reaction. The case in which histidine decarboxylation is catalyzed by Enterobacter aerogenes histidine decarboxylase is preferred.
    In one embodiment of the process of the invention, the enzymes that catalyze the process are added to the sample as part of a composition of the invention.
    In another embodiment, the enzymes that catalyze the steps of said process are synthesized in the sample from one of the expression vectors of the invention described above.
    The embodiment is preferred in which the enzymes that catalyze the reactions of the process of the invention are synthesized by a microorganism present in the
    20 sample or added to it. In that case, one possibility is that the coding sequences of enzymes synthesized by the microorganism are naturally present in its genome: this is what happens with Pseudomonas putida U (CECT 4848), whose presence in the sample in which it is wanted Reducing histamine content is one of the possible alternatives to perform the process of the invention.
    Another possible embodiment, for which particular preference is also taken, corresponds to the case in which the microorganism is one of the recombinant host organisms of the invention: this gives many possibilities to choose the exogenous genes introduced therein and, thereby , the steps of the process of the invention. On the other hand, the choice of the microorganism also grants a lot of versatility to choose the
    30 additional characteristics that the sample is to be endowed with, in addition to the decrease in histamine; This characteristic is of special interest for its application in the food industry.


    Accordingly, embodiments of the process of the invention are of particular interest, those in which the process of the invention is carried out within the framework of the food industry, the sample in which it is desired to decrease the content of the compound of the Formula I (histamine) a food or beverage intended for consumption by
    5 human beings or animals, a starting raw material for obtaining said food or drink or an intermediate product in obtaining the food or drink.
    A preferred case is one in which the food, raw material or intermediate product is selected from a dairy derivative, milk or milk mixture from any mammal, or an intermediate product of the transformation of the milk or milk mixture 10 as the Dairy derivatives, and cheeses in particular, are products in which histamine concentrations may appear that advise their decrease to make them more appropriate for human consumption, avoiding possible side effects. Therefore, in a preferred embodiment of the above, the food, raw material or intermediate product is selected from cheese, milk from cow, sheep or goat (the species of the 15 usually comes from the milk that is used as raw material in the cheese making), a mixture of milk from at least two of the above species; or an intermediate product of the transformation of milk or milk mixture into cheese. The case is preferred in which the enzymes that catalyze the process steps are synthesized by a microorganism present in the sample or added thereto. A specific case is one in which the microorganism is a recombinant host organism described above, preferably a bacterium capable of transforming a sugar into lactic acid, even more preferably belonging to the genus selected from Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptocuccus. This provides the advantage, on the one hand, that the transformation is carried out with a microorganism of a species of which naturally intervene in the process of food transformation; on the other, if the microorganism is added from the beginning, as an initiating culture, it will be present from the beginning of the process to control the histamine that is produced. Thus, another additional embodiment refers to the described process, in which the microorganism acts as a starter of the transformation of milk into a milk derivative. Another alternative embodiment of the process of the invention is that in which it is carried out in an alcoholic beverage, in which the beverage, raw material or intermediate product is selected from must, barley, malt, wine, beer, an intermediate product of the transformation of barley in beer, an intermediate product of the transformation of must into wine, any other alcoholic beverage that requires alcoholic fermentation by yeasts or any intermediate product of its transformation. Again


    he prefers the case in which the enzymes that catalyze the process steps are synthesized by a microorganism present in the sample or added to it, which is a recombinant host organism of the invention. In a particularly preferred embodiment, the beverage is wine and the microorganism is added to the wine during malolactic fermentation.
    Another alternative embodiment describes a process, in which the food, raw material or intermediate product is selected from a sausage; an intermediate product of the transformation of meat into sausage or beef; pig or deer or mixtures thereof intended for the preparation of a sausage.
    Another alternative embodiment is that relating to a process, in which the food, raw material or intermediate product is selected from sauerkraut; the raw material thereof or an intermediate product of obtaining sauerkraut.
    Even more preferred is an embodiment in which in the last two processes mentioned, the enzymes that catalyze the process steps are synthesized by a microorganism present in the sample or added thereto which is a recombinant host organism described above. The case in which the microorganism acts as an initiator (starter) of the transformation of the raw material into the sausage or sauerkraut is preferred.
    Because of its importance in the application in the food industry, a further aspect of the invention refers to the use of microorganisms that synthesize the enzymes suitable for carrying out the process of the invention to decrease the histamine content in a food or beverage intended for human or animal consumption A first possibility is that the organism synthesizes these enzymes naturally; Therefore, a possible embodiment is the use of Pseudomonas putida U CECT4848 to decrease the histamine content in a food or beverage intended for human or animal consumption, in the raw material used to obtain it or in an intermediate product of the transformation of the Raw material in the food or drink.
    The second possibility is that the microorganism is a recombinant microorganism, in which the appropriate genes have been introduced to synthesize the desired regulatory enzymes and polypeptides. Thus, a final aspect refers to the use of a recombinant host organism of the invention, to decrease the histamine content in a food or beverage intended for human or animal consumption, in the raw material used to obtain it or in an intermediate product of the transformation of the raw material into the food or drink.


    The control of starter microorganisms that initiate the processes of transformation of certain raw materials into foods or beverages (dairy products, sausages, alcoholic beverages, other foods in which fermentation processes such as sauerkraut, pickles or olives) It has gained great interest in recent years in the food industry, since it allows controlling the process conditions, providing the final products with desired characteristics and, in particular, facilitates the obtaining of final products whose characteristics are more homogeneous and identifiable by the consumer as the expected characteristics in a given brand, made more difficult to achieve when starting from the initiating organisms that contain the raw material 10 naturally. The use of initiators (starters) capable of carrying out the histamine transformation process of the invention is a further advantage within the process control, allowing the control of the concentration of these biogenic amines from the beginning of the process, as they go producing and, even, as the case may be, the same microorganisms that are capable of producing them that allow their elimination. That is why a preferred embodiment of the use of the invention is that in which the host organism acts as an initiator (starter) of the process of transformation of the raw material into the food or beverage intended for human or animal consumption. It is particularly preferred that the food is a dairy derivative (more preferably cheese), a beverage such as wine, a sausage, or a food during which
    Obtaining fermentation occurs such as sauerkraut, pickles or olives.
    A new catabolic pathway responsible for the degradation of the biogenic histamine amine has been characterized in the bacterium P. putida U (CECT 4848). The different genes that encode the enzymes that make up such a route have been identified by isolating different mutants of Pseudomonas putida U CECT4848 unable to grow in media containing only histamine or imidazoleacetic carbon (ImAA) sources. The mutagenic agent used was the transposon Tn5, which acts by randomly integrating itself into the chromosome of the bacteria (99,116). Through this procedure different mutants were isolated that were grouped into three types based on their ability to degrade different carbon sources. Type 1 mutants included those that were unable to grow in chemically defined media when they contained histamine as the sole source of carbon (Fig. 7 and Fig. 11). However, these mutants effectively degraded imidazoleacetic acid (ImAA), as well as many other carbon sources. The second type (type 2) included a group of mutants that did not grow in those media that contained only histamine carbon or imidazoleacetic acid sources (Fig. 7 and Fig. 11), but that they did in those others. that other carbon sources likely to be added


    used by the parental strain (P. putida U CECT 4848). The third group of mutants, those included in type 3, were characterized in that they could not grow in those culture media in which the amino acid Lhistidine was used as the sole source of carbon. However, these mutants grew effectively in those same media when this compound was replaced by other carbon sources that could be assimilated by the wild strain.
    The isolation of type 3 mutants clearly indicates that histidine and histamine degradation pathways are independent and do not possess common intermediaries. The existence of type 2 mutants indicates that the catabolic pathway responsible for the assimilation of histamine and ImAA is the same route or that they have at least common intermediaries. Additionally, the existence of type 1 mutants suggests that histamine must be converted into ImAA to be degraded, that is, ImAA is a catabolic intermediate in the degradation of histamine (Fig. 7). All these results indicated that in type 1 mutants the transposon had been integrated, either in a DNA sequence belonging to one of the genes necessary for the transformation of histamine into ImAA (Fig. 7), or in some area which affected the expression of any of those genes. In addition, the fact that these mutants grew well in media supplemented with ImAA suggested that in them the transposon was affecting genes that had to do with histamine deamination, but not later catabolic stages.
    In type 2 mutants, the transposon must have been in a DNA sequence corresponding to any of the genes encoding enzymes required for both histamine and ImAA catabolism.
    Type 3 mutants cannot catabolize the amino acid histidine but histamine and ImAA, indicating that in them the transposon is affecting the expression of some of the genes that encode the enzymes responsible for the degradation of that amino acid. The large number of defective mutants in the degradation of isolated histidine suggest that the histidine and histamine (and ImAA) degradation pathways are completely different routes and that they do not possess common stages or intermediates.
    The identification of the transposon insertion point in each of the mutants and the sequencing of adjacent areas has allowed us to identify all the genes necessary for the degradation of histamine and ImAA in P. putida U. We have identified two types of genes , some, are required for the degradation of histamine in any culture medium, even when there are other carbon sources that can support bacterial growth. Others are those that are required for the degradation of histamine or of


    ImAA only when either of these two compounds is the only source of carbon present in the medium.
    Unlike the genes involved in the degradation of 2-phenylethylamine, tyramine and dopamine in this same bacterium, in the case of the histamine degradation pathway the genes are located in different clusters (hin1 + hin2 + hinK + hin3) . The hin genes are all located in those hin1, hin2, hin3 and hinK clusters (Fig. 8). The cluster hin1 groups the genes required in P. putida U to transport histamine from the medium as well as for its transformation into Imidazolacetic (ImAA), this being the first evidence that this set of genes (hinEABCD) is involved in the deamination of histamine (Fig. 7 and Fig. 8). The hin2 cluster groups the genes (hinGHF) required for the transformation of ImAA into L-aspartic, this being the first time that the gene coding for the monooxygenase responsible for the hydroxylation of ImAA is identified, as well as the first description of the enzymatic activities that possess the HinG and HinH proteins as well as the sequence of their genes (hinG and hinH) (Fig. 7 and Fig. 8). The hinK gene, located elsewhere in the chromosome, encodes a regulator (HinK) that controls the expression of the genes included in the hin2 cluster (Fig. 8). Additionally, the hin3 cluster includes two genes (hinI and hinJ) that encode an aspartate amonioliase and a transcriptional activator respectively. These genes are responsible for the transformation of L-aspartic into fumaric, allowing the connection of the histamine and ImAA degradation pathway with that of the tricarboxylic acids (Krebs cycle) (Fig. 7 and Fig. 8).
    The fact that the P. putida U CECT4848 mutants affected in the hinI gene or in the hinJ gene are incapable of degrading histamine suggest that in this bacterium there are no other mechanisms for aspartic deamination, or at least, that they are not very effective and, particularly ineffective when histamine, L-aspartic acid or asparagine are the only carbon sources present in the culture medium. For this reason, the mutation of the hinI gene does not allow the formation of fumaric from L-aspartic, so, provided that this mutant (or the one affected in its transcriptional activator, that is, in the hinJ gene) is grown in a medium in which there is histamine and another carbon source that is not aspartic or asparagine (4-hydroxy-phenylacetic acid -4-OH-AFA-or other) L-aspartic acid accumulates in the culture broth (Fig. 9).
    As we have already indicated, there are other genes, which are required to degrade histamine (but not ImAA) when this amine is the only carbon source. In other cases (if an additional source that provides pyruvate and energy is supplemented), histamine is degraded. These genes correspond to two gene clusters (dadXAR clusters and


    coxBA-C). The dadXAR genes encode a D-amino acid dehydrogenase (DadA), a racemase (DadX) and a transcriptional regulator (DadR). Racemase uses the L-alanine generated in the histamine deamination reaction (catalyzed by HinC) and transforms it into D-Ala. Subsequently, this amino acid is deaminated by an FAD-dependent D-amino acid dehydrogenase (DadA) that generates the pyruvate required to deaminate another histamine molecule. During this oxidative deamination process, the FAD linked to DadA is reduced to FADH2 and, this, is coupled to an electronic transport chain in which, as we have indicated, a cytochrome C oxidase complex type aa3 (CoxBA-C) participates composed of three CoxA, CoxB and CoxC subunits and an assembly protein (Cox-) (Fig. 7). In this way the energy needed to transport histamine is generated when it is being used as the sole source of carbon.
    The fact that a mutation in the dadA gene prevents growth in histamine and L-Ala (Fig. 11) indicates that this pathway is the only one existing in P. putida U to degrade both compounds. For this reason, the mutation of the dadA gene does not allow the genesis of pyruvate from D-Alanine, so, provided that this mutant is grown in a medium in which there is histamine and pyruvate (or a source of pyruvate, as per For example, 4-hydroxyphenylacetic acid -4-OH-AFA-) accumulates alanine in the culture broth (Fig. 9).
    The sequences of all these genes are included in those indicated as SEQ ID No: 1, SEQ ID No: 2 SEQ ID No: 3 and SEQ ID No: 24. The sequences of the reactions required to degrade histamine in P. putida U are included in Fig. 7
    In summary, we have shown that in P. putida U the transformation of histamine and ImAA into fumaric acid requires the genes corresponding to the hin1, hin2, hin3 and hinK clusters, as well as that of others, dadXAR and coxBA-C, when the only Carbon source present in the growth medium is histamine. In addition, we have demonstrated for the first time that histamine degradation is a much more complex process than was supposed and that involves the participation of a large number of genes. This could be the reason why the route responsible for the degradation of this amine was unknown.
    The identification of the hin genes can have important biotechnological implications, since as we have indicated in the “State of the Art” section, the consumption of foods with a high histamine content can cause a large number of pharmacological effects. Therefore, obtaining a genetic construct that would allow carrier organisms to degrade this compound could be used to transform those microorganisms that participate in fermentation, thus preventing the accumulation of histamine in these foods.


    These recombinant strains capable of degrading histamine could even be used in the preparation of starter cultures, giving them greater efficiency, since the starters, although they are usually made up of non-producing strains of biogenic amines, not they can prevent the accumulation of the amines produced by the microbial flora present in the original raw materials. On the other hand, if these straters had the presence of bacteria capable of degrading histamine, they would avoid the accumulation of these compounds in food, regardless of the raw materials used in their preparation.
    In addition, analyzed from a strictly economic point of view, the possibility of providing bacterial strains with interest for the food industry with the capacity required to degrade histamine, may be essential for the development of new products and the opening of new markets since Many countries (Canada, Switzerland or the Netherlands, among others) are setting limits for the concentration of biogenic amines in imported foods, especially wine. The presence of amines in wine carries more risk than in other foods, since, by containing alcohol, the mechanisms of detoxification of the organism will be affected, increasing the possibilities of poisoning by amine intake.
    By transferring the hin1, hin2, hin3 and hinK gene clusters as isolated or tandem genetic cassettes (hin1 + hin2 + hinK clusters; or hin1 + hin2 + hinK + hin3) we could confer to any bacteria, both G + and G-, of the capacity required to degrade histamine, thus avoiding the accumulation of this compound in those foods whose production process involved such bacteria. The transfer of the indicated clusters to strains of Lactococcus lactis would prevent the accumulation of histamine (generated by decarboxylation of histidine by histidine decarboxylase present in this or other acid-lactic bacteria) in those cheeses and derivatives in which the bacterium is involved in the production process .
    Finally, through Metabolic Engineering, we have managed to establish a new useful route for the degradation of the amino acid L-histidine through the joint participation of the enzymes encoded by the hin1, hin2, hinK and hin3 clusters of P. putida U and the gene that encodes the histidine decarboxylase (hdc) of Enterobacter aerogenes (see Example 4). An interesting application of the confluence in the same microorganism of the hin1, hin2, hinK, hin3 and hdcA genes, which allow to catabolize L-histidine through the histamine intermediate, could be the preparation of foods with low content in this amino acid. In order to obtain these, starter cultures containing starters could be used


    microorganisms explicitly designed to express the enzymatic activities HinACDE and Hdc. Description of the figures
    Figure 1. Schematic representation of the biosynthetic pathway of catecholaminergic neurotransmitters.
    Figure 2. Schematic representation of the synthesis of histidine (A) from the amino acid histidine, and serotonin (B) from tryptophan.
    Figure 3. Structure of the most important “trace” amines. Β-Phenylethylamine, tyramine and tryptamine are synthesized by decarboxylation of the corresponding precursor amino acids by the action of an aromatic L-amino acid decarboxylase. Octopamine is synthesized by hydroxylation of tyramine by the action of a tyramine-β-hydroxylase.
    Figure 4. Main biogenic amines present in food and their precursor amino acids.
    Figure 5. Schematic representation of the reaction mechanism used by the QH-AmDH to oxidize the primary amines (modified from Sun et al, 2003, ref 82).
    Figure 6. Schematic representation of tyramine oxidation in Euphorbia characias.
    Figure 7. Scheme of the catabolic pathway required in Pseudomonas putida U CECT4848 for the degradation of the biogenic histamine amine.
    Figure 8. Organization of the hin genes required for histamine degradation in Pseudomonas putida CECT4848. The hinEABCD genes are included in the cluster hin1. In the hin2 cluster, the hinGHF genes are located. In the hin3 cluster, the hinI and hinJ genes are found. Another additional gene, located outside these indicated clusters, but required for histamine degradation is hinK.
    Figure 9. TLC plates (thin layer chromatography) showing (A) the Alanine (Ala) accumulated in the culture broth by the mutants P. putida U ΔdadA or P. putida U ΔdadX; and (B) Aspartic acid (Asp) accumulated by the mutants P. putida U ΔhinI or P.putida U ΔhinJ.
    Figure 10. Growth in plaque (A) and in liquid medium (B) of a mutant of P. putida U unable to degrade the amino acid histidine (P. putida U Δhis) and of the recombinant strain of that mutant (P. putida U Δhis pMChdc) in which the gene encoding the


    histidin decarboxylase (hdc) of Enterobacter aerogenes. In panel B the empty circles (white) correspond to the recombinant (P. putida U Δhis pMChdc) and the filled circles (black) to the pattern (P. putida U Δhis). In both cases the culture medium (MM) contained (in g / L): KH2PO4 (13.6); (NH4) 2SO4 (2.0); MgSO4.7H2O (0.25); FeSO4.7H2O (0.0005) and histidine (5mM). When it was a solid medium (plate) to that medium, purified agar (2.5% w / vol) was added.
    Figure 11. Growth of P. putida U CECT4848 (black circles and black triangles) when grown in MM (see foot of Fig 14 and patent text) containing as the sole source of carbon (10mM histamine, A; or imidazoleacetic acid -ImAA -, 10mM, B). The absence of growth of mutants in which Tn5 transposon had been integrated into any of the hinA, hinB, hinC, hinG, hinH, hinF, hinI, hinJ, hinK, daA, dadX, coxa, coxB, coxC or cox genes - when grown in MM + histamine (A) it is indicated with empty circles (white). The absence of growth of mutants in which Tn5 transposon had been integrated into any of the hinG, hinH, hinF, hinI, hinJ or hinK genes, when grown in MM + ImAA (B) is indicated by empty triangles (white ). The growth of those mutants in which the transposon had been inserted into the hinA, hinB, hinC, dadA, dadX, coxa, coxB, coxC or cox-when genes were grown in MM medium containing ImAA (10 mM) as the only one of Carbon was similar to the pattern (panel B, black triangles).
    Figure 12. HPLC chromatograms of histamine (panel A); imidazolacetic acid (panel B) as well as the products accumulated in the broths by the hinA, hinB, hinC mutants, when grown in MM medium containing histamine (10 mM) and 4-hydroxyphenylacetic acid (20 mM) (panel C); by the hinF and hinK mutants when grown in MM medium containing histamine (10 mM) and 4-hydroxyphenylacetic acid (20 mM) (panel E). When the P. putida DOC21 CECT8043 or E. coli W ATCC11105 strains into which the genes corresponding to the hin1 cluster were inserted were grown in different media containing 10mM histamine and other carbon sources (4-hydroxy-phenylacetic or succinic) at concentrations ranging from 20-40 mM, all of them transformed histamine into ImAA (panels C -0h-, D -6h- and E-30h-). However, parental strains that did not contain the hin1 cluster did not modify histamine. Examples EXAMPLE 1. Identification of the genes responsible for the degradation of histamine in P. putida U.


    Pseudomonas putida U (CECT 4848) is a bacterium that can grow using histamine (5 mM) or ImAA (5mM) as the only carbon sources, when grown in a liquid medium of defined chemical composition that contains (in g / L) KH2PO4 (13.6); (NH4) 2SO4 (2.0); MgSO4.7H2O (0.25); FeSO4.7H2O (0.0005). If the culture medium was solid, 2.5% agar (w / v) was also added. Incubations were performed on an orbital shaker, at 30 ° C and at 250 rpm, using 500 mL Erlenmeyer flasks containing 100 mL of medium (Fig. 15). When P. putida U CECT4848 was mutated with the Tn5 transposon, following the methodology described by us in other publications (83, 96, 98-102, 116), we isolated several mutants unable to degrade histamine (Fig 15) but, nevertheless They grew well when other sources of carbon (imidazoleacetic, 4-hydroxyphenylacetic, 3,4-dihydroxyphenylacetic, phenylacetic, benzoic, octanoic, glutamic, succinic, tyramine, dopamine, putrescine and 2-phenylethylamine) were added to the medium. The sequence location (99) of the transposon on the chromosome of the different mutants allowed us to verify that this mobile genetic element had been inserted in several open reading
    15 frames (ORFs) (hinABC genes) integrated into the hin1 cluster and encoding proteins (HinA, HinB and HinC) (Fig. 7, Fig. 8 and Fig. 9). These genes, according to these results, were essential for histamine catabolism in P. putida U.
    The additional sequencing of the areas adjacent to these genes allowed us to identify the ORFs indicated as hin1 and whose sequences are included in Fig. 9
    20 Following the same mutation procedure, we isolated another type of mutants unable to grow in the medium of defined composition indicated above, when histamine or ImAA carbon sources were used but which, however, grew well when other sources were added to the medium. carbon (acids, 4-hydroxyphenylacetic, 3,4-dihydroxyphenylacetic, phenylacetic, benzoic, octanoic, glutamic, succinic, tyramine,
    25 dopamine, putrescine and 2-phenylethylamine). The sequence location (99) of the transposon on the chromosome of these other mutants allowed us to verify that this mobile genetic element had been inserted into several open reading frames (ORFs) (hinGHF genes) integrated in the hin2 cluster and that encode proteins (HinF, HinH and HinF) (Fig. 7, Fig. 8 and Fig. 10. These genes, according to these results, were essential for the
    30 histamine and Imidazolacetic catabolism in P. putida U. Three other mutants belonging to this same group (unable to grow in media containing histamine or ImAA carbon source) presented mutations in two ORFs (hinIJ), not located in the hin2, but in another cluster we call hin3 and which encode the HinI and HinJ proteins (Fig. 7, Fig 8 and Fig.11). These mutants did not grow in the defined medium
    35 indicated above when the carbon source was histamine, ImAA, L-aspartic acid or asparagine,


    but if other sources of carbon were added to the medium (acids, 4-hydroxyphenylacetic, 3,4-dihydroxyphenylacetic, phenylacetic, benzoic, octanoic, glutamic, succinic, tyramine, dopamine, putrescine and 2-phenylethylamine).
    In another mutant, the transposon had been integrated into the hinK gene (Fig 8 and Fig.12), which encodes a regulator belonging to the LysR family (HinK), located outside the hin1, hin2 and hin3 clusters. This protein (HinK) activates the transcription of the hinGHK genes so that, when it is mutated, and whenever there is another carbon source in the medium that supports bacterial growth, histamine is transformed into ImAA, a compound that, when not continue degrading, accumulates as such in the broth (Fig. 16).
    A particular group of mutants were also isolated that were unable to degrade histamine only when this amine was the only carbon source. However, they grew well in a defined composition medium that contained ImAA as the sole source of carbon and also transformed histamine into intermediates of general catabolism if another source of carbon and energy existed in the medium in addition to histamine. In these mutants the transposon had been inserted into a cluster (dad) containing the dadXAR genes (which encode the DadX, DadA and DadR proteins) or into another cluster (coxBA-CoxC) that encodes the three subunits (CoxA, CoxB and CoxC ) and the assembly protein (Cox-) required to compose a cytochrome C oxidase type aa3 (Fig. 7).
    EXAMPLE 2. Identification of the minimum functional genetic unit required for oxidative deamination of histamine in P. putida U.
    The functional analysis of the genes that make up the hin1, hin2, hinK and hin3 clusters was performed by interrupting each of them following a procedure that involves a simple recombination event, and that is based on the use of an internal fragment of each gene as described in different publications (83, 99-102). When the disruption of any of these genes implied lack of function (absence of growth in media supplemented with histamine), a wild copy of the gene that had been affected in each mutant was cloned into a replicative plasmid in Pseudomonas (pMC) (83, 102 ) and expressed, in trans, in that mutant, establishing whether or not the effect observed after the mutation of that particular gene was reversed. Following this method, we found that in P. putida U the hinA, hinB and hinC genes were essential for histamine deamination to occur (Fig. 7 and Fig. 8).
    In other cases, the disruption of certain genes (hinD and hinE) did not impede the ability of different mutants to degrade histamine, but their transformation into ImAA was a bit


    slower, so we conclude that, at least in P. putida UCECT4848, these genes are not indispensable to assimilate that amine (probably because there are other genes in its genome that encode homologous enzymes).
    These results indicate that the genetic construction that possesses the minimum information necessary to catalyze the oxidative deamination of histamine in P. putida U, is hinABC.
    EXAMPLE 3. Obtaining a genetic construct that can degrade histamine
    or histamine and ImAA in other bacteria.
    In order to have a genetic construct that could be transferred to different microorganisms in such a way as to confirm the ability to partially or totally degrade histamine or ImAA, the genes that integrate the entire hin1 cluster into plasmids pK18 :: mob were cloned (replicative in E. coli and integrative in Pseudomonas) and hin2 in pJQ200RS (in E. coli and integrative in Pseudomonas), or in pMC (83, 99-102,117-118). With them, E. coli W ATCC11105 (a bacterium incapable of degrading histamine (63, 95) and P. putida DOC21 CECT8043, a wild strain isolated by us, which is incapable of degrading histamine and can assimilate different steroids. in Fig. 16 reveal that the recombinant E. coli strain (E. coli W pK18 :: mobhin1), unlike the parental strain (E. coli W transformed with the plasmid without insert -E. coli W pK18 :: mob) was able to transform histamine into ImAA when grown in defined media containing histamine and another source of carbon (4-OH-phenylacetic, succinic) or in more complex ones (LB). The same happened when P. putida DOC21 pK18 :: mobhin1 in the same media used for E. coli but at 30 ° C instead of at 37 ° C (Fig. 16) The disappearance of histamine from the culture broths and the consequent formation of ImAA was followed by HPLC (see conditions at the end of the example).
    Additionally, we verified that the expression in E. coli W and P. putida DOC21 of the pk18 :: mobhin1 and pMC-hin2 constructs led to the obtaining of recombinant strains
    (E. coli W pk18 :: mobhin1 pMC-3-hin2 and P. putida DOC21 pk18 :: mobhin1 pMC-3-hin2) that only transformed histamine into ImAA but were unable to degrade ImAA (Fig. 16). The lack of function of the enzymes encoded in the hin2 cluster could be due to the fact that in the absence of the HinK regulator the hinF, hinG and hinH genes were not being expressed. In fact, when the mutant in which the transposon had been inserted into the hinK gene, was grown in a medium of defined composition containing histamine and another carbon source, we found that histamine was transformed into ImAA and that this compound accumulated in the broth. This result suggests that the hin2 cluster genes are not expressed


    in the absence of HinK. For this reason we clone the hinK gene in plasmid pMC-5, replicative in P. putida DOC21 pk18 :: mobhin1 pMC-3-hin2 and use it to transform this bacterium. The analysis of the culture broth revealed that the recombinant strain (P. putida DOC21 pk18 :: mobhin1 pMC-3-hin2 pMC-5-hinK), unlike what happened in those that did not carry the hinK gene, degraded both the histamine like the ImAA. Analysis of the culture broths by high performance liquid chromatography (HPLC).
    The consumption of histamine and ImAA and other compounds, as well as the accumulation of the catabolic intermediates accumulated by the different mutants when grown in liquid medium, was carried out by HPLC analysis. For this, samples were taken from the culture broths (1 ml) at different times. These were centrifuged (31,000 x g, 20 minutes) to remove cell debris and filtered through Millipore filters (pore size of 0.22 µm). The different aliquots (10 µl) were analyzed using an HPLC device (Waters 600 Controller) equipped with a detector (Dual λ absorbance detector, Waters 2487), an integrator (Empower computing integrator) and a reverse phase column (Gemini 5µ C18 110A, 250mm x 4.6mm; Phenomenex Laboratories. USA). For the separation of the different molecules, an isocratic system consisting of H3PO4, 59.4 mM, 5.4 mM H2SO4 (pH. 1.0) was used as the mobile phase. The flow rate was 0.5 mL min-1 and the eluate was determined at 210 nm. Under these conditions the retention times (min) for the different molecules were: histamine, 4.9; N-formimino-L-aspartic acid, 5.5; imidazolcarboxaldehyde, 5.7; imidazolcarboxylic acid, 5.9; imidazolacetic acid (ImAA), 6.1; imidazollactic acid, 6.3; formamide, 6.5; N2-formylisoasparragine (FiAsn), 6.7;
    midazoletanol, 7.1; N-carbamoyl-aspartic acid, 7.8; N-formylaspartic acid (FA), 8.0; acid
    hydantoin-5-acetic acid, 9.2;acidimidazoldicarboxylic acid,10.3;dopamine,11.2;acid
    imidazolacrylic, 11.5; tyramine 15.0 and fumaric acid, 17).
    In other cases, especially when it comes to compounds with low or no absorption in the UV zone, the compounds were separated by thin layer chromatography (TLC). In such cases, we use TLC plates (TLC silical gel 60 aluminum sheets, 20 x 20 cm, Merck KgaA, Germany). The mobile phase used for the development of the plates was: ethanol (96%) water (7: 3, vol / vol.). Chromatographic spots were revealed by spraying the plaque with the ninhydrin-collidine reagent (119). Alanine and aspartic acid appear as purple spots with an Rf of 0.47 (Ala) and 0.55 (Asp) (Fig. 13).


    When carboxylic acids (ImAA, N-formimino-L-aspartic acid, N-carbamoylaspartic acid and others) were analyzed, the mobile phase used for TLC was: ethanol: water: ammonium (80: 16: 4 in vol). The spots were identified by spraying the TLC plates with a freshly prepared solution of bromophenol blue / bromocresol green (120). The plates were air dried and, after a few minutes, the organic acids appear as yellow spots on a blue-green background (121).
    EXAMPLE 4. Design of a metabolic drift protocol that gives different microorganisms the ability to degrade the amino acid L-histidine through the histamine pathway.
    The catabolism of the amino acid L-histidine (a proteinogenic amino acid precursor to histamine) has been studied in numerous living things. In certain bacteria (such as species belonging to the genus Pseudomonas) and in all eukaryotic cells, degradation takes place through a well-known route that involves: its oxidative deamination to generate urocanic acid; the hydration of this compound to generate imidazolon propionic acid; its subsequent hydrolysis giving rise to glutamic formimino which is finally hydrolyzed releasing glutamate. We have developed a procedure that allows combining the hin genes of P. putida U and the hdc (accession number in GenBank: M62745.1 sequence included in the ENTHDCA locus, 1327bp) of Enterobacter aerogenes (CECT684) (accession number in GenBank : M62745.1 sequence included in the ENTHDCA locus, 1327bp), which encodes the histidine decarboxylase of that bacterium. Thus, the expression of the hdc gene in P. putida (which already has the hin genes)
    or in different microorganisms (depending on their catabolic capacity) give the recipient microorganism the ability to degrade the amino acid histidine, via histamine, generating as a final product fumaric acid.
    P. Putida U CECT4848 is capable of degrading L-histidine by the route described above, however, those mutants in which it has been interrupted with transposon in any of the genes required for histidine degradation cannot catabolize this amino acid. However, all those P. putida U Δhis mutants have the functional Hin path. Therefore, if that bacterium were able to decarboxylate histidine to histamine, it could grow at the expense of this carbon source. To verify this, we cloned the Enterobacter aerogenes hdc gene in plasmids pK18 :: mob and pMC and those constructs were used to transform mutants incapable of degrading histidine (generically called P. putida U Δhis). The recombinant strains obtained (P. putida U Δhis pK18 :: mobhdc and P. putida U Δhis pMChdc) were able to efficiently degrade L


    histidine when grown in different media containing this amino acid as the sole source of carbon (Fig. 14).
    EXAMPLE 5. Design of a protocol to obtain mutants that accumulate ImAA from Histamine.
    As we have indicated in Example 1, the characterization of the histamine degradative pathway in Pseudomonas putida U CECT4848 was performed by analyzing the different mutants that had been obtained by inserting the Tn5 transposon into different genes. One of these mutants (P. putida U ΔhinF), lacked the monooxygenase activity responsible for the hydroxylation of imidazolacetic acid (HinF protein, Fig. 7) so
    10 could not grow in media containing histamine or imidazoleacetic as the only carbon sources. However, this mutant grew in all those media in which the wild strain (P. putida U CECT4848) did so as long as other carbon sources were supplemented. The analysis of the culture broths of this mutant when growing in medium containing different salts (in g / L KH2PO4 (13.6); (NH4) 2SO4 (2.0); MgSO4.7H2O (0.25);
    15 FeSO4.7H2O (0.0005) and to which histamine (usually 20 mM) and other necessary carbon sources were added to support bacterial growth (4-hydroxyphenylacetic acids, 3,4-dihydroxyphenylacetic, phenylacetic, benzoic, octanoic, glutamic, succinic and others, usually at a concentration of 20 or 30 mM) revealed that imidazocetic acid accumulated, and that the concentration of acid detected was practically equivalent to
    20 of the added histamine (Fig. 16). These results indicate that this mutant can be used for the removal of histamine from different media as well as to produce ImAA. Therefore, the alteration of the sequence of the hinF gene by any mechanism that prevents the expression of the monooxygenase activity inherent in the HinF protein is a useful procedure to obtain ImAA from histamine or to obtain ImAA derivatives.
    25 from histamine molecules that have undergone structural modifications that can be recognized by the Hin path proteins that catalyze the reactions that lead to ImAA.
    EXAMPLE 6. Design of a protocol to obtain mutants that accumulate the amino acid L-Aspartic acid from Histamine.
    As indicated in Example 5, the mutation with the Tn5 transposon led to the obtaining of different mutants affected in the degradation of Histamine, ImAA or both. In one of these mutants (P. putida U ΔhinI) the transposon had been integrated into the hinI gene (Fig. 8) that encodes a protein with aspartate amonioliase (HinI) activity (Fig. 7).


    In another mutant (P. putida U ΔhinJ) the transposon had been inserted into the hinJ gene (Fig. 8) that encodes the HinJ transcriptional activator (Fig. 7). None of these mutants could grow in media containing histamine or imidazoleacetic as the only carbon sources. However, if they grew in all those media in which the wild strain did (P. putida U CECT4848) as long as other sources of carbon were supplemented (except for aspartic acid, asparagine or those that lead directly to some of these two compounds). The analysis of the culture broths of these two mutants when grown in medium containing different salts (in g / L): KH2PO4 (13.6); (NH4) 2SO4 (2.0); MgSO4.7H2O (0.25); FeSO4.7H2O (0.0005) and to which histamine (usually 20 mM) and other carbon sources that support cell growth (4-hydroxyphenylacetic acids, 3,4-dihydroxyphenylacetic, phenylacetic, benzoic, octanoic, glutamic, succinic and others were added) , usually at a concentration of 20 or 30 mM) revealed that aspartic acid (or its salts) accumulated (Fig. 13). These results indicate that any of these two mutants (P. putida U ΔhinI and P. putida U ΔhinJ) could be used for the removal of histamine and ImAA from different media as well as to produce aspartic acid or its salts from histamine or ImAA. Therefore, the alteration of the sequences of the hinI and hinJ genes in Pseudomonas putida U (or in the organisms that possess them) by any mechanism that prevents the expression of the aspartate amonioliase activity inherent in the HinI protein, or that of the regulator (HinJ), is a useful procedure to obtain aspartic acid (or its salts) from histamine or from ImAA.
    EXAMPLE 7. Design of a protocol to obtain mutants that accumulate the amino acid Ala from Histamine.
    Within the different Pseudomonas putida U mutants unable to degrade histamine, we identified one that was mutated in the dadA gene. This gene belongs to the cluster dad in which there are three ORFs that respectively encode a racemase (DadX), a D-amino acid dehydrogenase (DadA) and a transcriptional regulator (DadR) that can act as an activator (linked to L-Ala) and also as repressor (122-127). The P. putida U UdadA mutant is unable to degrade histamine when this biogenic amine is the only carbon source present in the medium, however, unlike other mutants affected in the histamine catabolic pathway, when it exists in the culture medium Another source of additional carbon (4-hydroxyphenylacetic acid or other compounds) does assimilate histamine, making it general intermediaries. The reason that justifies this behavior is that the proteins encoded by the dad genes have the function of recovering the pyruvate used by histamine-pyruvate aminotransferase (HinC) for


    deaminate histamine, and to generate, by coupling to an electronic transport system, the energy needed to take histamine from the culture broth into the bacteria (Fig. 7). For this reason, this mutant does not grow in those media in which histamine is the only source of carbon. However, when, in addition to histamine, there is another source of carbon that generates pyruvate and that provides the energy needed to transport histamine from outside, the mutant P. putida U ΔdadA can catabolize this biogenic amine and remove it from the medium. The analysis of the culture broths of the P. putida U ΔdadA mutant when grown in a medium containing different salts (in g / L): KH2PO4 (13.6); (NH4) 2SO4 (2.0); MgSO4.7H2O (0.25); FeSO4.7H2O (0.0005) and to which histamine (usually 20 mM) and other carbon sources that support cell growth (4-hydroxyphenylacetic acids, 3,4-dihydroxyphenylacetic, phenylacetic, benzoic, octanoic, glutamic, succinic and others, usually added at a concentration of 20 or 30 mM) they revealed that Alanine accumulated (Fig. 13) and that the concentration (mM) of Ala detected was practically equivalent to that of the added histamine. These results indicate that the P. putida U ΔdadA mutant could be used for the elimination of histamine from different media as well as to produce the amino acid Ala from histamine. Therefore, the alteration of the sequences of the dadA gene in Pseudomonas putida U CECT4848 (or in the organisms that possess them and also have the hin genes - at least with hinC, hinB, and hinA-) by any mechanism that prevents The expression of the D-amino acid dehydrogenase activity inherent in the DadA protein is a useful procedure to obtain Alanine from histamine.
    Additionally, the mutation of the dadX gene encoding a racemase (DadX) (122-127) incapacitates Pseudomonas putida U to grow in both histamine and L-alanine. This mutant (P. putida U ΔdadX), for the reasons stated above, is unable to generate D-Alanine, a DadA substrate, and, therefore, cannot regenerate the pyruvate required to deaminate histamine (HinC catalyzed reaction) . Accordingly, the P. putida U ΔdadX mutant could be used for the removal of histamine from different media as well as for producing the amino acid Ala from histamine. Therefore, the alteration of the sequences of the dadX gene in Pseudomonas putida U CECT4848 (or in the organisms that possess them and also have the hin genes - at least with hinC, hinB, and hinA-) by any mechanism that prevents The expression of the racemase activity inherent in the DadX protein is a useful procedure to obtain Alanine from histamine.

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    权利要求:
    Claims (31)
    [1]
    1. An isolated nucleic acid molecule comprising sequences with at least 60% identity with the sequences represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO : 12.
    The nucleic acid molecule according to the preceding claim, wherein the sequences are coding sequences for the proteins represented by SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13.
    [3]
    3. The nucleic acid molecule according to any of the preceding claims, wherein
    The sequences are the sequences represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ 10 IDNO: 8, SEQIDNO: 10 and SEQIDNO: 12.
    [4]
    Four. The nucleic acid molecule according to any of the preceding claims, whose sequence is the sequence represented by SEQ ID NO: 1.
    [5]
    5. The nucleic acid molecule according to any of the preceding claims, further comprising: a sequence with at least 60% identity with the sequence
    15 represented by SEQ ID NO: 14; a coding sequence for the protein represented by SEQ ID NO: 15; or the sequence represented by SEQ ID NO: 14.
    [6]
    6. The nucleic acid molecule according to any of the preceding claims, further comprising: a sequence with at least 60% identity with the sequence represented by SEQ ID NO: 18; a protein coding sequence
    20 represented by SEQ ID NO: 19; or the sequence represented by SEQ ID NO: 18.
    [7]
    7. The nucleic acid molecule according to any of the preceding claims, further comprising: a sequence with at least 60% identity with the sequence represented by SEQ ID NO: 16; a coding sequence for the protein represented by SEQ ID NO: 17; or the sequence represented by SEQ ID NO: 16.
    The nucleic acid molecule according to any one of the preceding claims, the sequence of which comprises the sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2.
    [9]
    9. The nucleic acid molecule according to any of the preceding claims, further comprising: a sequence with at least 60% identity with the sequence

    represented by SEQ ID NO: 24; a coding sequence for the protein represented by SEQ ID NO: 25; or the sequence represented by SEQ ID NO: 24.
    [10]
    10. The nucleic acid molecule according to any of the preceding claims, further comprising: a sequence with at least 60% identity with the sequence
    5 represented by SEQ ID NO: 20; a coding sequence for the protein represented by SEQ ID NO: 21; or the sequence represented by SEQ ID NO: 20.
    [11]
    11. The nucleic acid molecule according to any of the preceding claims, further comprising: a sequence with at least 60% identity with the sequence represented by SEQ ID NO: 22; a protein coding sequence
    10 represented by SEQ ID NO: 23; or the sequence represented by SEQ ID NO: 22.
    [12]
    12. A microorganism capable of degrading histamine or a methylated derivative thereof, characterized in that it comprises the nucleic acid molecule according to any one of claims 1 to 11, wherein said molecule has been artificially introduced into said microorganism.
    13. A microorganism capable of degrading the imidazoleacetic acid characterized in that it comprises the nucleic acid molecule according to any of claims 5 to 11, wherein said molecule has been artificially introduced into said microorganism.
    [14]
    14. A procedure for the removal of histamine or a methylated derivative of the
    20 of a medium, characterized in that in said medium a microorganism comprising the nucleic acid molecule according to any one of claims 1 to 11 is cultured.
    [15]
    15. The procedure for the removal of histamine or a methylated derivative of the
    same according to the preceding claim, wherein the microorganism is strain 25 Pseudomonas putida U CECT4848.
    [16]
    16. The method for removing histamine or a methylated derivative thereof according to any of the two preceding claims, wherein the medium from which histamine is removed or the methylated derivative thereof is a fermented food that is treated with a starter culture of the microorganism.

    [17]
    17. The method for the removal of histamine or a methylated derivative thereof according to any of the three preceding claims, wherein the histamine or the methylated derivative thereof is transformed into imidazoleacetic acid and wherein the activity of the enzyme monooxygenase, that transforms imidazolacetic acid into acid
    5-Hydroxy-imidazol-5-acetic acid or its analogue, 4-imidazolon-5-acetic acid, is prevented by a mutation in the sequence represented by SEQ ID NO: 14.
    [18]
    18. The method for removing histamine or a methylated derivative thereof according to the preceding claim, wherein said mutation is introduced by the use of the Tn5 transposon.
    The method for the removal of histamine or a methylated derivative thereof according to any one of claims 14 to 16, wherein the histamine or the methylated derivative thereof is transformed into alanine and wherein the activity of enzyme D -amino acid dehydrogenase, which transforms alanine into pyruvate, is prevented by a mutation in the gene that gives; or where the activity of the enzyme racemase, which
    15 transforms L-alanine into D-alanine, it is prevented by a mutation in the dadX gene.
    [20]
    twenty. The method for removing histamine or a methylated derivative thereof according to the preceding claim, wherein said mutation is introduced by the use of the Tn5 transposon.
    [21]
    twenty-one. The procedure for the removal of histamine or a methylated derivative of the
    20 according to any of claims 14 to 16, wherein the histamine or the methylated derivative thereof is transformed into aspartic acid and where the activity of the enzyme aspartate ammonium lyase, which converts aspartate into fumarate, is prevented by a mutation in the sequence represented by SEQ ID NO: 20 or by a mutation in the sequence represented by SEQ ID NO: 22.
    22. The method for removing histamine or a methylated derivative thereof according to the preceding claim, wherein said mutation is introduced by the use of the transposon Tn5.
    [23]
    23. A process for the elimination of histamine or a methylated derivative thereof and imidazolacetic acid from a medium, characterized in that said medium is
    30 cultivates a microorganism comprising the nucleic acid molecule according to any one of claims 5 to 11.

    [24]
    24. The method for the removal of histamine or a methylated derivative thereof and of the imidazolacetic acid according to the preceding claim, wherein the microorganism is the strain Pseudomonas putida U CECT4848.
    [25]
    25. The procedure for the removal of histamine or a methylated derivative of the
    5 and of the imidazolacetic acid according to any of the two preceding claims, wherein the medium from which the histamine or a methylated derivative thereof and the imidazolacetic acid are removed is a fermented food that is treated with a microorganism starter culture.
    [26]
    26. A process for obtaining imidazoleacetic acid which comprises cultivating a
    A microorganism comprising the nucleic acid molecule according to any one of claims 1 to 4.
    [27]
    27. The process for obtaining imidazolacetic acid according to the preceding claim, wherein the activity of the enzyme monooxygenase, which transforms the imidazolacetic acid into 4-hydroxy-imidazol-5-acetic acid or its analogue, 4-imidazolon-5-acetic acid , is
    15 prevented by a mutation in the sequence represented by SEQ ID NO: 14.
    [28]
    28. The method for obtaining imidazoleacetic acid according to claim 26, wherein the expression and / or function of the HinK protein is prevented by a mutation in the sequence represented by SEQ ID NO: 24.
    [29]
    29. The procedure for obtaining imidazoleacetic acid according to either of the two
    20 previous claims, wherein the mutation is introduced through the use of the Tn5 transposon.
    [30]
    30. The method for obtaining imidazoleacetic acid according to any of the four preceding claims, wherein the microorganism is the strain Pseudomonas putida U CECT4848.
    A method for obtaining alanine which comprises culturing a microorganism comprising the nucleic acid molecule according to any one of claims 1 to 4.

    [32]
    32. The method for obtaining alanine according to the preceding claim, wherein the activity of the D-amino acid dehydrogenase, which transforms the alanine into pyruvate, is prevented by a mutation in the gene that gives.
    [33]
    33. The method for obtaining alanine according to the preceding claim, wherein said mutation is introduced through the use of the Tn5 transposon.
    [34]
    3. 4. The method for obtaining alanine according to any of the three preceding claims, wherein the microorganism is the strain Pseudomonas putida U CECT4848.
    [35]
    35 A process for obtaining aspartic acid which comprises cultivating a
    microorganism comprising the nucleic acid molecule according to any one of claims 1 to 4.
    [36]
    36. The method for obtaining aspartic acid according to the preceding claim, wherein the activity of the enzyme aspartate ammonium lyase, which converts aspartate into fumarate, is prevented by a mutation in the sequence represented by SEQ ID NO: 20 or by a mutation in the sequence represented by SEQ ID NO: 22.
    The method for obtaining aspartic acid according to the preceding claim, wherein said mutation is introduced by the use of the Tn5 transposon.
    [38]
    38. The method of obtaining aspartic acid according to any of the three preceding claims, wherein the microorganism is the strain Pseudomonas putida U CECT4848.
    A method for removing L-histidine from a medium, characterized in that in said medium a microorganism comprising the nucleic acid molecule according to any one of claims 1 to 11 and additionally comprising a nucleic acid molecule is cultivated which encodes an enzyme with histidine decarboxylase activity.
    The method for eliminating the L-histidine according to the preceding claim, wherein the medium from which the L-histidine is removed is a fermented food that is treated with a microorganism starter culture.









































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