RECOMBINANT CYANOBACTERIA OVER-PRODUCING SACAROSE (Machine-translation by Google Translate, not lega

专利摘要:
Recombinant sucrose overproducing cyanobacteria. The present invention refers to a sucrose overproducing recombinant cyanobacteria in the absence of osmotic stress, which comprises the overexpression of a set of genes, and the use thereof for the generation of synthetic bacterial consortia composed of both halophilic and non-halophilic heterotrophic bacteria. . (Machine-translation by Google Translate, not legally binding)
公开号:ES2844298A1
申请号:ES202030042
申请日:2020-01-21
公开日:2021-07-21
发明作者:Enrique Juan Nogales；López José Luis García；Sánchez Igor Martínez
申请人:Consejo Superior de Investigaciones Cientificas CSIC；
IPC主号:

专利说明:

[0002] Recombinant sucrose overproducing cyanobacteria
[0004] The present invention relates to a recombinant sucrose overproducing cyanobacteria in the absence of osmotic stress, and its use in the production of value-added products such as biofuels and bioplastics in consortium with other microorganisms. Therefore, the present invention falls within the field of biotechnology, in particular, in the use of recombinant microorganisms in industrial processes.
[0006] BACKGROUND OF THE INVENTION
[0008] Advances in microbial engineering for the production of biofuels, chemicals and therapeutics have stimulated investment in the production of a wide variety of products from biological sources. Heterotrophic microbes comprise the vast majority of microorganisms currently used for the generation of these products and require a source of carbohydrates to obtain carbon and energy that can represent a significant proportion (<60%) of the cost of inputs. These carbohydrates are typically obtained from agricultural crops, mainly sugar cane, sugar beet and corn, although lignocellulosic materials are being extensively investigated as an alternative raw material source. While biologically produced fuels and chemicals promise greater sustainability and a lower CO ² footprint, current sources of feedstock put biotech processes on a par with the use of agricultural farmland and food trading. Therefore, the development of biological alternatives to standard petroleum-based fuels and chemicals have been criticized for their increased cost and the instability of the food they produce. In fact, in recent years, sugar prices have risen and fluctuated enormously in food globally, due in part to the increased demand for it in biofuel production.
[0010] Photosynthetic microorganisms (cyanobacteria and algae) have been proposed as alternative sources for the creation of compounds similar to biofuels or industrial raw materials, in part because they have many advantages over traditional land plants with respect to the production of specific metabolites.
[0011] For example, the photosynthetic efficiency of cyanobacteria is even higher than that of plants, and cyanobacteria do not require supporting tissues that further reduce yield and productivity (eg roots / stems). Furthermore, cyanobacteria are genetically tractable, allowing rapid modification and selection of desirable strains. Lastly, cyanobacteria are aquatic microbes with minimal nutritional requirements and therefore can be grown in places that do not compete with traditional agricultural crops. Even so, only a small number of reports have been published on the extraction of homogeneous simple carbohydrates from cyanobacterial species.
[0013] Previous strategies that have been used to achieve high rates of sucrose production with cyanobacteria involve overproduction of the sucrose transporter CscB [Ducat et al. (2012) Appl Environ Microbiol 78 (8): 2660-2668]. Other strategies include the simultaneous overexpression of said transporter and the gene encoding sucrose synthase (Sps) under the control of an inducible promoter [Duan et al. (2016) J Ocean Univ China 15 (5): 890-896]. To achieve significant sucrose production, both strategies require the cultivation of cyanobacteria under osmotic stress (NaCl concentration> 150 mM). More reports have been published that describe the production of sucrose, however, in all these cases the induction of an osmotic stress is mandatory for said production to occur. Therefore, current approaches have several limitations including:
[0014] i) culturing the cyanobacteria under suboptimal culture conditions, including high salt osmotic stress,
[0015] ii) requirement of the two-phase process (first biomass accumulation and then sucrose production), and
[0016] iii) the obligatory presence of halophytic bacteria as partners in artificial microbial consortia, since cyanobacteria must be used as suppliers of sucrose to facilitate the growth of said artificial microbial consortia.
[0018] Therefore, there is a need in the state of the art to develop a new sucrose production method that overcomes the drawbacks of the state of the art mentioned above.
[0019] DESCRIPTION OF THE INVENTION
[0020] The present invention relates to a recombinant cyanobacteria that overproduces sucrose and is not dependent on osmotic stress to trigger the production of said sucrose. The production of sucrose in this cyanobacteria is generated in a manner coupled with growth. This characteristic allows the bacteria to be cultivated in the presence of non-halophilic microorganisms, forming consortiums of microorganisms useful in industrial processes. Additionally, said independence from osmotic stress allows the cyanobacteria to be cultivated in a bioreactor, both in continuous culture and in discontinuous culture, simultaneously maintaining growth and sucrose production during all the growth phases of the cyanobacteria (phase of adaptation, exponential phase and stationary phase), which avoids having to divide the process into two stages: one for biomass accumulation and another for sucrose production.
[0022] The inventors developed this cyanobacterium, in particular, Synechococcus elongatus, by overexpression of a set of genes, some homologous and others heterologous to the cyanobacterium genome, through the introduction of two operons comprising the genes in question at the insertion sites. NSI and NSII of the cyanobacteria genome (Example 1). The overexpressed genes were the following:
[0023] (i) the gene encoding the enzyme glucose-6-phosphate isomerase ( pgi) from Eschenchia coli,
[0024] (ii) the gene encoding the enzyme phosphoglucomutase ( pgmT), from E. coli
[0025] (iii) the gene encoding the enzyme UTP-glucose-1-phosphate uridylyltransferase ( galU) from E. coli ,
[0026] (iv) the gene encoding the sucrose phosphate synthase (sps) enzyme from S. elongatus, (v) the gene encoding the sucrose permease enzyme (CSCB cscB) from E. coli, and (vi) the gene encoding the Repressor protein of an E. coli metabolite-inducible operon ( lacI).
[0028] Contrary to what currently exists in the state of the art, and to achieve higher productivity, the recombinant strain of the present invention can be used additionally:
[0030] 1) Both in batch and continuous cultivation for long-term production of sucrose in a bioreactor. More than 4 g / L can be produced in 10 days on a laboratory scale. When compared to the strains of the state of the art, the recombinant cyanobacteria of the present invention maintain similar production rates but for longer periods of time. Therefore, the present invention provides the first growth coupled sucrose overproducing photosynthetic strain capable of maintaining high sucrose production rates for at least 12 days (see Example 2).
[0032] 2) As a sucrose overproducing strain in a synthetic association with other microorganisms without the presence of high concentrations of salt and, therefore, without the need to use halophilic microorganisms, highly tolerant to salt, to create industrial microbial consortia between the Recombinant cyanobacteria and the other microorganisms (see Example 3).
[0034] Therefore, in one aspect, the invention relates to a recombinant cyanobacterium, hereinafter "cyanobacterium of the invention", comprising the following nucleotide sequences
[0035] (i) the nucleotide sequence encoding the enzyme glucose-6-phosphate isomerase (PGI) or a fragment thereof,
[0036] (ii) the nucleotide sequence encoding the enzyme phosphoglucomutase (PGMT) or a fragment thereof,
[0037] (iii) the nucleotide sequence encoding the enzyme UTP-glucose-1-phosphate uridylyltransferase (GalU), or a fragment thereof,
[0038] (iv) the nucleotide sequence encoding the enzyme sucrose phosphate synthase (SPS), or a fragment thereof; Y
[0039] (v) the nucleotide sequence encoding a sucrose transporter, or a fragment thereof,
[0040] where:
[0041] - the nucleotide sequences (i) to (v) are overexpressed with respect to a non-recombinant or wild type cyanobacterium, and
[0042] - the nucleotide sequences (i), (ii), (iii) and (v) are heterologous.
[0044] In the present invention, "cyanobacteria" is understood as an organism of the Bacteria domain that is capable of oxygenic photosynthesis, that is, it is a photoautotrophic organism capable of using CO2 as the sole carbon source and light as a source. of energy. Cyanobacteria use the same CO ² fixation pathway as eurokaryotic cells as algae and higher plants (the Calvin cycle, C3). Cyanobacteria are also known as blue-green algae.
[0046] Examples of cyanobacteria that can be used in the context of the present invention include, but are not limited to, cyanobacteria belonging to the genus Chamaesiphon, Chroococcus, Cyanobacterium, Cyanobium, Dactylococcopsis, Gloeobacter, Gloeocapsa, Gloeothecece, Microcystis, Prochlorococcus, Sychonecochloroncusidio, Sychonecochloroncusidio, Sychonecochloroncusidio , Cyanocystis, Dermocarpella, Myxosarcina, Pleurocapsis, Stanieria, Xenococcus, Arthrospira, Borzia, Crinalium, Geitlerinema, Halospirulina, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus, Oscillatoria, Planktothrix, Prosechlorothinabaena, Starychria, Picholamploy, Procholorium, Starchorium , Anabaenopsis, Aphanizomenon, Calothrix, Cyanospira, Cylindrospermopsis, Cylindrospermum, Nodularia, Nostoc, Chlorogloeopsis, Fischerella, Geitleria, Nostochopsis, Iyengariella, Stigonema, Rivularia, Scytonema, and Tolypothri. In a particular embodiment, the cyanobacterium belongs to the genus Synechococcus sp. which, in another more particular embodiment, is Synechococcus elongatus which, in yet another more particular embodiment, is Synechococcus elongatus PCC 7942.
[0048] In the present invention, "recombinant cyanobacteria" is understood as that cyanobacterium that has been genetically modified, thus distinguishing itself from the "mother", "parental" or wild type strain. In the context of the present invention, the terms "recombinant cyanobacteria" and "transgenic cyanobacteria" are equivalent terms and mean the same thing.
[0050] The cyanobacterium of the invention is a sucrose overproducer and is not dependent on osmotic stress thanks to the fact that it has several nucleotide sequences inserted in its genome, hereinafter "nucleotide sequences of the invention", which code for different enzymes or proteins:
[0052] - the enzyme glucose-6-phosphate isomerase or phosphoglucose isomerase or PGI (in English GPI or Glucose-6-phosphate isomerase) is an enzyme that catalyzes the reversible reaction of glucose-6-phosphate to fructose-6-phosphate. In a particular embodiment, the PGI enzyme is the PGI enzyme from E. coli. In another particular embodiment, the enzyme PGI comprises an amino acid sequence that has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 1. In another particular embodiment, the PGI enzyme comprises, or consists of, an amino acid sequence exhibiting 100% sequence identity with the sequence SEQ ID NO: 1.
[0054] SEQ ID NO: 1:
[0055] MKNINPTQTAAWQALQKHFDEMKDVTIADLFAKDGDRFSKFSATFDDQMLVDYSKNRITEE TLAKLQDLAKECDLAGAIKSMFSGEKINRTENRAVLHVALRNRSNTPILVDGKDVMPEVNA VLEKMKTFSEAIISGEWKGYTGKAITDVVNIGIGGSDLGPYMVTEALRPYKNHLNMHFVSN VDGTHIAEVLKKVNPETTLFLVASKTFTTQETMTNAHSARDWFLKAAGDEKHVAKHFAALS TNAKAVGEFGIDTANMFEFWDWVGGRYSLWSAIGLSIVLSIGFDNFVELLSGAHAMDKHFS TTPAEKNLPVLLALIGIWYNNFFGAETEAILPYDQYMHRFAAYFQQGNMESNGKYVDRNGN VVDYQTGPIIWGEPGTNGQHAFYQL1HQGTKMVPCDFIAPAITHNPLSDHHQKLLSNFFAQ TEALAFGKSREVVEQEYRDQGKDPATLDYVVPFKVFEGNRPTNSILLREITPFSLGALIAL YEHKIFTQGVILNIFTFDQWGVELGKQLANRILPELKDDKEISSHDSSTNGLINRYKAWRG
[0057] In the present invention, all amino acid sequences that have a sequence identity of at least 70% with the sequence SEQ ID NO: 1 are considered functionally equivalent variants of the PGI protein, that is, although they comprise a different sequence of amino acids, are capable of catalyzing the reversible reaction of glucose-6-phosphate to fructose-6-phosphate. An assay to determine whether a given protein is a functionally equivalent variant of PGI includes, but is not limited to, an assay for monitoring the EC 5.3.1.9 reaction (responsible for the reversible isomerization of glucose-6-phosphate and fructosesphosphate) by the protein. Dadaist. Carrying out the monitoring of the EC 5.3.1.9 reaction by an enzyme is routine practice for the person skilled in the art.
[0059] In another particular embodiment, the nucleotide sequence that encodes the PGI enzyme comprises a nucleotide sequence that has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 2. In another particular embodiment, the nucleotide sequence encoding the PGI enzyme comprises, or consists of, the nucleotide sequence SEQ ID NO: 2. However, as understood by the person skilled in the art, in many times the nucleotide sequence has to be optimized for its expression in a cellular system different from the original one. For this reason, in another particular embodiment, the gene that encodes the PGI enzyme comprises, or consists of, a sequence of nucleotides, optimized for its correct expression in Synechococcus sp, in particular S. elongatus, more in particular S. elongatus PCC7942 , which exhibits 100% sequence identity with the sequence SEQ ID NO: 3.
[0061] SEQ ID NO: 2: atgaaaaacatcaatccaacgcagaccgctgcctggcaggcactacagaaacacttcgat gaaatgaaagacgttacgatcgccgatctttttgctaaagacggcgatcgtttttctaag ttctccgcaaccttcgacgatcagatgctggtggattactccaaaaaccgcatcactgaa gagacgctggcgaaattacaggatctggcgaaagagtgcgatctggcgggcgcgattaag tcgatgttctctggcgagaagatcaaccgcactgaaaaccgcgccgtgctgcacgtagcg ctgcgtaaccgtagcaataccccgattttggttgatggcaaagacgtaatgccggaagtc aacgcggtgctggagaagatgaaaaccttctcagaagcgattatttccggtgagtggaaa ggttataccggcaaagcaatcactgacgtagtgaacatcgggatcggcggttctgacctc ggcccatacatggtgaccgaagctctgcgtccgtacaaaaaccacctgaacatgcacttt gtttctaacgtcgatgggactcacatcgcggaagtgctgaaaaaagtaaacccggaaacc acgctgttcttggtagcatctaaaaccttcaccactcaggaaactatgaccaacgcccat agcgcgcgtgactggttcctgaaagcggcaggtgatgaaaaacacgttgcaaaacacttt gcggcgctttccaccaatgccaaagccgttggcgagtttggtattgatactgccaacatg ttcgagttctgggactgggttggcggccgttactctttgtggtcagcgattggcctgtcg attgttctctccatcggctttgataacttcgttgaactgctttccggcgcacacgcgatg gacaagcatttctccaccacgcctgccgagaaaaacctgcctgtactgctggcgctgatt ggcatctggt acaacaatttctttggtgcggaaactgaagcgattctgccgtatgaccag tatatgcaccgtttcgcggcgtacttccagcagggcaatatggagtccaacggtaagtat gttgaccgtaacggtaacgttgtggattaccagactggcccgattatctggggtgaacca ggcactaacggtcagcacgcgttctaccagctgatccaccagggaaccaaaatggtaccg tgcgatttcatcgctccggctatcacccataacccgctctctgatcatcaccagaaactg ctgtctaacttcttcgcccagaccgaagcgctggcgtttggtaaatcccgcgaagtggtt gagcaggaatatcgtgatcagggtaaagatccggcaacgcttgactacgtggtgccgttc aaagtattcgaaggtaaccgcccgaccaactccatcctgctgcgtgaaatcactccgttc agcctgggtgcgttgattgcgctgtatgagcacaaaatctttactcagggcgtgatcctg aacatcttcaccttcgaccagtggggcgtggaactgggtaaacagctggcgaaccgtatt ctgccagagctgaaagatgataaagaaatcagcagccacgatagctcgaccaatggtctg attaaccgctataaagcgtggcgcggttaa
[0062] SEQ ID NO: 3:
[0063] atgaaaaacatcaatccaacgcagaccgctgcctggcaggcactacagaaacacttcgatg aaatgaaagacgttacgatcgccgatctttttgctaaagacggcgatcgcttttctaagtt ctccgcaaccttcgacgatcagatgctggtggattactccaaaaaccgcatcactgaagag acgctggcgaaattacaggatctggcgaaagagtgcgatctggcgggcgcgattaagtcga tgttctcgggcgagaagatcaaccgcactgaaaaccgcgccgtgctgcacgtggcgctgcg taaccgcagcaataccccgattttggttgatggcaaagacgtcatgccggaagtcaacgcg gtgctggagaagatgaaaaccttctcagaagcgattatttccggtgagtggaaaggttata ccggcaaagcaatcactgacgtagtgaacatcgggatcggcggttcggacctcggcccata catggtgaccgaagctctgcggccgtacaaaaaccacctgaacatgcactttgtttctaac gtcgatgggactcacatcgcggaagtgctgaaaaaagtgaacccggaaaccacgctgttct tggtagcatcgaaaaccttcaccactcaggaaactatgaccaacgcccatagcgcgcgtga ctggttcctgaaagcggcaggtgatgaaaaacacgttgcaaaacactttgcggcgctttcc accaatgccaaagccgttggcgagtttggtattgatactgccaacatgttcgagttctggg actgggttggcggccggtactctttgtggtcagcgattggcctgtcgattgttctctccat cggctttgataacttcgttgaactgctttccggcgcacacgcgatggacaagcatttctcc accacgcctgccgagaaaaacctgcctgtcctgctggcgctgattggcatctggtacaaca atttcttt ggtgcggaaactgaagcgattctgccgtatgaccagtatatgcaccgtttcgc ggcgtacttccagcagggcaatatggagtccaacggtaagtatgttgaccgtaacggtaac gttgtggattaccagactggcccgattatctggggtgaaccaggcactaacggtcagcacg cgttctaccagctgatccaccagggaaccaaaatggtgccgtgcgatttcatcgctccggc tatcacccataacccgctctctgatcatcaccagaaactgctgtcgaacttcttcgcccag accgaagcgctggcgtttggtaaatcccgcgaagtggttgagcaggaatatcgcgatcagg gtaaagatccggcaacgcttgactacgtggtgccgttcaaagtattcgaaggtaaccgccc gaccaactccatcctgctgcgtgaaatcactccgttcagcctgggtgcgttgattgcgctg tatgagcacaaaatctttactcagggcgtgatcctgaacatcttcaccttcgaccagtggg gcgtggaactgggtaaacagctggcgaaccgtattctgccagagctgaaagatgataaaga aatcagcagccacgatagctcgaccaatggtctgattaaccgctataaagcgtggcgcggt taa
[0065] - The enzyme phosphoglucomutase or PGMT (in English PGMT or phosphoglucomutase) is an enzyme that catalyzes the transfer of the phosphate group from carbon 1 of glucose-1-phosphate to carbon 6 of glucose-6-phosphate. In a particular embodiment, the PGMT enzyme is the PGMT enzyme from E. coli. In another particular embodiment, the PGMT enzyme comprises an amino acid sequence that has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 4. In another particular embodiment, the PGMT enzyme comprises, or consists of, an amino acid sequence that presents 100% sequence identity with the sequence SEQ ID NO: 4.
[0067] SEQ ID NO: 4:
[0069] MA1HNRAGQPAQQSDLINVAQLTAQYYVLKPEAGNAEHAVKFGTSGHRGSAARHSFNEPHI LAIAQAIAEERAKNGITGPCYVGKDTHALSEPAFISVLEVLAANGVDVIVQENNGFTPTPA VSNAILVHNKKGGPLADGIVITPSHNPPEDGGIKYNPPNGGPADTNVTKVVEDRANALLAD GLKGVKRISLDEAMASGHVKEQDLVQPFVEGLADIVDMAAIQKAGLTLGVDPLGGSGIEYW KRIGEYYNLNLTIVNDQVDQTFRFMHLDKDGAIRMDCSSECAMAGLLALRDKFDLAFANDP DYDRHGIVTPAGLMNPNHYLAVAINYLFQHRPQWGKDVAVGKTLVSSAMIDRVVNDLGRKL VEVPVGFKWFVDGLFDGSFGFGGEESAGASFLRFDGTPWSTDKDGIIMCLLAAEITAVTGK NPQEHYNELAKRFGAPSYNRLQAAATSAQKAALSKLSPEMVSASTLAGDPITARLTAAPGN GASIGGLKVMTDNGWFAARPSGTEDAYKIYCESFLGEEHRKQIEKEAVEIVSEVLKNA-
[0071] In the present invention, all amino acid sequences that have a sequence identity of at least 70% with the sequence SEQ ID NO: 4 are considered functionally equivalent variants of the PGMT protein, that is, although they comprise a different sequence of amino acids, are capable of catalyzing the transfer of the phosphate group from carbon 1 of glucose-1-phosphate to carbon 6 of glucose-6-phosphate. An assay to determine whether a given protein is a functionally equivalent variant of PGMT includes, but is not limited to, an EC 5.4.2.2 reaction monitoring assay (responsible for the conversion of D-glucose 1-phosphate to D-glucose 6- phosphate) for the given protein. Carrying out the monitoring of the EC 5.4.2.2 reaction by an enzyme is routine practice for the person skilled in the art.
[0073] In another particular embodiment, the nucleotide sequence that encodes the PGMT enzyme comprises a nucleotide sequence that has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 5. In another particular embodiment, the nucleotide sequence encoding the PGMT enzyme comprises, or consists of, the nucleotide sequence SEQ ID NO: 5. However, as understood by the person skilled in the art, in many times the nucleotide sequence has to be optimized for its expression in a system cell other than that of origin. For this reason, in another particular embodiment, the gene that encodes the PGMT enzyme comprises, or consists of, a sequence of nucleotides, optimized for its correct expression in Synechococcus sp, in particular S. elongatus, more in particular S. elongatus PCC7942 , which exhibits 100% sequence identity with the sequence SEQ ID NO: 6.
[0075] SEQ ID NO: 5: atggcaatccacaatcgtgcaggccaacctgcacaacagagtgatttgattaacgtcgcc caactgacggcgcaatattatgtactgaaaccagaagcagggaatgcggagcacgcggtg aaattcggtacttccggtcaccgtggcagtgcagcgcgccacagctttaacgagccgcac attctggcgatcgctcaggcaattgctgaagaacgtgcgaaaaacggcatcactggccct tgctatgtgggtaaagatactcacgccctgtccgaacctgcattcatttccgttctggaa gtgctggcagcgaacggcgttgatgtcattgtgcaggaaaacaatggcttcaccccgacg cctgccgtttccaatgccatcctggttcacaataaaaaaggtggcccgctggcagacggt atcgtgattacaccgtcccataacccgccggaagatggtggaatcaaatacaatccgcca aatggtggcccggctgataccaacgtcactaaagtggtggaagacagggccaacgcactg ctggccgatggcctgaaaggcgtgaagcgtatctccctcgacgaagcgatggcatccggt catgtgaaagagcaggatctggtgcagccgttcgtggaaggtctggccgatatcgttgat atggccgcgattcagaaagcgggcctgacgctgggcgttgatccgctgggcggttccggt atcgaatactggaagcgtattggcgagtattacaacctcaacctgactatcgttaacgat caggtcgatcaaaccttccgctttatgcaccttgataaagacggcgcgatccgtatggac tgctcctccgagtgtgcgatggcgggcctgctggcactgcgtgataagttcgatctggcg tttgctaacgacccggattatgaccgtcacggtatcgtcactccggcaggtttgatgaat ccgaaccact acctggcggtggcaatcaattacctgttccagcatcgtccgcagtggggc aaagatgttgccgtcggtaaaacgctggtttcatctgcgatgatcgaccgtgtggtcaac gacttgggccgtaaactggtagaagtcccggtaggtttcaaatggtttgtcgatggtctg ttcgacggcagcttcggctttggcggcgaagagagtgcaggggcttccttcctgcgtttc gacggcacgccgtggtccaccgacaaagacggcatcatcatgtgtctgctggcggcggaa atcaccgctgtcaccggtaagaacccgcaggaacactacaacgaactggcaaaacgcttt ggtgcgccgagctacaaccgtttgcaggcagctgcgacttccgcacaaaaagcggcgctg tctaagctgtctccggaaatggtgagcgccagcaccctggcaggtgacccgatcaccgcg cgcctgactgctgctccgggcaacggtgcttctattggcggtctgaaagtgatgactgac aacggctggttcgccgcgcgtccgtcaggcacggaagacgcatataagatctactgcgaa agcttcctcggtgaagaacatcgcaagcagattgagaaagaagcggttgagattgttagc gaagttctgaaaaacgcgtaa
[0076] SEQ ID NO: 6: atggcaatccacaatcgcgcaggccaacctgcacaacagagtgatttgattaacgtcgccc aactgacggcgcaatattatgtgctgaaaccagaagcagggaatgcggagcacgcggtgaa attcggtacttccggtcaccggggcagtgcagcgcgccacagctttaacgagccgcacatt ctggcgatcgctcaggcaattgctgaagaacgcgcgaaaaacggcatcactggcccttgct atgtgggtaaagatactcacgccctgtccgaacctgcattcatttccgttctggaagtgct ggcagcgaacggcgttgatgtcattgtgcaggaaaacaatggcttcaccccgacgcctgcc gtttccaatgccatcctggttcacaataaaaaaggtggcccgctggcagacggtatcgtga ttacaccgtcccataacccgccggaagatggtggaatcaaatacaatccgccaaatggtgg cccggctgataccaacgtcactaaagtggtggaggaccgggccaacgcactgctggccgat ggcctgaaaggcgtgaagcgtatctccctcgacgaagcgatggcatccggtcatgtgaaag agcaggatctggtgcagccgttcgtggaaggtctggccgatatcgttgatatggccgcgat tcagaaagcgggcctgacgctgggcgttgatccgctgggcggttccggtatcgaatactgg aagcgcattggcgagtattacaacctcaacctgactatcgttaacgatcaggtcgatcaaa ccttccgctttatgcaccttgataaagacggcgcgatccgtatggactgctcctccgagtg tgcgatggcgggcctgctggcactgcgtgataagttcgatctggcgtttgctaacgacccg gattatgaccgccacggtatcgtcactccggcaggtttgatgaatccgaaccacta CCTGG cggtggcaatcaattacctgttccagcatcgtccgcagtggggcaaagatgttgccgtcgg taaaacgctggtttcatcggcgatgatcgaccgtgtggtcaacgacttgggccgtaaactg gtagaagtcccggtaggtttcaaatggtttgtcgatggtctgttcgacggcagcttcggct ttggcggcgaagagagtgcaggggcttccttcctgcgtttcgacggcacgccgtggtccac cgacaaagacggcatcatcatgtgtctgctggcggcggaaatcaccgctgtcaccggtaag aacccgcaggaacactacaacgaactggcaaaacgctttggtgcgccgagctacaaccgtt tgcaggcagctgcgacttccgcacaaaaagcggcgctgtctaagctgtctccggaaatggt gagcgccagcaccctggcaggtgacccgatcaccgcgcgcctgactgctgctccgggcaac ggtgcttctattggcggtctgaaagtgatgactgacaacggctggttcgccgcgcgtccgt caggcacggaagatgcatataagatctactgcgaaagtttcctcggtgaagaacatcgcaa gcagattgagaaagaagcggttgagattgttagcgaagttctgaaaaacgcgtaa
[0078] - The enzyme UTP-glucose-1-phosphate uridylyltransferase or GalU (in English GalU or UTP-glucose-1-phosphate urídylyltransferase, UDP-glucose pyrophosphorylase, or glucose-1-phosphate urídylyltransferase) is an enzyme that catalyzes the reaction between Glucose -1-phosphate and UTP to form UDP-glucose-2. In a particular embodiment, the GalU enzyme is the GalU enzyme from E. coli. In another particular embodiment, the GalU enzyme comprises an amino acid sequence that has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with the sequence SEQ ID NO: 7. In another particular embodiment, the GalU enzyme comprises, or consists of, an amino acid sequence that presents 100% sequence identity with the sequence SEQ ID NO: 7.
[0080] SEQ ID NO: 7:
[0081] MAAINTKVKKAVIPVAGLGTRMLPATKAIPKEMLPLVDKPLIQYVVNECIAAGITEIVLVT HSSKNSIENHFDTSFELEAMLEKRVKRQLLDEVQSICPPHVTIMQVRQGLAKGLGHAVLCA HPVVGDEPVAVILPDVILDEYESDLSQDNLAEMIRRFDETGHSQIMVEPVADVTAYGVVDC KGVELAPGESVPMVGVVEKPKADVAPSNLAIVGRYVLSADIWPLLAKTPPGAGDEIQLTDA IDMLIEKETVEAYHMKGKSHDCGNKLGYMQAFVEYGIRHNTLGTEFKAWLEEEMGIKK-
[0083] In the present invention, all amino acid sequences that have a sequence identity of at least 70% with the sequence SEQ ID NO: 7 are considered functionally equivalent variants of the GalU protein, that is, although they comprise a different sequence of amino acids, are capable of catalyzing the reaction between Glucose-1-phosphate and UTP to form UDP-glucose-2. An assay to determine whether a given protein is a functionally equivalent variant of GalU includes, but is not limited to, an assay for monitoring the EC 2.7.7.9 reaction (responsible for the transfer of a UTP group to glucose-1-phosphate) by the given protein. Carrying out the monitoring of the EC 2.7.7.9 reaction by an enzyme is routine practice for the person skilled in the art.
[0085] In another particular embodiment, the nucleotide sequence that encodes the GalU enzyme comprises a nucleotide sequence that has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 8. In another particular embodiment, the nucleotide sequence encoding the GalU enzyme comprises the nucleotide sequence SEQ ID NO: 8. However, as understood by those skilled in the art, on many occasions the sequence of nucleotides have to be optimized for their expression in a cellular system other than the one of origin. For this reason, in another particular embodiment, the gene encoding the GalU enzyme comprises, or consists of, a sequence of nucleotides, optimized for its correct expression in Synechococcus sp, in particular S. elongatus, more in particular S. elongatus PCC7942 , which exhibits 100% sequence identity with the sequence SEQ ID NO: 9.
[0086] SEQ ID NO: 8: atggctgccattaatacgaaagtcaaaaaagccgttatccccgttgcgggattaggaacc aggatgttgccggcgacgaaagccatcccgaaagagatgctgccacttgtcgataagcca ttaattcaatacgtcgtgaatgaatgtattgcggctggcattactgaaattgtgctggtt acacactcatctaaaaactctattgaaaaccactttgataccagttttgaactggaagca atgctggaaaaacgtgtaaaacgtcaactgcttgatgaagtgcagtctatttgtccaccg cacgtgactattatgcaagttcgtcagggtctggcgaaaggcctgggacacgcggtattg tgtgctcacccggtagtgggtgatgaaccggtagctgttattttgcctgatgttattctg gatgaatatgaatccgatttgtcacaggataacctggcagagatgatccgccgctttgat gaaacgggtcatagccagatcatggttgaaccggttgctgatgtgaccgcatatggcgtt gtggattgcaaaggcgttgaattagcgccgggtgaaagcgtaccgatggttggtgtggta gaaaaaccgaaagcggatgttgcgccgtctaatctcgctattgtgggtcgttacgtactt agcgcggatatttggccgttgctggcaaaaacccctccgggagctggtgatgaaattcag ctcaccgacgcaattgatatgctgatcgaaaaagaaacggtggaagcctatcatatgaaa gggaagagccatgactgcggtaataaattaggttacatgcaggccttcgttgaatacggt attcgtcataacacccttggcacggaatttaaagcctggcttgaagaagagatgggcatt aagaagtaa
[0088] SEQ ID NO: 9: atggctgccattaatacgaaagtcaaaaaagccgttatccccgttgcgggattgggaaccc ggatgttgccggcgacgaaagccatcccgaaagagatgctgccactggtcgataagccatt gattcaatacgtcgtgaatgaatgtattgcggctggcattactgaaattgtgctggttaca cactcatcgaaaaactctattgaaaaccactttgataccagttttgaactggaagcaatgc tggaaaaacgcgtaaaacgtcaactgcttgatgaagtgcagtcgatttgtccaccgcacgt gactattatgcaagttcgccagggtctggcgaaaggcctgggacacgcggtgttgtgtgct cacccggtcgtgggtgatgaaccggtggctgttattttgcctgatgttattctggatgaat atgaatccgatttgtcacaggataacctggcagagatgatccgccgctttgatgaaacggg tcatagccagatcatggttgaaccggttgctgatgtgaccgcatatggcgttgtggattgc aaaggcgttgaattagcgccgggtgaaagcgtgccgatggttggtgtggtcgaaaaaccga aagcggatgttgcgccgtctaatctcgctattgtgggtcgttacgtactcagcgcggatat ttggccgttgctggcaaaaacccctccgggagctggtgatgaaattcagctcaccgacgca attgatatgctgatcgaaaaagaaacggtggaagcctatcatatgaaagggaagagccatg actgcggtaataaattaggttacatgcaggccttcgttgaatacggtattcgtcataacac ccttggcacggaatttaaagcctggcttgaagaagagatgggcattaagaagtaa
[0090] - The enzyme sucrose phosphate synthase or SPS (in English SPS or sucrose-phosphate synthase) is a multifunctional enzyme that possesses: i) glucosyltransferase activity that synthesizes sucrose-6-phosphate from UDP-Glc and / or ADP-Glc and Fru-6-P, and ii) specific phosphohydrolase activity, sucrose-phosphate -phosphatase (SPP); both activities, encoded in the same polypeptide chain, are responsible for the net synthesis of sucrose. In a particular embodiment, the SPS enzyme is the SPS enzyme from S. elongatus. In another particular embodiment, the SPS enzyme comprises an amino acid sequence that has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 10. In another particular embodiment, the SPS enzyme comprises, or consists of, an amino acid sequence that presents 100% identity with the sequence SEQ ID NO: 10.
[0092] SEQ ID NO: 10:
[0093] MAAQNLYILHIQTHGLLRGQNLELGRDADTGGQTKYVLELAQAQAKSPQVQQVDIITRQIT DPRVSVGYSQAIEPFAPKGRIVRLPFGPKRYLRKELLWPHLYTFADAILQYLAQQKRTPTW IQAHYADAGQVGSLLSRWLNVPLIFTGHSLGRIKLKKLLEQDWPLEEIEAQFNIQQRIDAE EMTLTHADWIVASTQQEVEEQYRVYDRYNPERKLVIPPGVDTDRFRFQPLGDRGVVLQQEL SRFLRDPEKPQILCLCRPAPRKNVPALVRAFGEHPWLRKKANLVLVLGSRQDINQMDRGSR QVFQEIFHLVDRYDLYGSVAYPKQHQADDVPEFYRLAAHSGGVFVNPALTEPFGLTILEAG SCGVPVVATHDGGPQEILKHCDFGTLVDVSRPANIATALATLLSDRDLWQCYHRNGIEKVP AHYSWDQHVNTLFERMETVALPRRRAVSFVRSRKRLIDAKRLVVSDIDNTLLGDRQGLENL MTYLDQYRDHFAFGIATGRRLDSAQEVLKEWGVPSPNFWVTSVGSE1HYGTDAEPDISWEK HINRNWNPQRIRAVMAQLPFLELQPEEDQTPFKVSFFVRDRHETVLREVRQHLRRHRLRLK SIYSHQEFLDILPLAASKGDAIRHLSLRWRIPLENILVAGDSGNDEEMLKGHNLGVVVGNY SPELEPLRSYERVYFAEGHYANGILEALKHYRFFEAIA-
[0095] In the present invention, all amino acid sequences that have a sequence identity of at least 70% with the sequence SEQ ID NO: 10 are considered functionally equivalent variants of the SPS protein, that is, although they comprise a different sequence of amino acids, are capable of acting as a glucosyltransferase that synthesizes sucrose-6-phosphate from UDP-Glc and / or ADP-Glc and Fru-6-P. An assay to determine whether a given protein is a functionally equivalent variant of SPS includes, but is not limited to, an assay for monitoring the activities EC 2.4.1.12 and EC 3.1.3.24 in the same polypeptide chain (responsible for the production of sucrose a from fructose-6-P and ADP / UDP-glucose) for the given protein. Carrying out the monitoring of activities EC 2.4.1.12 and EC 3.1.3.24 by an enzyme is routine practice for those skilled in the art.
[0097] In another particular embodiment, the nucleotide sequence that encodes the SPS enzyme comprises a nucleotide sequence that has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 11. In another particular embodiment, the nucleotide sequence encoding the SPS enzyme comprises the nucleotide sequence SEQ ID NO: 11. However, as understood by the person skilled in the art, on many occasions the sequence of nucleotides have to be optimized for expression in a cellular system. For this reason, in another particular embodiment, the gene that encodes the PSP enzyme comprises, or consists of, a sequence of nucleotides, optimized for its correct expression in Synechococcus sp, in particular S. elongatus, more in particular S. elongatus PCC7942 , which exhibits 100% sequence identity with the sequence SEQ ID NO: 12.
[0099] SEQ ID NO: 11: gtggcagctcaaaatctctacattctgcacattcagacccatggtctgctgcgagggcag aacttggaactggggcgagatgccgacaccggcgggcagaccaagtacgtcttagaactg gctcaagcccaagctaaatccccacaagtccaacaagtcgacatcatcacccgccaaatc accgacccccgcgtcagtgttggttacagtcaggcgatcgaaccctttgcgcccaaaggt cggattgtccgtttgccttttggccccaaacgctacctccgtaaagagctgctttggccc catctctacacctttgcggatgcaattctccaatatctggctcagcaaaagcgcaccccg acttggattcaggcccactatgctgatgctggccaagtgggatcactgctgagtcgctgg ttgaatgtaccgctaattttcacagggcattctctggggcggatcaagctaaaaaagctg ttggagcaagactggccgcttgaggaaattgaagcgcaattcaatattcaacagcgaatt gatgcggaggagatgacgctcactcatgctgactggattgtcgccagcactcagcaggaa gtggaggagcaataccgcgtttacgatcgctacaacccagagcgcaagcttgtcattcca ccgggtgtcgataccgatcgcttcaggtttcagcccttgggcgatcgcggtgttgttctc caacaggaactgagccgctttctgcgcgacccagaaaaacctcaaattctctgcctctgt cgccccgcacctcgcaaaaatgtaccggcgctggtgcgagcctttggcgaacatccttgg ctgcgcaaaaaagccaaccttgtcttagtactgggcagccgccaagacatcaaccagatg gatcgcggcagtcggcaggtgttccaagagattttccatctggtcgatcgctacgacctc tacggcagc gtcgcctatcccaaacagcatcaggctgatgatgtgccggagttctatcgc ctagcggctcattccggcggggtattcgtcaatccggcgctgaccgaaccttttggtttg acaattttggaggcaggaagctgcggcgtgccggtggtggcaacccatgatggcggcccc caggaaattctcaaacactgtgatttcggcactttagttgatgtcagccgacccgctaat atcgcgactgcactcgccaccctgctgagcgatcgcgatctttggcagtgctatcaccgc aatggcattgaaaaagttcccgcccattacagctgggatcaacatgtcaataccctgttt gagcgcatggaaacggtggctttgcctcgtcgtcgtgctgtcagtttcgtacggagtcgc aaacgcttgattgatgccaaacgccttgtcgttagtgacatcgacaacacactgttgggc gatcgtcaaggactcgagaatttaatgacctatctcgatcagtatcgcgatcattttgcc tttggaattgccacggggcgtcgcctagactctgcccaagaagtcttgaaagagtggggc gttccttcgccaaacttctgggtgacttccgtcggcagcgagattcactatggcaccgat gctgaaccggatatcagctgggaaaagcatatcaatcgcaactggaatcctcagcgaatt cgggcagtaatggcacaactaccctttcttgaactgcagccggaagaggatcaaacaccc ttcaaagtcagcttctttgtccgcgatcgccacgagactgtgctgcgagaagtacggcaa catcttcgccgccatcgcctgcggctgaagtcaatctattcccatcaggagtttcttgac attctgccgctagctgcctcgaaaggggatgcgattcgccacctctcactccgctggcgg attcctcttgagaacattttggtggcaggcgattctggtaacgatgaggaaatgctcaag ggccataatctcggcgttgtagttggcaattactcaccggaattggagccactgcgcagc tacgagcgcgtctattttgctgag ggccactatgctaatggcattctggaagccttaaaa cactatcgcttttttgaggcgatcgcttaa
[0101] SEQ ID NO: 12:
[0102] ATGGCAGCTCAAAatctctacattctgcacattcagacccatggtctgctgcgagggcaga acttggaactggggcgagatgccgacaccggcgggcagaccaagtacgtcttagaactggc tcaagcccaagctaaatccccacaagtccaacaagtcgatatcatcacccgccaaatcacc gacccccgcgtcagtgttggttacagtcaggcgatcgaaccctttgcgcccaaaggtcgga ttgtccgtttgccttttggccccaaacgctacctccgtaaagagctgctttggccccatct ctacacctttgcggatgcaattctccaatatctggctcagcaaaagcgcaccccgacttgg attcaggcccactatgctgatgctggccaagtgggatcactgctgagtcgctggttgaatg taccgctaattttcacagggcattctctggggcggatcaagctaaaaaagctgttggagca agactggccgcttgaggaaattgaagcgcaattcaatattcaacagcgaattgatgcggag gagatgacgctcactcatgctgactggattgtcgccagcactcagcaggaagtggaggagc aataccgcgtttacgatcgctacaacccagagcgcaagctggtcattccaccgggtgtcga taccgatcgcttcaggtttcagcccttgggcgatcgcggtgttgttctccaacaggaactg agccgctttctgcgcgacccagaaaaacctcaaattctctgcctctgtcgccccgcacctc gcaaaaatgtaccggcgctggtgcgagcctttggcgaacatccttggctgcgcaaaaaagc caaccttgtcttagtactgggcagccgccaagacatcaaccagatggatcgcggcagtcgg caggtgttccaagagattttccatctggtcgatcgctacgacctctacggcagcgtcgcct atcccaaacagcatcaggctgatgatgtgccggagttctatcgcctagcggctcattccgg cggggtattcgtcaatccggcgctgaccgaaccttttggtttgacaattttggaggcagga agctgcggcgtgccggtggtggcaacccatgatggcggcccccaggaaattctcaaacact gtgatttcggcactttagttgatgtcagccgacccgctaatatcgcgactgcactcgccac cctgctgagcgatcgcgatctttggcagtgctatcaccgcaatggcattgaaaaagttccc gcccattacagctgggatcaacatgtcaataccctgtttgagcgcatggaaacggtggctt tgcctcgtcgtcgtgctgtcagtttcgtacggagtcgcaaacgcttgattgatgccaaacg ccttgtcgttagtgacatcgacaacacactgttgggcgatcgtcaaggactcgaaaattta atgacctatctcgatcagtatcgcgatcattttgcctttggaattgccacggggcgtcgcc tagactctgcccaagaagtcttgaaagagtggggcgttccttcgccaaacttctgggtgac ttccgtcggcagcgagattcactatggcaccgatgctgaaccggatatcagctgggaaaag catatcaatcgcaactggaatcctcagcgcattcgggcagtaatggcacaactaccctttc ttgaactgcaaccggaagaggatcaaacacccttcaaagtcagcttctttgtccgcgatcg ccacgagactgtgctgcgagaagtacggcaacatcttcgccgccatcgcctgcggctgaag tcaatctattcccatcaggagtttcttgacattctgccgctggctgcctcgaaaggggatg cgattcgccacctctcactccgctggcggattcctcttgagaacattttggtggcaggcga ttctggta acgatgaggaaatgctcaagggccataatctcggcgttgtagttggcaattac tcaccggaattggagccactgcgcagctacgagcgcgtctattttgctgagggccactatg ctaatggcattctggaagccttaaaacactatcgcttttttcttaggcgatc
[0104] Alternatively, the PSP enzyme can be replaced by the enzymes responsible for carrying out each of the enzymatic activities presented by the PSP enzyme, that is, an enzyme with glucosyltransferase activity that synthesizes sucrose-6-phosphate from UDP-Glc and / or ADP-Glc and Fru-6-P, and another enzyme with specific phosphohydrolase activity. Enzymes with glucosyltransferase activity, as well as enzymes with phosphohydrolase activity, are known in the state of the art, and likewise the nucleotide sequence that encodes them can be optimized for their expression in Synechococcus sp., In particular S. elongatus, more in particular , S. elongatus PCC7942.
[0106] - A sucrose transporter across the cell membrane. Examples of sucrose transporters include, but are not limited to, SoSUT1 (sucrose transporter from spinach ( Spinacea olerácea)), OsSUT1, OsSUT2, OsSUT3, OsSUT4 and OsSUT5 (sucrose transporters from rice ( Oryza sativa)), HvSUT1 and HvSUT2 (sucrose transporters from barley ( Hordeum vulgare)), TaSUT1A, TaSUT1B and TaSUT1D (transporters of sucrose from wheat ( Triticum aestivum)), AcSUTI (transporter of sucrose from pineapple ( Anana comosus)), BoSUTI (transporter of sucrose from bamboo ( Bambusa oldhamii)), and SbSUT1 and SbSUT4 (transporters of sucrose from sorghum ( Sorghum bicolor)). The nucleotide and amino acid sequences corresponding to these sucrose transporters are available in public databases accessible to those skilled in the art.
[0108] In a particular embodiment, the sucrose transporter is sucrose permease or CSCB. Sucrose permease or CSCB (in English CSCB or sucrose permease) is responsible for the transport of sucrose together with the simultaneous import of protons. This transporter has the advantage that it is functional under alkaline pH conditions, which makes it the optimal sucrose transporter for use by cyanobacteria, in particular, by S. elongatus PCC7942. In a particular embodiment, the CSCB protein is the E. coli CSCB protein. In another particular embodiment, the CSCB protein comprises an amino acid sequence that has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 13. In another particular embodiment, the CSCB protein comprises, or consists of, a nucleotide sequence that presents 100% sequence identity with the sequence SEQ ID NO: 13.
[0110] SEQ ID NO: 13:
[0111] MALNIPFRNAYYRFASSYSFLFFISWSLWWSLYAIWLKGHLGLTGTELGTLYSVNQFTSIL FMMFYGIVQDKLGLKKPLIWCMSFILVLTGPFMIYVYEPLLQSNFSVGLILGALFFGLGYL AGCGLLDSFTEKMARNFHFEYGTARAWGSFGYAIGAFFAGIFFSISPHINFWLVSLFGAVF MMINMRFKDKDHQCVAADAGGVKKEDFIAVFKDRNFWVFVIFIVGTWSFYNIFDQQLFPVF YSGLFESHDVGTRLYGYLNSFQVVLEALCMAIIPFFVNRVGPKNALLIGVVIMALRILSCA LFVNPWIISLVKLLHAIEVPLCVISVFKYSVANFDKRLSSTIFLIGFQIASSLGIVLLSTP TGILFDHAGYQTVFFAISGIVCLMLLFGIFFLSKKREQIVMETPVPSAI-
[0113] In the present invention, all amino acid sequences that have a sequence identity of at least 70% with the sequence SEQ ID NO: 13 are considered functionally equivalent variants of the CSCB protein, that is, although they comprise a different sequence of amino acids, are capable of transporting sucrose together with the simultaneous importation of protons. An essay Determining whether a given transporter is a functionally equivalent variant of the CSCB transporter includes, but is not limited to, an assay for monitoring the transport reaction 2.A.1.5.3 for the given protein. Carrying out the monitoring of the transport reaction 2.A.1.5.3 by a carrier protein is routine practice for the person skilled in the art.
[0115] In another particular embodiment, the nucleotide sequence that encodes the CSCB protein comprises a nucleotide sequence that has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 14. In another particular embodiment, the nucleotide sequence encoding the CSCB protein comprises, or consists of, the nucleotide sequence SEQ ID NO: 14. However, as understood by the person skilled in the art, in many times the nucleotide sequence has to be optimized for its expression in a cellular system other than the one of origin. For this reason, in another particular embodiment, the gene that encodes the CSCB enzyme comprises, or consists of, a sequence of nucleotides, optimized for its correct expression in Synechococcus sp, in particular S. elongatus, more in particular, S. elongatus PCC7942 , which presents 100% sequence identity with the sequence SEQ ID NO: 15.
[0117] SEQ ID NO: 14 atggcactgaatattccattcagaaatgcgtactatcgttttgcatccagttactcattt ctcttttttatttcctggtcgctgtggtggtcgttatacgctatttggctgaaaggacat ctagggttgacagggacggaattaggtacactttattcggtcaaccagtttaccagcatt ctatttatgatgttctacggcatcgttcaggataaactcggtctgaagaaaccgctcatc tggtgtatgagtttcatcctggtcttgaccggaccgtttatgatttacgtttatgaaccg ttactgcaaagcaatttttctgtaggtctaattctgggggcgctattttttggcttgggg tatctggcgggatgcggtttgcttgatagcttcaccgaaaaaatggcgcgaaattttcat ttcgaatatggaacagcgcgcgcctggggatcttttggctatgctattggcgcgttcttt gccggcatattttttagtatcagtccccatatcaacttctggttggtctcgctatttggc gctgtatttatgatgatcaacatgcgttttaaagataaggatcaccagtgcgtagcggca gatgcgggaggggtaaaaaaagaggattttatcgcagttttcaaggatcgaaacttctgg gttttcgtcatatttattgtggggacgtggtctttctataacatttttgatcaacaactt tttcctgtcttttattcaggtttattcgaatcacacgatgtaggaacgcgcctgtatggt tatctcaactcattccaggtggtactcgaagcgctgtgcatggcgattattcctttcttt gtgaatcgggtagggccaaaaaatgcattacttatcggagttgtgattatggcgttgcgt atcctttcctgcgcgctgttcgttaacccctggattatttcattagtgaagttgttacat gccattgaggttccactttgtgtcatatccgtcttcaaatacagcgtggcaaactttgat aagcgcctgtcgtcgacgatctttctgattggttttcaaattgccagttcgcttgggatt gtgctgctttcaacgccgactgggatactctttgaccacgcaggctaccagacagttttc ttcgcaatttcgggtattgtctgcctgatgttgctatttggcattttcttcttgagtaaa aaacgcgagcaaatagttatggaaacgcctgtaccttcagcaatatag
[0119] SEQ ID NO: 15 atggcactgaatattccattccgaaatgcgtactatcgttttgcatccagttactcgtttc tcttttttatttcctggtcgctgtggtggtcgttgtacgctatttggctgaaaggacatct agggttgacagggacggaattaggtacactgtattcggtcaaccagtttaccagcattcta tttatgatgttctacggcatcgttcaggataaactcggtctgaagaaaccgctcatctggt gtatgagtttcatcctggtcttgaccggaccgtttatgatttacgtttatgaaccgttgct gcaaagcaatttttctgtgggtctaattctgggggcgctattttttggcttggggtatctg gcgggatgcggtttgcttgatagcttcaccgaaaaaatggcgcgaaattttcatttcgaat atggaacagcgcgcgcctggggatcttttggctatgctattggcgcgttctttgccggcat cttttttagtatcagtccccatatcaacttctggttggtgtcgctatttggcgctgtattt atgatgatcaacatgcgttttaaagataaggatcaccagtgcgtagcggcagatgcgggag gggtcaaaaaagaggattttatcgcagttttcaaggatcgaaacttctgggttttcgtcat ctttattgtggggacgtggtctttctataacatttttgatcaacaactgtttcctgtcttt tattcaggtttattcgaatcgcacgatgtaggaacgcgcctgtatggttatctcaactcat tccaggtggtcctcgaagcgctgtgcatggcgattattcctttctttgtgaatcgggtggg gccaaaaaatgcattacttatcggagttgtgattatggcgttgcgtatcctgtcctgcgcg ctgttcgttaacccctggattatttcattggtgaagttgttacatgccattgaggt tccac tttgtgtcatctccgtgttcaaatacagcgtggcaaactttgataagcgcctgtcgtccac gatctttctgattggttttcaaattgccagttcgcttgggattgtgctgctttcgacgccg actgggattctctttgaccacgcaggctaccagacagttttcttcgcaatttcgggtattg tctgcctgatgttgctatttggcattttcttcttgagtaaaaaacgcgagcaaatcgttat ggaaacgcctgtaccttcagcaatctag
[0121] In the present invention, the term "variant" means a polypeptide or protein that has the same activity as a reference polypeptide or protein, but that comprises an alteration in the amino acid sequence, that is, a substitution, insertion and / or deletion. , in one or more (eg, multiple) positions. A substitution means the replacement of the amino acid that occupies a position with a different amino acid; a deletion means the deletion of the amino acid that occupies a position; and an insert means to add an amino acid adjacent to and immediately after the amino acid occupying a position. The amino acid changes may be of a minor nature, that is, conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the polypeptide / protein; small deletions, typically 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing the net charge or other function, such as a polyhistidine tail, an antigenic epitope, or a binding domain. Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine, and histidine), amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.
[0123] Fragments of the proteins encoded by the nucleotide sequences of the invention are also contemplated in the context of the present invention, as long as said fragments can perform the function of the complete protein. Thus, the term "fragment" refers to a protein or polypeptide with one or more (eg, several) amino acids absent from the amino and / or carboxyl terminus of the protein or polypeptide; where the fragment has the activity of the entire protein. Assays to find out if a protein fragment has the same activity / function as the complete protein have been described in previous paragraphs for the case of protein variants.
[0125] In the present invention, "identity" or "sequence identity" is understood as the degree of similarity between two nucleotide or amino acid sequences obtained by optimal alignment of the two sequences. Depending on the number of common residues between the aligned sequences, a degree of identity expressed as a percentage will be obtained. The degree of identity between two amino acid sequences can be determined by conventional methods, for example, by standard sequence alignment algorithms known in the state of the art, such as BLAST. BLAST programs, eg, BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, are in the public domain on the website of The National Center for Biotechonology Information (NCBI).
[0127] A characteristic of the cyanobacterium of the invention is that the nucleotide sequences (i) to (v) mentioned above are overexpressed with respect to a non-recombinant or wild type cyanobacterium, and the nucleotide sequences (i), (ii), ( iii) and (v) are heterologous.
[0129] In the present invention, "heterologous" is understood to mean that nucleotide (or amino acid) sequence that derives or comes from a species other than the reference species. In the context of the present invention, the reference species is S. elongatus .
[0131] As indicated in previous paragraphs, the codons of the nucleotide sequences of the invention can be optimized for their expression in cyanobacteria, that is, the nucleotide sequence has been altered with respect to the original nucleotide sequence so that one or more codons of the nucleotide sequence have been changed to a different codon encoding the same amino acid, but which is used more frequently by the host cell (in the present invention, a cyanobacterium) than the original codon. The degeneracy of the genetic code causes all amino acids except methionine and tryptophan to be encoded by more than one condom. For example, arginine, leucine, and serine are encoded by 6 different codons, but many organisms use certain codons more frequently than others. Since the nucleotide sequences of the invention are heterologous to the host cell, it is possible to optimize the nucleotide sequence for its expression in cyanobacteria.
[0133] The nucleotide sequences of the invention can form part of one or more genetic constructs that will be introduced into a cyanobacterium to obtain the cyanobacterium of the invention. The introduction of the nucleotide sequences of the invention or of the gene construct (s) comprising said nucleotide sequences can be carried out by using a vector, for example, an expression vector. Therefore, the present invention also relates to a gene construct that comprises at least one nucleotide sequence of the invention. The gene construct may further comprise the elements necessary for the expression of the nucleotide sequence (s) of the invention. Such elements include, but are not limited to, promoters, transcription terminators, selection markers, origins of replication, expression enhancers ( enchancers), and ribosome binding sites (RBS). Alternative transcription terminators include the E. coli rrnB operon T1 and lamda phage T0, T7 coliphage TE, among others. RBS alternatives include those from the Anderson library, BBa_J61100 through BBa_J61139.
[0135] One or more of the nucleotide sequences of the invention can be operably linked with a promoter that can act in host cyanobacteria. Preferably, the promoter can act efficiently in cyanobacteria and bacteria, such as E. coli . Promoter selection can allow expression of a desired gene product under a variety of conditions. Promoters can be selected for optimal function in a cyanobacterium, such that the vector construct is inserted. Promoters can also be selected based on their regulatory characteristics. Examples of such characteristics include enhancement of transcription activity and inducibility.
[0137] The promoter can be an inducible promoter. For example, the promoter can be induced according to temperature, pH, a hormone, a metabolite (for example, lactose, mannitol, an amino acid), light (for example, specific wavelength), a heavy metal, or a antibiotic. Numerous standard inducible promoters are known to those of skill in the art.
[0139] The promoter can be a temperature inducible promoter. For example, the Lambda promoter is a temperature-inducible promoter that can act in cyanobacteria. In cyanobacteria, the Lambda promoter reaches its maximum activity at approximately 30 ° C to 35 ° C, an ideal growth temperature range for cyanobacteria and a range much less than optimal expression of the Lambda promoter in E. coli . Thus, the Lambda promoter provides effective expression of disaccharide biosynthetic activity in cyanobacteria.
[0141] Examples of promoters that can be inserted into the gene construct include, but are not limited to, promoters of the carB, nirA, psbAII, cpcB, cpt genes and their synthetic variants, dnaK and kaiA. In a particular embodiment, the promoter is the Ptrc promoter. The Ptrc promoter is a hybrid promoter of the lac and trp promoters, comprising the lacO operator sequence to which the LacI repressor binds. The repressor binds to the operator and with high specificity and inhibits expression until inducer such as allolactose or IPTG is added to the medium. The inducer binds to the repressor and the repressor cannot bind to the operator, allowing RNA polymerase to bind to the promoter and initiate transcription of the nucleotide sequence (s) controlled by the promoter.
[0143] Selection markers are widely known in the state of the art. These selection markers, or selection genes, can be antibiotic resistance genes, reporter genes, such as the gene encoding the beta-galactosidase of the lactose operon, etc. Genes that confer antibiotic resistance include, but are not limited to, ampicillin, tetracycline, kanamycin, hygromycin, gentamicin, etc. The markers allow the selection of successfully transformed cells that grow in a medium containing the corresponding antibiotic because they carry the appropriate resistance gene. In a particular embodiment of the present invention, the construction comprises the gentamicin resistance gene or the chloramphenicol resistance gene.
[0145] On the other hand, the nucleotide sequences of the invention can be grouped to be under the control of the same promoter, thus giving rise to operons. As explained above, in the present invention, "operon" is understood as the functional genetic unit formed by a group of genes capable of regulating their own expression by means of the substrates with which the proteins encoded by your genes. Thus, in a particular embodiment of the cyanobacterium of the invention, the overexpression of the nucleotide sequences (i), (ii) and (iii) of the invention is under the control of an "A" operon comprising a promoter functional in cyanobacteria, and / or the overexpression of the nucleotide sequences (iv), and (v) is under the control of a "B" operon comprising a promoter functional in cyanobacteria (promoter the same or different from the previous one). In a particular embodiment, the "A" operon comprises, or consists of, the nucleotide sequence SEQ ID NO: 19 and / or in another particular embodiment, the "B" operon comprises, or consists of, the nucleotide sequence SEQ ID NO: 20.
[0147] SEQ ID NO: 19:
[0148] aggagctccaccgatcaagcttttgacataagcctgttcggttcgtaaactgtaatgcaagtag cgtatgcgctcacgcaactggtccagaaccttgaccgaacgcagcggtggtaacggcgcagtgg cggttttcatggcttgttatgactgtttttttgtacagtctatgcctcgggcatccaagcagca agcgcgttacgccgtgggtcgatgtttgatgttatggagcagcaacgatgttacgcagcagcaa cgatgttacgcagcagggcagtcgccctaaaacaaagttaggtggctcaagtatgggcatcatt cgcacatgtaggctcggccctgaccaagtcaaatccatgcgggctgctcttgatcttttcggtc gtgagttcggagacgtagccacctactcccaacatcagccggactccgattacctcgggaactt gctccgtagtaagacattcatcgcgcttgctgccttcgaccaagaagcggttgttggcgctctc gcggcttacgttctgcccaggtttgagcagccgcgtagtgagatctatatctatgatctcgcag tctccggcgagcaccggaggcagggcattgccaccgcgctcatcaatctcctcaagcatgaggc caacgcgcttggtgcttatgtgatctacgtgcaagcagattacggtgacgatcccgcagtggct ctctatacaaagttgggcatacgggaagaagtgatgcactttgatatcgacccaagtaccgcca cctaacaattcgttcaagccgagatcggcttcccggccgcggagttgttcggtaaattgtcaca acgccgcggccggatccggtaccctgaaatgagctgttgacaattaatcatccggctcgtataa tgtgtggaattgtgagcggataacaatttcacactactagagtagtggaggttactaaatgaaa aacatcaatccaacgcagaccgctg cctggcaggcactacagaaacacttcgatgaaatgaaag acgttacgatcgccgatctttttgctaaagacggcgatcgcttttctaagttctccgcaacctt cgacgatcagatgctggtggattactccaaaaaccgcatcactgaagagacgctggcgaaatta caggatctggcgaaagagtgcgatctggcgggcgcgattaagtcgatgttctcgggcgagaaga tcaaccgcactgaaaaccgcgccgtgctgcacgtggcgctgcgtaaccgcagcaataccccgat tttggttgatggcaaagacgtcatgccggaagtcaacgcggtgctggagaagatgaaaaccttc tcagaagcgattatttccggtgagtggaaaggttataccggcaaagcaatcactgacgtagtga aaaccacctgaacatgcactttgtttctaacgtcgatgggactcacatcgcggaagtgctgaaa acatcgggatcggcggttcggacctcggcccatacatggtgaccgaagctctgcggccgtacaa aaagtgaacccggaaaccacgctgttcttggtagcatcgaaaaccttcaccactcaggaaacta tgaccaacgcccatagcgcgcgtgactggttcctgaaagcggcaggtgatgaaaaacacgttgc aaaacactttgcggcgctttccaccaatgccaaagccgttggcgagtttggtattgatactgcc aacatgttcgagttctgggactgggttggcggccggtactctttgtggtcagcgattggcctgt cgattgttctctccatcggctttgataacttcgttgaactgctttccggcgcacacgcgatgga caagcatttctccaccacgcctgccgagaaaaacctgcctgtcctgctggcgctgattggcatc tggtacaacaatttctttggtgcggaaactgaagcgattctgccgtatga ccagtatatgcacc gtttcgcggcgtacttccagcagggcaatatggagtccaacggtaagtatgttgaccgtaacgg taacgttgtggattaccagactggcccgattatctggggtgaaccaggcactaacggtcagcac gcgttctaccagctgatccaccagggaaccaaaatggtgccgtgcgatttcatcgctccggcta tcacccataacccgctctctgatcatcaccagaaactgctgtcgaacttcttcgcccagaccga agcgctggcgtttggtaaatcccgcgaagtggttgagcaggaatatcgcgatcagggtaaagat ccggcaacgcttgactacgtggtgccgttcaaagtattcgaaggtaaccgcccgaccaactcca tcctgctgcgtgaaatcactccgttcagcctgggtgcgttgattgcgctgtatgagcacaaaat ctttactcagggcgtgatcctgaacatcttcaccttcgaccagtggggcgtggaactgggtaaa cagctggcgaaccgtattctgccagagctgaaagatgataaagaaatcagcagccacgatagct cgaccaatggtctgattaaccgctataaagcgtggcgcggttaataaaatgagagtagtggagg ttactaaatggcaatccacaatcgcgcaggccaacctgcacaacagagtgatttgattaacgtc gcccaactgacggcgcaatattatgtgctgaaaccagaagcagggaatgcggagcacgcggtga aattcggtacttccggtcaccggggcagtgcagcgcgccacagctttaacgagccgcacattct ggcgatcgctcaggcaattgctgaagaacgcgcgaaaaacggcatcactggcccttgctatgtg ggtaaagatactcacgccctgtccgaacctgcattcatttccgttctggaagtgctggcagcga acggcgttgatgtcattgtgcaggaaaacaatggcttcaccccgacgcctgccgtttccaatgc catcctggttcacaataaaaaaggtggcccgctggcagacggtatcgtgattacaccgtcccat aacccgccggaagatggtggaatcaaatacaatccgccaaatggtggcccggctgataccaacg tcactaaagtggtggaggaccgggccaacgcactgctggccgatggcctgaaaggcgtgaagcg tatctccctcgacgaagcgatggcatccggtcatgtgaaagagcaggatctggtgcagccgttc gtggaaggtctggccgatatcgttg atatggccgcgattcagaaagcgggcctgacgctgggcg ttgatccgctgggcggttccggtatcgaatactggaagcgcattggcgagtattacaacctcaa cctgactatcgttaacgatcaggtcgatcaaaccttccgctttatgcaccttgataaagacggc gcgatccgtatggactgctcctccgagtgtgcgatggcgggcctgctggcactgcgtgataagt tcgatctggcgtttgctaacgacccggattatgaccgccacggtatcgtcactccggcaggttt gatgaatccgaaccactacctggcggtggcaatcaattacctgttccagcatcgtccgcagtgg ggcaaagatgttgccgtcggtaaaacgctggtttcatcggcgatgatcgaccgtgtggtcaacg acttgggccgtaaactggtagaagtcccggtaggtttcaaatggtttgtcgatggtctgttcga cggcagcttcggctttggcggcgaagagagtgcaggggcttccttcctgcgtttcgacggcacg ccgtggtccaccgacaaagacggcatcatcatgtgtctgctggcggcggaaatcaccgctgtca ccggtaagaacccgcaggaacactacaacgaactggcaaaacgctttggtgcgccgagctacaa ccgtttgcaggcagctgcgacttccgcacaaaaagcggcgctgtctaagctgtctccggaaatg gtgagcgccagcaccctggcaggtgacccgatcaccgcgcgcctgactgctgctccgggcaacg gtgcttctattggcggtctgaaagtgatgactgacaacggctggttcgccgcgcgtccgtcagg cacggaagatgcatataagatctactgcgaaagtttcctcggtgaagaacatcgcaagcagatt gagaaagaagcggttgagattgttagcgaagttctgaaaaacgcgtaata aaggtagagtagtg gaggttactaaatggctgccattaatacgaaagtcaaaaaagccgttatccccgttgcgggatt gggaacccggatgttgccggcgacgaaagccatcccgaaagagatgctgccactggtcgataag ccattgattcaatacgtcgtgaatgaatgtattgcggctggcattactgaaattgtgctggtta cacactcatcgaaaaactctattgaaaaccactttgataccagttttgaactggaagcaatgct ggaaaaacgcgtaaaacgtcaactgcttgatgaagtgcagtcgatttgtccaccgcacgtgact attatgcaagttcgccagggtctggcgaaaggcctgggacacgcggtgttgtgtgctcacccgg tcgtgggtgatgaaccggtggctgttattttgcctgatgttattctggatgaatatgaatccga tttgtcacaggataacctggcagagatgatccgccgctttgatgaaacgggtcatagccagatc atggttgaaccggttgctgatgtgaccgcatatggcgttgtggattgcaaaggcgttgaattag cgccgggtgaaagcgtgccgatggttggtgtggtcgaaaaaccgaaagcggatgttgcgccgtc taatctcgctattgtgggtcgttacgtactcagcgcggatatttggccgttgctggcaaaaacc cctccgggagctggtgatgaaattcagctcaccgacgcaattgatatgctgatcgaaaaagaaa cggtggaagcctatcatatgaaagggaagagccatgactgcggtaataaattaggttacatgca ggccttcgttgaatacggtattcgtcataacacccttggcacggaatttaaagcctggcttgaa gaagagatgggcattaagaagtaataaaggtccaggcatcaaataaaacgaaaggctcagtcga aagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactgg ctcaccttcgggtgggcctttctgcgtttatagtcgacctcgagggggggcccggtaccttctg
[0150] SEQ ID NO: 20:
[0151] GATCTGTTTTTGTTCCTGCAATGACCATTGCTGAGGAGTTCCCATGAAAATCAAGTCATCGATT TCTGTGCTCGCTGCGATATTTTCCTGCTTAACCGCTGAAAGTACGTCCATATAAATGCCTCTAT TAGTTAGCGCTATCGCGCGAAAAGAATGGTGATATAAGGGGCATCGCTGCCCCTTCAGCATCAG TTAAACGTATTTAAATGGCCACTAGTAGGTGGGGTACCCTGAAATGAGCTGTTGACAATTAATC ATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACTACTAGAGTAGTGGA GGTTACTAAATGGCAGCTCAAAatctctacattctgcacattcagacccatggtctgctgcgag ggcagaacttggaactggggcgagatgccgacaccggcgggcagaccaagtacgtcttagaact ggctcaagcccaagctaaatccccacaagtccaacaagtcgatatcatcacccgccaaatcacc gacccccgcgtcagtgttggttacagtcaggcgatcgaaccctttgcgcccaaaggtcggattg tccgtttgccttttggccccaaacgctacctccgtaaagagctgctttggccccatctctacac ctttgcggatgcaattctccaatatctggctcagcaaaagcgcaccccgacttggattcaggcc cactatgctgatgctggccaagtgggatcactgctgagtcgctggttgaatgtaccgctaattt tcacagggcattctctggggcggatcaagctaaaaaagctgttggagcaagactggccgcttga ggaaattgaagcgcaattcaatattcaacagcgaattgatgcggaggagatgacgctcactcat gctgactggattgtcgccagcactcagcaggaagtggaggagcaataccgcgtttacgatcgct acaacccagagcgcaagctggtcat tccaccgggtgtcgataccgatcgcttcaggtttcagcc cttgggcgatcgcggtgttgttctccaacaggaactgagccgctttctgcgcgacccagaaaaa cctcaaattctctgcctctgtcgccccgcacctcgcaaaaatgtaccggcgctggtgcgagcct ttggcgaacatccttggctgcgcaaaaaagccaaccttgtcttagtactgggcagccgccaaga catcaaccagatggatcgcggcagtcggcaggtgttccaagagattttccatctggtcgatcgc tacgacctctacggcagcgtcgcctatcccaaacagcatcaggctgatgatgtgccggagttct atcgcctagcggctcattccggcggggtattcgtcaatccggcgctgaccgaaccttttggttt gacaattttggaggcaggaagctgcggcgtgccggtggtggcaacccatgatggcggcccccag gaaattctcaaacactgtgatttcggcactttagttgatgtcagccgacccgctaatatcgcga ctgcactcgccaccctgctgagcgatcgcgatctttggcagtgctatcaccgcaatggcattga aaaagttcccgcccattacagctgggatcaacatgtcaataccctgtttgagcgcatggaaacg gtggctttgcctcgtcgtcgtgctgtcagtttcgtacggagtcgcaaacgcttgattgatgcca aacgccttgtcgttagtgacatcgacaacacactgttgggcgatcgtcaaggactcgaaaattt aatgacctatctcgatcagtatcgcgatcattttgcctttggaattgccacggggcgtcgccta gactctgcccaagaagtcttgaaagagtggggcgttccttcgccaaacttctgggtgacttccg tcggcagcgagattcactatggcaccgatgctgaaccggatatcagctgggaaaagcatatcaa tcgcaactggaatcctcagcgcattcgggcagtaatggcacaactaccctttcttgaactgcaa ccggaagaggatcaaacacccttcaaagtcagcttctttgtccgcgatcgccacgagactgtgc tgcgagaagtacggcaacatcttcgccgccatcgcctgcggctgaagtcaatctattcccatca ggagtttcttgacattctgccgctggctgcctcgaaaggggatgcgattcgccacctctcactc cgctggcggattcctcttgagaacattttggtggcaggcgattctggtaacgatgaggaaatgc tcaagggccataatctcggcgttgtagttggcaattactcaccggaattggagccactgcgcag ctacgagcgcgtctattttgctgag ggccactatgctaatggcattctggaagccttaaaacac tatcgcttttttgaggcgatcgcttaataaaatgagagtagtggaggttactaaatggcactga atattccattccgaaatgcgtactatcgttttgcatccagttactcgtttctcttttttatttc ctggtcgctgtggtggtcgttgtacgctatttggctgaaaggacatctagggttgacagggacg gaattaggtacactgtattcggtcaaccagtttaccagcattctatttatgatgttctacggca tcgttcaggataaactcggtctgaagaaaccgctcatctggtgtatgagtttcatcctggtctt gaccggaccgtttatgatttacgtttatgaaccgttgctgcaaagcaatttttctgtgggtcta attctgggggcgctattttttggcttggggtatctggcgggatgcggtttgcttgatagcttca ccgaaaaaatggcgcgaaattttcatttcgaatatggaacagcgcgcgcctggggatcttttgg ctatgctattggcgcgttctttgccggcatcttttttagtatcagtccccatatcaacttctgg ttggtgtcgctatttggcgctgtatttatgatgatcaacatgcgttttaaagataaggatcacc agtgcgtagcggcagatgcgggaggggtcaaaaaagaggattttatcgcagttttcaaggatcg aaacttctgggttttcgtcatctttattgtggggacgtggtctttctataacatttttgatcaa caactgtttcctgtcttttattcaggtttattcgaatcgcacgatgtaggaacgcgcctgtatg gttatctcaactcattccaggtggtcctcgaagcgctgtgcatggcgattattcctttctttgt gaatcgggtggggccaaaaaatgcattacttatcggagttgtgattatgg cgttgcgtatcctg tcctgcgcgctgttcgttaacccctggattatttcattggtgaagttgttacatgccattgagg ttccactttgtgtcatctccgtgttcaaatacagcgtggcaaactttgataagcgcctgtcgtc cacgatctttctgattggttttcaaattgccagttcgcttgggattgtgctgctttcgacgccg actgggattctctttgaccacgcaggctaccagacagttttcttcgcaatttcgggtattgtct gcctgatgttgctatttggcattttcttcttgagtaaaaaacgcgagcaaatcgttatggaaac gcctgtaccttcagcaatctagtaaaggtcgttgacaccatcgaatggtgcaaaacctttcgcg gtatggcatgatagcgcccggaagagagtcaattcagggtggtgaatatgaaaccagtgacgtt gtacgatgtcgcagagtatgccggtgtctcgtatcagaccgtttcccgcgtggtgaaccaggcc agccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattc ccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccag tctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatcgcgcgccgatcaactgggt gccagcgtggtggtgtcgatggtggaacgaagcggcgtcgaagcctgtaaagcggcggtgcaca atcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccat tgctgtggaagctgcctgcactaatgttccggcgttgtttcttgatgtctcggaccagacaccc atcaacagtattattttctcccatgaggacggtacgcgactgggcgtggagcatctggtcgcat tgggtcaccagcaaatcgcgctgttggcgggcccattaagttctgtctcggcgcgcctgcgtct ggctggctggcataaatatctcactcgcaatcaaattcagccgatcgcggaacgggaaggcgac tggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactg cgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggct gcgcgttggtgcggatatctcggtagtgggatacgacgataccgaagatagctcatgttatatc ccgccgttaaccaccatcaaacagg attttcgcctgctggggcaaaccagcgtggaccgcttgc tgcaactctcgcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaacg aaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcgttaatg cagctggcacgacaggtttcccgactggaaagcgggcagtgataaaggtccaggcatcaaataa aacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctct ctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatagtcgacctcgagct agcaagcttagatcgatccgtcacacgggataataccgcgccacatagcagaactttaaaagtg ctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatcca gttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttc tgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgt tgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatga gcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccg aaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgt atcacgaggccctttcgtcttcgaataaatacctgtgacggaagatcacttcgcagaataaata aatcctggtgtccctgttgataccgggaagccctgggccaacttttggcgaaaatgagacgttg atcggcacgtaagaggttccaactttcaccataatgaaataagatcacta ccgggcgtattttt tgagttatcgagattttcaggagctaaggaagctaaaatggagaaaaaaatcactggatatacc accgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaat gtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataa gcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattc cgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttt tccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtt tctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaaggg tttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaa acgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaagg cgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtttgtgatggcttccatgtc ggcagaatgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgtaattttttt aaggcagttattggtgcccttaaacgcctggtgctacgcctgaataagtgataataagcggatg aatggcagaaattcgaaagcaaattcgacccggtcgtcggttcagggcagggtcgttaaatagc cgcttatgtctattgctggtttaccggtttattgactaccggaagcagtgtgaccgtgtgcttc tcaaatgcctgaggccagtttgctcaggctctccccgtggaggtaataattgacgatatgatcg ACGT
[0153] The operons can be comprised within a gene construct such as that described in the previous paragraphs. In the present invention, an operon is understood to be the genetic construct, preferably a DNA construct, where the genetic material of interest is introduced to be expressed in the cell. The synthetic operon comprises all the elements necessary for the expression of the nucleotide sequences of the genes of interest included in it. The elements comprised within the genetic construct are arranged in the correct reading frame in such a way that the expression of the coding sequence (s) takes place under conditions compatible with the other elements or sequences present in the construct. In the present invention, the nucleotide sequences of the genes comprised in the synthetic operon are preferably arranged in tandem.
[0155] In the present invention, "inducible operon" is understood to mean the operon that is not expressed under normal conditions, but is activated in response to an inducing agent that functions as an activator. The moment the inducer binds to the operator, the promoter is activated and transcription of the structural genes begins. In the particular case of the lactose operon, any lactose analog can be used as an inducer. Examples of inducers of the lactose operon include, but are not limited to, IPTG (isopropyl-pD-thiogalactoside), phenyl-Gal (phenyl-pD-galactose), ONPG (ortho-nitrophenyl-pD-galactopyranoside) and X-gal (5-bromo -4-chloro-3-indolyl-pD-galactoside). In a particular embodiment, the inducer of the lactose operon is IPTG.
[0157] In a particular embodiment, the "B" operon comprises a nucleotide sequence encoding a protein of a metabolite-inducible operon. Hereinafter, this nucleotide sequence is referred to as the nucleotide sequence (vi) of the invention. The nucleotide sequence (vi) of the invention may be heterologous and / or overexpressed relative to a "stem" or wild type cell. Examples of metabolite-inducible operons include, but are not limited to, the lactose operon, the maltose operon, and the arabinose operon In a particular embodiment, the metabolite-inducible operon is the lactose operon, therefore the repressor protein is LacI.
[0159] Thus, in a more particular embodiment, the repressor protein of the metabolite-inducible operon is the LacI protein from E.coli. In another particular embodiment, the LacI protein comprises an amino acid sequence that has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 16. In another particular embodiment, the LacI protein comprises, or consists of, an amino acid sequence that presents 100% sequence identity with the sequence SEQ ID NO: 16.
[0161] SEQ ID NO: 16:
[0162] MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLI GVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPL DDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSV SARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRA ITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLP VSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ-
[0164] In another particular embodiment, the nucleotide sequence that encodes the LacI protein comprises a nucleotide sequence that has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 17. In another particular embodiment, the nucleotide sequence encoding the LacI protein comprises, or consists of, the nucleotide sequence SEQ ID NO: 17.
[0166] SEQ ID NO: 17: gtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtt tcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcg gcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacag tcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtc gcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaa cgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagt gggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcctgc actaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattatt ttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccag caaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtctggctggc tggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactgg agtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccact gcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtcc gggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaagacagctca tgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaaccagc gtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgccc gtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgc gcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcag tga
[0168] In another particular embodiment, the gene encoding the LacI protein comprises, or consists of, a nucleotide sequence, optimized for its correct expression in Synechococcus sp, in particular S. elongatus, more in particular S. elongatus PCC7942, which has a 100% sequence identity SEQ ID NO: 18.
[0170] SEQ ID NO: 18: atgaaaccagtgacgttgtacgatgtcgcagagtatgccggtgtctcgtatcagaccgtttccc gcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggc ggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgatt ggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatcgc gcgccgatcaactgggtgccagcgtggtggtgtcgatggtggaacgaagcggcgtcgaagcctg taaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctg gatgaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttgtttcttgatg tctcggaccagacacccatcaacagtattattttctcccatgaggacggtacgcgactgggcgt ggagcatctggtcgcattgggtcaccagcaaatcgcgctgttggcgggcccattaagttctgtc tcggcgcgcctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatcg cggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatga gggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgcc attaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaag atagctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaac cagcgtggaccgcttgctgcaactctcgcagggccaggcggtgaagggcaatcagctgttgccc gtctcactggtgaaacgaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgt tggccgattcgttaatcgaggcaggcagg
[0172] Thus, as indicated above, in a particular embodiment, the nucleotide sequence of Operon "B" comprises the nucleotide sequence SEQ ID NO: 20. In a particular embodiment, the recombinant cyanobacterium of the invention comprises the nucleotide sequence SEQ ID NO: 19 and / or the nucleotide sequence SEQ ID NO: 20.
[0174] The nucleotide sequence of the invention, or the gene constructs comprising the nucleotide sequences of the invention, can for example be inserted into a vector or plasmid to carry out their insertion into the genome of the cyanobacteria.
[0176] In the present description, the term "vector" refers to a nucleic acid molecule that can be used to transport or transfer a nucleotide sequence into a cell. A vector can contain different functional elements including, but not limited to , transcription control elements, such as promoters or operators, regions or enhancers for binding to transcription factors, and control elements for initiating and terminating transcription. Vectors include, but are not limited to: plasmids, cosmids, viruses , phages, recombinant expression cassettes, and transposons. Some vectors are capable of autonomously replicating or dividing after being introduced into the host cell, such as bacterial vectors with a bacterial origin of replication. Other vectors can integrate into the genome of the host cell and thus replicate along with the cellular genome.
[0178] In general, an expression vector comprises, in addition to at least 1, 2, 3, 4, 5 or 6 of the nucleotide sequences of the invention, a promoter that directs its transcription (for example, pT7, plac, pire, ptac , pBAD, laugh, etc.), to which it is operatively linked, and other necessary or appropriate sequences that control and regulate said transcription and, where appropriate, the translation of the product of interest, for example, transcription initiation and termination signals ( tlt2, etc.), polyadenylation signal, origin of replication, ribosome binding sequences (RBS), transcriptional regulator coding sequences, ( enhancers), transcriptional silencers ( silencers), repressors, etc. Examples of appropriate expression vectors can be selected according to the conditions and needs of each specific case among expression plasmids, viral vectors (DNA or RNA), cosmids, artificial chromosomes, etc. which may also contain markers that can be used to select cells transfected or transformed with the nucleotide sequences of the invention. The choice of vector will depend on the host cell and the type of use to be made. In a particular embodiment of the invention, the vector used is a "suicide" vector, that is, it does not replicate in cyanobacteria, but rather the entire construction comprising the nucleotide sequences of the invention is inserted into the cyanobacterial chromosome. Examples of vectors of this type are shown in Example 1 of the present description.
[0180] Obtaining said vector can be carried out by conventional methods known to those skilled in the art, as well as for the transformation of cyanobacteria, different widely known methods can be used - chemical transformation, liposomes, transfection, electroporation, particle bombardment, gene bullets ( "gene gun"), microinjection, etc. - described in various manuals widely known to those skilled in the art.
[0182] The nucleotide sequences (i) to (iv) of the invention can be inserted into any part of the genome of the cyanobacteria as long as said insertion does not interrupt the expression of a gene essential for the viability of the cyanobacterium. Thus, the preferred insertion sites in the cyanobacterial genome are the Neutral Site I (NSI), Neutral Site II (NSII) and / or Neutral Site III (NSIII) sites.
[0184] In a particular embodiment of the cyanobacterium of the invention, the "A" operon is inserted into the NSI of the cyanobacterium genome and the "B" operon is inserted into the NSII of the cyanobacterium genome. As understood by the person skilled in the art, other alternatives are also possible in the context of the present invention, such as the "B" operon being inserted in the NSI of the cyanobacteria genome and the "A" operon being inserted in the Neutral Site. II (NSII) of the cyanobacteria genome. The characteristics of the "A" operon and the "B" operon have been described in previous paragraphs.
[0186] In any case, it is essential that the co-expression of the nucleotide sequences of the invention (i) to (v) occurs within the cyanobacteria to obtain a Sucrose overproducing recombinant cyanobacteria in the absence of osmotic stress. As understood by the person skilled in the art, this can be achieved by using the same metabolite-inducible promoter in the expression of the nucleotide sequences of the invention.
[0188] In another aspect, the present invention relates to a composition, hereinafter "composition of the invention", comprising the cyanobacterium of the invention, including alone or in combination all the particular embodiments described in previous paragraphs. Additionally, the composition of the invention (see Example 1) can comprise other microorganisms in addition to the cyanobacteria of the invention, such as heterotrophic microorganisms and / or non-halophytic microorganisms. Therefore, in another aspect, the present invention relates to a composition comprising a synthetic bacterial consortium, also called "composition of the invention", comprised of the cyanobacterium of the invention as defined throughout the present description, and at least one heterotrophic microorganism and / or one non-halophilic microorganism. In a particular embodiment, these heterotrophic microorganisms are capable of consuming the sucrose produced by the cyanobacterium of the invention either to grow and multiply, and / or to produce products derived from the assimilation and metabolization of said sucrose. In Example 3 of the present description a consortium of microorganisms is shown in which heterotrophic bacteria grow from the sucrose produced by the cyanobacterium of the invention. Within the present invention, the production of metabolites or products of industrial interest, such as biofuels and bioplastics, by cultivating a consortium of microorganisms as described in the previous paragraph, where the heterotrophic bacteria comprise, for example, and without limiting to, a biofuel producing microorganism (eg, isobutanol) and / or an alcohol producing microorganism.
[0190] Therefore, in another aspect, the present invention relates to the use of the composition of the invention for the production of metabolites with high added value or products of industrial interest, where the composition comprises a phosphate concentration greater than 0 , 2, 0.5, 1, 1.5 or 2 mM. In another particular embodiment, said high added value metabolites or products of industrial interest are butanol or Polyhydroxyalkanoates.
[0191] In another aspect, the invention relates to the use of the cyanobacterium of the invention for the production of sucrose in the absence of osmotic stress, hereinafter, use of the invention (Example 2). In the present invention, it is considered that there is an absence of osmotic stress when the concentration of salts in the culture medium is less than 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 0.5 mM. Thus, in one aspect, the present invention relates to the use of the cyanobacteria of the invention or the composition of the invention, for the production of sucrose in a salt concentration lower than 150, 100, 90, 80, 70, 60 , 50, 40, 30, 20, 10 or 0.5 mM. In a particular embodiment of the use of the invention, the salt is sodium chloride.
[0193] In another particular embodiment, the production of sucrose is carried out in a batch culture or continuous culture bioreactor.
[0195] In another aspect, the present invention relates to a method for producing sucrose in the absence of osmotic stress, hereinafter "method for producing sucrose of the invention", which comprises cultivating the recombinant cyanobacterium of the invention, or the composition of the invention, in a salt concentration lower than 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 0.5 mM.
[0197] In case the promoter that regulates the nucleotide sequences of the invention in the recombinant bacterium of the invention is a metabolite-inducible promoter, the method of the invention comprises (i) cultivating the recombinant cyanobacteria of the invention in a concentration of salts less than 150 mM until the cyanobacteria exceeds the growth phase, which usually occurs when the optical density is 1, and (ii) adding the appropriate inducer to the promoter that regulates the nucleotide sequences of the invention to promote the expression of the nucleotides of the invention. Additionally, the method of the invention can comprise a step that comprises the centrifugation of the culture broth to eliminate the cyanobacteria and isolate the supernatant comprising sucrose, which can be used as a source of carbon and energy by heterotrophic microorganisms, in particular , heterotrophic microorganisms that produce metabolites or products of industrial interest or of high added value in industry.
[0199] In a particular embodiment of the method for producing sucrose of the invention, the concentration of salts in the culture is less than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 0.5 mM.
[0201] In another particular embodiment of the method for producing sucrose of the invention, the culture of the cyanobacteria of the invention is carried out for at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days .
[0203] In another aspect, the present invention relates to a method for producing products of industrial interest from sucrose comprising:
[0204] (i) cultivating the recombinant cyanobacterium of the invention in a salt concentration lower than 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 0.5 mM in combination with a producing heterotrophic bacterium of the product of industrial interest that uses sucrose as a carbon source, where the culture medium comprises a phosphate concentration greater than 0.2 mM, and
[0205] (ii) obtain the product of industrial interest from the supernatant resulting from the culture of step (i).
[0207] The culture conditions of a cyanobacteria, such as nutrients, pH, temperature, etc., necessary for its growth, are widely known to those skilled in the art. In a particular embodiment, the culture medium comprises a phosphate concentration greater than 0.5, 1, 1.5 or 2 mM.
[0209] In the state of the art there are a great variety of products of industrial interest that can be produced by heterotrophic microorganisms. Examples of metabolites of industrial interest include, but are not limited to, enzymes, antibiotics, biofuel amino acids (such as biobutanol, bioethanol, propanol, etc.), organic acids, bioplastic, biomass, and Polyhydroxyalkanoates.
[0211] Examples of heterotrophic microorganisms that produce products of industrial interest, and that can be cultivated in consortium with the cyanobacteria of the invention, include, without limitation, Pseudomonas sp. (eg, Pseudomonas putida), Bacillus sp. (eg Bacillus subtilis, Bacillus thuringiensis ), Salmonella sp. (for example, Salmonella tiphymurium), Bortedella sp. (eg Bortedella pertusis), Clostridium sp. (eg Clostridium tetani), Corynebacterium sp. (eg Corynebacterium diphteriae), Propionebacterium sp., Penicillium sp. (eg, Penicillium chrysogenum), Streptomyces sp. (for example, Streptomyces chrestomyceticus), Rhizopus sp. (for example, Rhizopus nigricans), Curvularia sp. (for example Curvularia lunata), Aspergillus sp. ( Aspergillus ochraceus), E. coli, Saccharomyces sp. ( Saccharomyces cervisiae), Streptococcus sp. (eg Streptococcus lactis), Lactobacillus sp. (eg Lactobacillus bulgaricus), Brevibacterium sp., Aspergillus sp. (eg Aspergillus niger), Clostridium sp. (eg Clostridium acetobutilicum), Thiobacillus sp. (eg Thiobacillus thioxidans), Trichoderma sp. (eg, Trichoderma reseei) and Xanthomonas sp. (for example, Xanthomonas campestris).
[0213] Likewise, the heterotrophic microorganism that produces the product of industrial interest that is going to form a consortium with the cyanobacterium of the invention can be genetically modified so that it is capable of using sucrose as an energy source. This modification can be carried out using genetic engineering tools widely known to those skilled in the art, and the genetic modification can comprise the inclusion in the heterotrophic microorganism of the cscA and / or cscB genes that respectively encode sucrose hydrolase and the transporter of saccharose. These heterologous genes could be introduced into heterotrophic macro-organisms that do not naturally use sucrose as a carbon and energy source, both in replicative plasmids and integrated into the genome.
[0215] Alternatively, in the context of the present invention, the possibility of genetically modifying the heterotrophic microorganism that is naturally capable of using sucrose as a carbon source is also contemplated, so that it is a producer of a product of industrial interest such as those described in previous paragraphs. .
[0217] Within the context of the present invention, the combination of all the particular embodiments of the different inventive aspects described in this description is contemplated. Also, throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
[0218] BRIEF DESCRIPTION OF THE FIGURES
[0220] Figure 1. Diagram of the metabolic pathway of sucrose production in Synechococcus elongatus PCC7942 indicating, in black boxes, the genes to be overexpressed in the present invention.
[0222] Figure 2 . Graphic representation of the result of the integration of the M1 and M2 operons in the chromosome of Synechococcus elongatus PCC 7942.
[0224] Figure 3 . Growth of a recombinant cyanobacteria using air enriched in CO ²
[0226] Figure 4 . Sucrose production coupled to the growth of a recombinant cyanobacterial strain using air enriched in CO ²
[0228] Figure 5 . Average accumulated biomass Dcwt + vcwt- i by the strain of the invention (gray
[0229] dark) and a reference recombinant cyanobacteria strain [Ducat et al. (2012) Appl Environ Microbiol 78 (8): 2660-2668] (light gray).
[0231] Figure 6 . Daily sucrose production using a recombinant cyanobacterial strain developed in the present invention (dark gray) and a reference recombinant cyanobacterial strain (light gray)
[0233] Figure 7 . Growth of E. coli W in culture broths of a recombinant cyanobacteria under induction (+ IPTG) and non-induction (-IPTG) conditions.
[0235] EXAMPLES
[0237] The invention will now be illustrated by means of tests carried out by the inventors, which show the effectiveness of the product of the invention.
[0239] EXAMPLE 1
[0240] CONSTRUCTION OF A RECOMBINANT STRAIN OF CYANOBACTERIA OVER EXPRESSING THE GENES pgi, pgmt, galU, sps, cscB and lacl
[0241] 1. MATERIALS AND METHODS
[0243] 1.1. Strain
[0244] The parental strain is Synechococcus elongatus PCC7942 (PCC or Pasteur Culture collection of Cyanobacteria).
[0246] 1.2. Overexpressed genes
[0247] The following heterologous genes have been expressed in the recombinant strain of Synechococcus elongatus:
[0248] • pgi Glucose-6-phosphate isomerase (SEQ ID NO: 3)
[0249] • pgmT Phosphoglucomutase (SEQ ID NO: 6)
[0250] • galU Mannose-1-phosphate guanylyltransferase (SEQ ID NO: 9)
[0251] • cscB Sucrose permease (SEQ ID NO: 15)
[0252] • lacI Repressor LacI (SEQ ID NO: 18)
[0254] Likewise, the following homologous gene has been overexpressed:
[0255] • sps Sucrose phosphate synthase (SEQ ID NO: 12)
[0257] 1.3. Molecular Biology Techniques
[0259] 1.3.1. DNA assembly techniques
[0260] Modular Cloning (MoClo) is a DNA assembly method based on the Golden Gate system (Engler et al, 2014, ACS Synth Biol, 3 (11): 839-43) that is based on the use of type II restriction enzymes -S, like BsaI and / or BbsI, which recognize a specific DNA site but cut at a different sequence. Therefore, it is possible to use a single enzyme, in a single reaction, to generate a variety of fusion sites at the same time. The DNA fragments generated by digestion, flanked by BsaI sites, are assembled in a receptor vector, interrupting the lacZ gene, allowing the selection by color of the positive clones (white color). The protocol performs several combined cycles of digestion and ligation, so that the fragments generated by digestion are joined by ligation into a receptor vector in the same reaction, and in a specific order defined by the sequence of the fusion sites. This method can be used to generate transcriptional units that, in turn, can be combined to generate different genetic circuits using the same protocol and changing the restriction enzyme (BbsI, in this case). In this way, the enzymes BsaI and BbsI they alternate each round of assembly. This method allows assembling DNA fragments in a short time (less than 3 hours) using a very simple protocol, the product of which can be directly transformed into cloning strains.
[0262] In this example, the CIDAR MoClo library (Iverson et al., 2016, ACS Synth Biol. 5 (1): 99-103) has been used as a source of DNA parts (Promoters, ribosome binding sites, coding sequences, terminators, receptor vectors, etc.). The MoClo system has been used in this example for the construction of a genetic circuit that contains the different overexpressed genes.
[0264] 1.3.2. Synechococcus elongatus genetic manipulation
[0265] Synechococcus elongatus PCC7942 is naturally transformed with plasmids whose genes of interest are flanked by sequences of homology to certain neutral sites of its genome, allowing a double recombination that can be selected by resistance to antibiotics.
[0267] 1.4. Culture media
[0268] The different strains of S. elongatus have been grown in a modified version of the BG11 culture medium (Stanier et al. 1971. Bacteriol Rev. 35 (2): 171-205), called BG11-HP with a higher concentration of phosphate ( 2 mM K ² HPO ⁴ ) and with 10 mM HEPES buffer previously adjusted to a pH of 7.8 with NaOH. In this way, the composition of the BG11-HP medium is as follows:
[0269] • 1.5 g NaNO ³ / L,
[0270] • 0.348 g K ² HPO ⁴ / L,
[0271] • 0.075 g MgSO ⁴ - ⁷ H ² O / L,
[0272] • 0.036 g CaCl ² - ² H ² O / L,
[0273] • 6.56 mg citric acid monohydrate / L,
[0274] • ⁶ mg ferric ammonium citrate / L,
[0275] • 1.04 mg Na ² ED TA ² H ² O / L,
[0276] • ^{0, 0 2} g Na ² CO ³ / L,
[0277] • 2.86 mg H ³ BO ³ / L,
[0278] • 1.81 mg MnCl ² - ⁴ H ² O / L,
[0279] • ^{0.2 2 2} mg ZnSO ⁴ ' ⁷ H ² O / L,
[0280] • 0.39 mg Na ² MoO ⁴ - ² H ² O / L,
[0281] • 0.079 mg CuSO ⁴ - ⁵ H ² O / L,
[0282] • 0.022 mg CoCh / L,
[0283] • HEPES buffer 10 mM pH 7.8 / NaOH.
[0285] 1.5. Growing conditions
[0286] The Synechococcus elongatus strains have been grown in 500 mL Erlenmeyer flasks with 50 mL of BG11-HP medium. The growth conditions are: 30 ° C in an orbital incubator at 200 rpm with an illumination of approximately 6000 lux.
[0288] 2. RESULTS
[0290] 2.1. Identification of genes to overexpress
[0291] An in silico analysis was performed using the S. elongatus metabolic model and the (GDLS) algorithm to optimize growth coupled sucrose production. The results indicated that overexpression of the galU, sps, pgi pgmt genes may be necessary for the overproduction of sucrose in the absence of salt stress. In addition, the cscB gene is necessary for the secretion of sucrose to the exterior of the cell as previously demonstrated in [Ducat et al. (2012) Appl Environ Microbiol 78 (8): 2660-2668]. A diagram of the sucrose production pathway in Synechococcus elongatus PCC7942 and of the genes to be overexpressed is represented in Figure 1.
[0293] 2.2. Construction of synthetic operons
[0294] The sequences of the genes to be overexpressed have been selected from the Eschenchia coli genome (SEQ ID NO: 2, 5, 8, 14), except for the sps gene, which has been selected from Synechococcus elongatus PCC7942 (SEQ ID NO: 11). The sequences of all the genes to be overexpressed have been edited to optimize their expression in Synechococcus and to avoid the cleavage sites of the Bsal and Bbsl enzymes (SEQ ID NO: 3, 6, 9, 12, 15)
[0296] For overexpression of genes, they have been organized into two different operons, both controlled by the Ptrc promoter and preceded by a ribosome binding site commonly used in cyanobacteria.
[0298] To control the activity of the Ptrc promoter by adding the inducer IPTG, the lacI gene (SEQ ID NO: 17) has been overexpressed, also optimized for its expression in Synechococcus (SEQ ID NO: 18).
[0299] The construction of the operons has been carried out using the MoClo method of DNA assembly, detailed in the Materials and Methods section.
[0301] 2.2.1. Construction of the operon Ptrc, pgi, pgmt, galU-T1T7 (M1)
[0302] The individual parts were assembled into the DVA_AE vector from the CIDAR kit (Addgene, 490 Arsenal Way, Suite 100, Watertown, MA 02472, USA). The resulting construct has been named M1-DVA. The plasmid M1-DVA was then digested with the enzymes BamHI and XhoI and the resulting operon was ligated into the vector pMSM230, which is integrated into the neutral site 1 (NSI) of the Synechococcus elongatus PCC7942 chromosome. This new vector has been named pMSM230-M1. The nucleotide sequence integrated into neutral site 1 is SEQ ID NO: 19
[0304] 2.2.2. Construction of the operon Ptrc-sps-cscB, PlacIq-lacI-T1T7 (M2)
[0305] The individual parts were assembled in the vector DVA_AE from the CIDAR kit (Addgene). The resulting construct has been named M2-DVA. The plasmid M2-DVA was then digested with the enzymes KpnI and XhoI and the resulting operon was ligated into the vector pMSM249, which is integrated into the neutral site 2 (NSII) of the Synechococcus elongatus PCC7942 chromosome. This new vector has been named pMSM249-M2. The nucleotide sequence integrated into neutral site 2 is SEQ ID NO: 20
[0307] 2.3. Integration of synthetic operons into the chromosome of Synechococcus elongatus
[0308] Following the manipulation strategy described in Materials and Methods, the genomic integration of the M1 ( Ptrc-pgi-pgmt-galü) and M2 ( Ptrcsps-cscB-Plaqlq-lacl) operons has been carried out, both individually and in combination.
[0310] First, Synechococcus elongatus PCC7942 was transformed with the plasmid pMSM230-M1, selecting the recombinant clones for resistance to gentamicin. Said strain was transformed with the plasmid pMSM249-M2, selecting the recombinant clones for resistance to chloramphenicol and gentamicin.
[0312] Figure 2 shows a graphic representation of the result of the integration of the M1 and M2 operons in the Synechococcus elongatus PCC7942 chromosome.
[0314] EXAMPLE 2
[0315] PROCESS FOR THE PRODUCTION OF GROWTH-COUPLED SACROSE FROM CO ² USING A RECOMBINANT STRAIN OF CYANOBACTERIA
[0317] 1. MATERIALS AND METHODS
[0319] 1.1. Strain
[0320] The strain used is the recombinant cyanobacterium detailed in Example 1
[0322] 1.2. Culture media
[0323] The culture medium is detailed in Example 1.
[0324] For the induction of overexpressed genes, the medium is supplemented with 1 mM IPTG.
[0326] 1.3. Growing conditions
[0327] Standard growth conditions are those detailed in Example 1.
[0328] For the production of sucrose, 500 mL kitasate-type flasks have been used with 50 mL of BG11-HP medium supplemented with 1 mM IPTG through which a stream of air previously bubbled in carbonate buffer (K ² CO ³ 2M KHCO ³ , in a 1: 1 ratio). The sucrose production conditions are: 30 ° C in an orbital incubator at 200 rpm with an illumination of approximately 6000 lux.
[0330] 1.4. Determination of growth
[0331] Samples were taken during growth for analysis of optical density at 720 nm (OD720). Said value of OD720 was transformed into data of grams of dry weight per liter (gDCW / L) using the following relationship:
[0332] gDCW / L = OD ⁷²⁰ x 0.3342
[0334] 1.5. Sucrose analysis
[0335] Samples were taken during growth for analysis of sucrose concentration. For this, the samples were centrifuged at 12000g for 1 minute and the supernatant was collected.
[0337] The analysis of the sucrose concentration in the supernatant was carried out using the sucrose / D-glucose kit from the company Megazyme. (https://www.megazyme.com/sucrose-fructose-d-glucose-assay-kit).
[0339] 2. RESULTS
[0340] The culture of the recombinant cyanobacteria was maintained under the growth conditions defined for the production of sucrose for 12 days. In the growth analysis (Figure 3) a constant linear growth is observed during the first 10 days.
[0342] Sucrose production shows an evolution clearly associated with the growth of the recombinant cyanobacteria (Figures 3-4) until at least day 12. The maximum sucrose production is 4.22 g / L.
[0344] These results constitute the first and best test of sucrose production using recombinant cyanobacteria in the absence of high salt concentrations in the medium.
[0346] Comparing the sucrose production of the recombinant cyanobacterium of the present invention with the best results obtained to date ([Ducat et al. (2012) Appl Environ Microbiol 78 (8): 2660 - 2668]), it is observed how the cyanobacterium of The present invention presents a growth and a higher production of sucrose, maintained for a longer time, and using a medium without high salt concentration (Figures 5-6).
[0348] EXAMPLE 3
[0349] CULTURE OF NON-HALOPHITE BACTERIAL STRAINS IN GROWTH BROTHS OF A RECOMBINANT CYANOBACTERIA STRAIN
[0351] 1. MATERIALS AND METHODS
[0353] 1.1. Strains
[0354] The sucrose-producing strain is the recombinant cyanobacterium of the present invention detailed in Example 1, producing sucrose following Example 2.
[0356] The non-halophytic bacterial strain is Escheríchia coli W (American Type Culture Collection Access number ATCC 9637).
[0357] 1.2. Culture media
[0358] The culture media are those detailed in Examples 1 and 2.
[0360] 1.3. Growing conditions
[0361] The growth conditions of the recombinant cyanobacteria are those detailed in Examples 1 and 2.
[0363] For the growth of E. coli W, the media has been supplemented with 19 mM NH ⁴ CL
[0365] 1.4. Escherichia coli W growth analysis
[0366] For the analysis of the growth of E. coli W, the optical density at 600 nm (OD600) was determined.
[0368] 2. RESULTS
[0369] Escherichia coli W has been grown in culture broths of S. elongatus, both IPTG-induced and uninduced.
[0371] For this, S. elongatus was grown for ⁶ days in a culture induced with IPTG and in another culture without inducing. In the induced culture 2 g / L of sucrose was produced (Example 2). Both growth broth culture of the bacteria was removed by centrifugation and subsequent filtration with a membrane of ^{0, 2 2} mm.
[0373] The culture broths, thus obtained and purified, were supplemented with 19 mM NH ⁴ Cl and used as culture medium for the growth of E. coli W. 20 mL of the growth broths of S. elongatus (induced and without induce) were inoculated with E. coli W at an initial OD600 of 0.1. After 24 hours of culture at 37 ° C with orbital shaking at 170 rpm, the growth of E. coli W was analyzed by measuring its OD600. As a control, BG11-HP medium supplemented with 2 g / L sucrose was used.
[0375] As seen in Figure 7, significant growth of E. coli W occurs using IPTG-induced Synechococcus culture supernatant. This growth is comparable to that achieved by E. coli W in fresh medium using 2 g / L of exogenous sucrose. The result confirms that the sucrose produced by S. elongatus is sufficient to support the growth of a non-halophytic strain.

权利要求:
Claims (33)
[1]
1. A recombinant cyanobacterium comprising the following nucleotide sequences:
(i) the nucleotide sequence encoding the enzyme glucose-6-phosphate isomerase (PGI) or a fragment thereof,
(ii) the nucleotide sequence encoding the enzyme phosphoglucomutase (PGMT) or a fragment thereof,
(iii) the nucleotide sequence encoding the enzyme UTP-glucose-1-phosphate uridylyltransferase (GalU), or a fragment thereof,
(iv) the nucleotide sequence encoding the enzyme sucrose phosphate synthase (SPS), or a fragment thereof;
(v) the nucleotide sequence encoding sucrose permease (CSCB), or a fragment thereof, and optionally,
(vi) the nucleotide sequence encoding the repressor protein of a metabolite-inducible operon,
where:
- the nucleotide sequences (i) to (vi) are overexpressed with respect to a non-recombinant or wild type cyanobacterium, and
- the nucleotide sequences (i), (ii), (iii), (v) and (vi) are heterologous.
[2]
2. Cyanobacteria according to claim 1, wherein the repressor protein of a metabolite-inducible operon is the LacI protein of the lactose operon.
[3]
Cyanobacteria according to claim 1 or 2, wherein the overexpression of the nucleotide sequences (i), (ii) and (iii) is under the control of an "A" operon comprising a promoter functional in cyanobacteria.
[4]
Cyanobacteria according to any one of claims 1 to 4, wherein the overexpression of the nucleotide sequences (iv), (v) and (vi) is under the control of a "B" operon comprising a promoter functional in cyanobacteria .
[5]
5. Cyanobacteria according to claim 3 or 4, wherein the "A" operon is inserted in the Neutral Site I (NSI) of the cyanobacterium genome and the "B" operon is inserted in the Neutral Site II (NSII) of the genome. cyanobacteria, or alternatively, the operon B is inserted into the NSI and operon A is inserted into the NSII.
[6]
Cyanobacteria according to any one of claims 1 to 5, wherein the PGI enzyme comprises an amino acid sequence exhibiting a sequence identity of at least 70, 75, 80, 85, 90, 95, 96, 97 98.99% with the amino acid sequence SEQ ID NO: 1.
[7]
Cyanobacteria according to any one of claims 1 to 6, wherein the nucleotide sequence encoding the PGI enzyme comprises a nucleotide sequence exhibiting a sequence identity of at least 70, 75, 80, 85, 90 , 95, 96, 97, 98, 99% with the nucleotide sequence SEQ ID NO: 2.
[8]
8. Cyanobacteria according to any one of claims 1 to 7, wherein the nucleotide sequence encoding the PGI enzyme comprises the sequence SEQ ID NO: 3.
[9]
Cyanobacteria according to any one of claims 1 to 8, wherein the PGMT enzyme comprises an amino acid sequence exhibiting a sequence identity of at least 70, 75, 80, 85, 90, 95, 96, 97 98.99% with the amino acid sequence SEQ ID NO: 4.
[10]
10. Cyanobacterium according to any one of claims 1 to 9, wherein the nucleotide sequence encoding the PGMT enzyme comprises a nucleotide sequence exhibiting a sequence identity of at least 70, 75, 80, 85, 90 , 95, 96, 97, 98, 99% with the sequence SEQ ID NO: 5
[11]
11. Cyanobacteria according to any one of claims 1 to 10, wherein the nucleotide sequence encoding the PGMT enzyme comprises a nucleotide sequence exhibiting 100% sequence identity with the sequence SEQ ID NO: 6
[12]
12. Cyanobacterium according to any one of claims 1 to 11, wherein the GalU enzyme comprises an amino acid sequence exhibiting a sequence identity of at least 70, 75, 80, 85, 90, 95, 96, 97 98.99% with the sequence SEQ ID NO: 7.
[13]
13. Cyanobacteria according to any one of claims 1 to 12, wherein the nucleotide sequence encoding the GalU enzyme comprises a nucleotide sequence exhibiting a sequence identity of at least 70, 75, 80, 85, 90 , 95, 96, 97, 98, 99% with the sequence SEQ ID NO: 8.
[14]
14. Cyanobacterium according to any one of claims 1 to 13, wherein the nucleotide sequence encoding the Gal-U enzyme comprises a nucleotide sequence exhibiting 100% sequence identity with the sequence SEQ ID NO: 9.
[15]
15. Cyanobacterium according to any one of claims 1 to 14, wherein the SPS enzyme comprises an amino acid sequence exhibiting a sequence identity of at least 70, 75, 80, 85, 90, 95, 96, 97 98.99% with the sequence SEQ ID NO: 10.
[16]
16. Cyanobacteria according to any one of claims 1 to 15, wherein the nucleotide sequence encoding the SPS enzyme comprises a nucleotide sequence having a sequence identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% with the sequence SEQ ID NO: 11
[17]
17. Cyanobacterium according to any one of claims 1 to 16, wherein the nucleotide sequence encoding the SPS enzyme comprises a nucleotide sequence exhibiting 100% sequence identity with the sequence SEQ ID NO: 12.
[18]
18. Cyanobacterium according to any one of claims 1 to 17, wherein the CSCB enzyme comprises an amino acid sequence exhibiting a sequence identity of at least 70, 75, 80, 85, 90, 95, 96, 97 98.99% with the sequence SEQ ID NO: 13.
[19]
Cyanobacteria according to any one of claims 1 to 18, wherein the nucleotide sequence encoding the CSCB enzyme comprises a nucleotide sequence exhibiting a sequence identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% with the sequence SEQ ID NO: 14.
[20]
20. Cyanobacterium according to any one of claims 1 to 19, wherein the nucleotide sequence encoding the CSCB enzyme comprises a nucleotide sequence exhibiting 100% sequence identity with the sequence SEQ ID NO: 15.
[21]
21. Cyanobacteria according to any one of claims 1 to 20, wherein the repressor protein of a metabolite-inducible operon comprises an amino acid sequence exhibiting a sequence identity of at least 70, 75, 80, 85, 90 , 95, 96, 97, 98, 99% with the sequence SEQ ID NO: 16.
[22]
22. Cyanobacterium according to any one of claims 1 to 21, wherein the nucleotide sequence encoding the repressor protein of a metabolite-inducible operon comprises a nucleotide sequence exhibiting a sequence identity of at least 70.75 , 80, 85, 90, 95, 96, 97, 98, 99% with the sequence SEQ ID NO: 17.
[23]
23. Cyanobacterium according to any one of claims 1 to 22, wherein the nucleotide sequence encoding the repressor protein of a metabolite-inducible operon comprises a nucleotide sequence exhibiting 100% sequence identity with the sequence SEQ ID NO : 18.
[24]
24. Cyanobacteria according to any one of claims 3 to 23, wherein the operon integrated into the chromosome at neutral site 1 comprises the nucleotide sequence SEQ ID NO: 19.
[25]
25. Cyanobacterium according to any of claims 4 to 24, wherein the operon integrated into the chromosome at neutral site 2 comprises the nucleotide sequence SEQ ID NO: 20.
[26]
26. Cyanobacterium according to any one of claims 1 to 25, wherein the cyanobacterium belongs to the genus Synechococcus sp. or Synechocystis sp.
[27]
27. Cyanobacterium according to claim 26, wherein the cyanobacterium is Synechococcus elongatus.
[28]
28. A composition comprising a cyanobacterium according to any one of claims 1 to 27.
[29]
29. A composition comprising a synthetic bacterial consortium comprised of a cyanobacterium according to any one of claims 1 to 27 and at least one heterotrophic microorganism.
[30]
30. Use of a composition according to claim 28 or 29, for the production of metabolites or products of industrial interest, wherein the composition comprises a phosphate concentration greater than 0.2, 0.5, 1, 1.5 or 2 mM.
[31]
31. Use according to claim 30, wherein the metabolites or products of industrial interest are butanol or Polyhydroxyalkanoates.
[32]
32. Use of a cyanobacterium according to any one of claims 1 to 27, or of a composition according to claim 28, for the production of sucrose in a salt concentration lower than 150, 100, 80, 60, 40, 20, 10 or 0.5 mM.
[33]
33. Use according to claim 32, wherein the salt is sodium chloride.

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ES2844298B2|2022-01-17|

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ES202030042A|ES2844298B2|2020-01-21|2020-01-21|SUCROSE OVERPRODUCING RECOMBINANT CYANOBACTERIA|ES202030042A| ES2844298B2|2020-01-21|2020-01-21|SUCROSE OVERPRODUCING RECOMBINANT CYANOBACTERIA|

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PCT/ES2021/070030| WO2021148693A1|2020-01-21|2021-01-20|Sucrose-overproducing recombinant cyanobacterium|