Methods and compositions for the improved production of fatty acids and derivatives thereof

专利摘要:

公开号:ES2808287T9
申请号:ES12719914T
申请日:2012-04-02
公开日:2021-03-05
发明作者:Derek L Greenfield；Louis G Hom；Fernando A Sanchez-Riera；Zhihao Hu；Vikranth Arlagadda；Eli S Groban；Scott A Frykman
申请人:Genomatica Inc；
IPC主号:

专利说明:

[0002] Methods and compositions for the improved production of fatty acids and derivatives thereof
[0004] Background of the invention
[0006] Crude oil is a limited natural resource, found on Earth in liquid, gaseous and solid forms. Although crude oil is a valuable resource, it is discovered and extracted from the Earth at considerable financial and environmental cost. Furthermore, in its natural form, crude oil extracted from the Earth has few commercial uses. Crude oil is a mixture of hydrocarbons (eg, paraffins (or alkanes), olefins (or alkenes), alkynes, naphthenes (or cycloalkanes), aliphatic compounds, aromatic compounds, etc.) of varying length and complexity. Additionally, crude oil contains other organic compounds (eg, organic compounds containing nitrogen, oxygen, sulfur, etc.) and impurities (eg, sulfur, salt, acid, metals, etc.). Thus, crude oil must be refined and purified at considerable cost before it can be used commercially.
[0008] Crude oil is also a primary source of raw materials for the production of petrochemicals. The two main classes of petroleum-derived raw materials are short-chain olefins (eg, ethylene and propylene) and aromatics (eg, benzene and xylene isomers). These feedstocks are derived from the longer chain hydrocarbons of crude oil by cracking the same at considerable cost using a variety of methods, such as catalytic cracking, steam cracking, or catalytic reforming. These raw materials can be used to make petrochemicals such as monomers, solvents, detergents, and adhesives, which cannot otherwise be directly refined from crude oil.
[0010] Petrochemicals, in turn, can be used to make specialty chemicals, such as plastics, resins, fibers, elastomers, pharmaceuticals, lubricants, and gels. Special chemicals that can be produced from petrochemical raw materials include fatty acids, hydrocarbons (e.g. long chain, branched chain, saturated, unsaturated, etc.), fatty aldehydes, fatty alcohols, esters, ketones, lubricants, etc.
[0012] Due to the inherent challenges that oil poses, there is a need for a renewable oil source that does not need to be explored, extracted, transported long distances, or substantially refined like crude oil. There is also a need for a renewable oil source that can be produced economically without creating the kind of environmental damage that the oil industry and the burning of petroleum-based fuels do. For similar reasons, there is also a need for a renewable source of chemicals that are normally derived from petroleum.
[0014] One method of producing renewable oil is by engineering microorganisms to produce renewable oil products. Some microorganisms have long been known to possess a natural ability to produce petroleum products (eg, yeast to produce ethanol). More recently, the development of advanced biotechnologies has made it possible to metabolically engineer an organism to produce bioproducts and biofuels. Bioproducts (eg chemicals) and biofuels (eg biodiesel) are renewable alternatives to chemicals and petroleum-based fuels, respectively. Bioproducts and biofuels can be derived from renewable sources, such as plant matter, animal matter, and organic waste matter, collectively known as biomass.
[0016] Biofuels can replace any petroleum-based fuel (eg gasoline, diesel, jet fuel, heating oil, etc.) and offer several advantages over petroleum-based fuels. Biofuels do not require expensive and risky exploration or extraction. Biofuels can be produced locally and therefore do not require long-distance transport. Furthermore, biofuels can be manufactured directly and require little or no additional refining. Furthermore, the combustion of biofuels causes a lower burden on the environment since the amount of harmful emissions (e.g. greenhouse gases, air pollution, etc.) that is released during combustion is reduced compared to combustion. of petroleum-based fuels. Furthermore, biofuels maintain a balanced carbon cycle because biofuels are produced from biomass, a renewable natural resource. Although the combustion of biofuels releases carbon (for example, in the form of carbon dioxide), this carbon will be recycled during biomass production (for example, growing crops), thus balancing the carbon cycle, which is not achieves with the use of petroleum-based fuels.
[0018] Biologically derived chemicals offer advantages over petrochemicals similar to those that biofuels offer over petroleum-based fuels. In particular, biologically derived chemicals can be converted from biomass into the desired chemical directly without the need for extensive refining, unlike petrochemicals, which must be produced by refining crude oil to recover raw materials that are later further processed into the desired petrochemical product.
[0020] Hydrocarbons have many commercial uses. For example, shorter chain alkanes are used as fuels. Methane and ethane are the main components of natural gas. Longer chain alkanes (eg, five to sixteen carbons) are used as transportation fuels (eg, gasoline, diesel, or jet fuel). Alkanes that have more than sixteen carbon atoms are important components of fuel oils and lubricating oils. Even longer alkanes, which are solid at room temperature, can be used, for example, as paraffin wax. Alkanes containing approximately 35 carbons are found in bitumen, which is used for asphalting roads. In addition, longer chain alkanes can be cracked to produce commercially useful shorter chain hydrocarbons.
[0022] Like short chain alkanes, short chain alkenes are used in transportation fuels. Longer chain alkenes are used in plastics, lubricants, and synthetic lubricants. In addition, alkenes are used as raw materials to produce alcohols, esters, plasticizers, surfactants, tertiary amines, enhanced oil recovery agents, fatty acids, thiols, alkenylsuccinic anhydride epoxides, chlorinated alkanes, chlorinated alkenes, waxes, fuel additives and drag flow reducers.
[0024] Esters have many commercial uses. For example, biodiesel, an alternative fuel, is made up of esters (eg, fatty acid methyl ester, fatty acid ethyl esters, etc.). Some low molecular weight esters are volatile with a pleasant odor that makes them useful as fragrances or flavoring agents. In addition, esters are used as solvents for lacquers, paints and varnishes. Also, some naturally occurring substances, such as waxes, fats, and oils, are made up of esters. Esters are also used as softening agents in resins and plastics, plasticizers, flame retardants, and additives in gasoline and oil. Additionally, esters can be used in the manufacture of polymers, films, fabrics, colorants, and pharmaceuticals.
[0025] Aldehydes are used to produce many specialty chemicals. For example, aldehydes are used to produce polymers, resins (eg, bakelite), colorants, flavors, plasticizers, perfumes, pharmaceuticals, and other chemicals, some of which can be used as solvents, preservatives, or disinfectants. Also, certain natural and synthetic compounds, such as vitamins and hormones, are aldehydes, and many sugars contain aldehyde groups. Fatty aldehydes can be converted to fatty alcohols by chemical or enzymatic reduction.
[0027] Fatty alcohols have many commercial uses. Annual worldwide sales of fatty alcohols and their derivatives exceed $ 1 billion. Shorter chain fatty alcohols are used in the cosmetic and food industries as emulsifiers, emollients and thickeners. Due to their amphiphilic nature, fatty alcohols behave as nonionic surfactants, which are useful in household and personal care products, such as, for example, detergents. In addition, fatty alcohols are used in pharmaceutical waxes, gums, resins, ointments and lotions, lubricating oil additives, textile antistatic and finishing agents, plasticizers, cosmetics, industrial solvents, and fat solvents.
[0029] Acyl-CoA synthase (ACS) esterifies free fatty acids to acyl-CoA through a two-step mechanism. The free fatty acid is first converted to an acyl-AMP intermediate (an adenylate) through pyrophosphorolysis of ATP. The activated carbonyl carbon of the adenylate then couples with the thiol group of CoA, releasing AMP and the final acyl-CoA product (Shockey et al., Plant. Physiol., 129: 1710-1722 (2002)).
[0031] FadR is a key regulatory factor involved in fatty acid degradation and fatty acid biosynthesis pathways (Cronan et al., Mol. Microbiol., 29 (4): 937-943 (1998)). The ACS enzyme from E. coli FadD and the fatty acid transport protein Fadl are essential components of a fatty acid uptake system. Fadl mediates the transport of fatty acids to the bacterial cell, and FadD mediates the formation of acyl-CoA esters. When no other carbon source is available, exogenous fatty acids are taken up by bacteria and converted to acyl-CoA esters, which can bind to the transcription factor FadR and derepress the expression of the fad genes that encode the proteins responsible for transport ( Fadl), activation (FadD) and p-oxidation of fatty acids (FadA, FadB, FadE and FadH). When alternative carbon sources are available, bacteria synthesize fatty acids such as acyl-ACP, which are used for phospholipid synthesis, but are not substrates for poxidation. Therefore, acyl-CoA and acyl-ACP are independent sources of fatty acids that can give rise to different end products (Caviglia et al., J. Biol. Chem., 279 (12): 1163-1169 (2004)). US 2010/242345 describes genetically engineered microorganisms that produce fatty acid biosynthetic pathway products (derivatives of fatty acids) and methods for their use.
[0033] There remains a need for methods and compositions to enhance the production of biologically derived chemicals, such as fatty acids and fatty acid derivatives. The present invention provides such methods and compositions. The invention further provides fatty acid derivatives and derivatives thereof produced by the methods described herein, such as fuels, surfactants, and detergents.
[0035] Brief summary of the invention
[0037] The invention provides improved methods for producing a fatty acid or a fatty acid derivative thereof in a bacterial host cell. The method consists of (a) providing a bacterial host cell engineered that comprises a heterologous promoter and / or ribosome binding site operably linked to a polynucleotide sequence encoding a FadR polypeptide, said promoter and / or ribosome binding site causing overexpression of the FadR polypeptide in said cell, (b) cultivating the genetically engineered bacterial host cell in a culture medium under conditions that allow the production of a fatty acid or a fatty acid derivative thereof and (c) isolating the fatty acid or a fatty acid derivative thereof same from the genetically engineered bacterial host cell, wherein the fatty acid or fatty acid derivative thereof is a fatty acid, an acyl-ACP, an acyl-CoA, a fatty aldehyde, a short-chain alcohol, an alcohol long chain, a fatty alcohol, a hydrocarbon or an ester. As a result of this method, one or more of the titer, yield, or productivity of the fatty acid or fatty acid derivative produced by the engineered bacterial host cell is increased relative to that of the corresponding wild-type host cell.
[0039] Also disclosed are fatty acids and fatty acid derivatives, such as an acyl-CoA, a fatty aldehyde, a short-chain alcohol, a long-chain alcohol, a fatty alcohol, a hydrocarbon or an ester, produced by the methods of the invention. Biofuel compositions and surfactant compositions comprising a fatty acid or a fatty acid derivative produced by the methods of the invention are further disclosed.
[0041] Brief description of the different views of the drawings
[0043] FIG. 1 is a table of example genes suitable for use in the practice of the invention. The registration numbers of polypeptides and / or polynucleotides are from the database of the National Center for Biotechnology Information (NCBI) and the CE numbers of enzymes are from the Nomenclature Committee of the International Union of Biochemistry and Biology Molecular (NC-IUBMB).
[0044] FIG. 2 is a graph of fatty species production in a control E. coli strain (ALC310) or the transposon insertion strain, D288.
[0045] FIG. 3 is a diagram depicting the location of the transposon insertion in strain D288.
[0046] FIG. 4 is a bar graph of total fat species (FA) titers from the expression library of E. coli strains that have altered expression of wild-type FadR or mutant FadR [S219N] in comparison with FA titers of the control E. coli strain (ALC487).
[0047] FIG. 5 is a bar graph of total fat species (FA) titers in three separate shake flask (SF) fermentations of E. coli strain D512 having altered expression of wild-type FadR compared to fA titers in control strain ALC487.
[0048] FIG. 6 is a bar graph of total yield of fat species on carbon in shake flask fermentations of control strain ALC487 or E. coli strain D512 having altered expression of wild type FadR.
[0049] FIG. 7 is a graph of fatty acid and fatty alcohol production and total yield of fatty species in 5 L bioreactor fermentations of control strain ALC487 fed at a glucose rate of 10 g / L / h or of strain D512 that has altered expression of wild-type FadR fed at a glucose rate of 10 g / l / h or 15 g / l / h. The bars represent the fatty alcohol or fatty acid titer, and the circles represent the total yield of fatty species on carbon.
[0050] FIG. 8 is a graph of fatty acid and fatty alcohol production and total yield of fatty species in shake flask fermentations of strain D512 or a strain D512 in which the entD gene was deleted. Bars represent fatty acid or fatty alcohol titer and circles represent fatty acid yield. FIG. 9 is a graph of total fat species (fatty acids and FAME) yields and titers in two strains of E. coli from the ribosome binding site (RBS) library that have altered expression of mutant FadR [S219N] (en ie, P1A4 and P1G7) compared to the total fat species titers and yields in the parental E. coli strain (DAM1-pDS57) in shake flask (SF) fermentations at 32 ° C. The bars represent the total fatty species titers after 56 hours of culture and the squares represent the total yield of fatty species after 56 hours of culture.
[0051] FIG. 10 is a line graph of combined FAME and free fatty acid (FFA) titers in parental DAM1 strain pDS57 or RBS P1A4 or P1G7 library strains in bioreactor fermentations at various time points after induction of FAME production and FFA, where DAM1 P1A4 and DAM1 P1G7 express FadR and DAM1 pDS57 does not express FadR.
[0052] FIG. 11 is a line graph of combined yields of FAME and FFA in parental strain DAM1 pDS57 or RBS P1A4 or P1G7 library strains in bioreactor fermentations at various time points after induction of FAME and FFA production, where DAM1 P1A4 and DAM1 P1G7 express FadR and DAM1 pDS57 does not express FadR.
[0054] Detailed description of the invention
[0056] The invention is based, at least in part, on the discovery that altering the level of FadR expression in a host cell facilitates enhanced production of fatty acids and fatty acid derivatives by the host cell.
[0058] The invention provides improved methods of producing a fatty acid or a fatty acid derivative in a cell. bacterial host. The method consists of (a) providing an engineered bacterial host cell comprising a heterologous promoter and / or ribosome binding site operably linked to a polynucleotide sequence encoding a FadR polypeptide, eliciting said promoter and / or site of ribosome binding the overexpression of the FadR polypeptide in said cell, (b) culturing the genetically engineered bacterial host cell in a culture medium under conditions that allow the production of a fatty acid or a derivative thereof, and (c) isolating the fatty acid or a derivative thereof from the genetically engineered bacterial host cell, wherein the fatty acid or derivative thereof is a fatty acid, an acyl-ACP, an acyl-CoA, a fatty aldehyde, a chain alcohol short, long chain alcohol, fatty alcohol, hydrocarbon, or ester. As a result of this method, one or more of the titer, yield, or productivity of the fatty acid or fatty acid derivative produced by the engineered bacterial host cell is increased relative to that of the corresponding wild-type host cell.
[0060] Definitions
[0062] As used in this specification and in the appended claims, the singular forms "a", "an" and "the" or "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a recombinant host cell" includes two or more such recombinant host cells, reference to "a fatty alcohol" includes one or more fatty alcohols or mixtures of fatty alcohols, reference to "a nucleic acid coding sequence "includes one or more nucleic acid coding sequences, reference to" an enzyme "includes one or more enzymes, and the like.
[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although other methods and materials similar or equivalent to those described herein may be used in the practice of the present invention, preferred materials and methods are described herein.
[0066] In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.
[0068] Registration Numbers: Sequence registration numbers throughout this description were obtained from databases provided by the NCBI (National Center for Biotechnology Information) maintained by the National Institutes of Health, USA (which is identified in this document as "NCBI Registration Numbers" or, alternatively, as "GenBank Registration Numbers"), and from the UniProt knowledge base (UniProtKB) and the Swiss-Prot databases provided by the Institute Swiss Bioinformatics (identified herein as "UniProtKB Registration Numbers").
[0070] Enzyme Classification Numbers (CE): CE numbers are established by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), a description of which is available on the IUBMB Enzyme Nomenclature website at the World Wide Web. EC numbers classify enzymes according to the reaction catalyzed.
[0072] The term "FadR polypeptide" refers to a polypeptide having a biological activity corresponding to that of FadR derived from E. coli MG1655 (SEQ ID NO: 1).
[0074] As used herein, the term "fatty acid or derivative thereof" means a "fatty acid" or a "fatty acid derivative". The term "fatty acid" means a carboxylic acid having the formula RCOOH. R represents an aliphatic group, preferably an alkyl group. R can comprise from about 4 to about 22 carbon atoms. Fatty acids can be saturated, monounsaturated, or polyunsaturated. In a preferred embodiment, the fatty acid is prepared from a fatty acid biosynthetic pathway. A "fatty acid derivative" is a product prepared in part from the fatty acid biosynthetic pathway of the production host organism. "Fatty acid derivatives" include products prepared in part from acyl-ACP or acyl-ACP derivatives. Exemplary fatty acid derivatives include, for example, acyl-CoA, fatty acids, fatty aldehydes, long and short chain alcohols, hydrocarbons, fatty alcohols, esters (eg, waxes, fatty acid esters or fatty esters), terminal olefins, internal olefins and ketones.
[0076] A "fatty acid derivative composition", as referred to herein, is produced by a recombinant host cell and typically comprises a fatty acid derivative mixture. In some cases, the mixture includes more than one type of product (for example, fatty acids and fatty alcohols, fatty acids and esters of fatty acids or alkanes and olefins). In other cases, the fatty acid derivative compositions may comprise, for example, a mixture of fatty alcohols (or other fatty acid derivative) with various chain lengths and saturation or branching characteristics. In still other cases, the fatty acid derivative composition comprises a mixture of both more than one type of product and products with various chain lengths and saturation or branching characteristics.
[0078] As used herein "acyl-CoA" refers to an acylthioester formed between the carbon atom carbonyl of the alkyl chain and the sulfhydryl group of the 4'-phosphopantethionyl moiety of coenzyme A (CoA), having the formula RC (O) S-CoA, where R is any alkyl group having at least 4 carbon atoms carbon.
[0080] As used herein "acyl-ACP" refers to an acyl thioester formed between the carbonyl carbon of an alkyl chain and the sulfhydryl group of the phosphopantethenyl moiety of an acyl carrier protein (ACP).
[0081] The phosphopantethenyl moiety binds post-translationally to a conserved serine residue in ACP through the action of holo-acyl carrier protein synthase (ACPS), a phosphopantethenyl transferase. In some embodiments, an acyl-ACP is an intermediate in the synthesis of fully saturated acyl-ACP. In other embodiments, an acyl-ACP is an intermediate in the synthesis of unsaturated acyl-ACPs. In some embodiments, the carbon chain will have approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 carbons. Each of these acyl-ACPs are substrates for the enzymes that convert them into fatty acid derivatives.
[0083] As used herein, the term "fatty acid biosynthetic pathway" means a biosynthetic pathway that produces fatty acids. The fatty acid biosynthetic pathway includes fatty acid synthases that can be engineered to produce fatty acids and, in some embodiments, can be expressed with additional enzymes to produce fatty acids that have the desired carbon chain characteristics.
[0085] As used herein, "fatty aldehyde" means an aldehyde having the formula RCHO characterized by a carbonyl group (C = O). In some embodiments, the fatty aldehyde is any aldehyde prepared from a fatty acid or fatty acid derivative.
[0087] As used herein, "fatty alcohol" means an alcohol having the formula ROH. In some embodiments, the fatty alcohol is any alcohol made from a fatty acid or fatty acid derivative.
[0089] In certain embodiments, the R group of a fatty acid, fatty aldehyde, or fatty alcohol has at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19, carbons in length. Alternatively, or in addition, the R group has 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, or 6 or less carbon atoms in length. Thus, the R group may have an R group limited by any two of the above endpoints. For example, the R group can be 6-16 carbon atoms in length, 10-14 carbon atoms in length, or 12-18 carbon atoms in length. In some embodiments, the fatty acid, fatty aldehyde, or fatty alcohol is a C6, C ⁷ , Cs, C ⁹ , C ¹⁰ , C ¹¹ , C ¹² , C ¹³ , C ¹⁴ , C ^{15 fatty acid, fatty aldehyde, or fatty alcohol.} , C ¹⁶ , C ¹⁷ , C ¹⁸ , C ¹⁹ ,
[0090] C ²⁰ , C ²¹ , C ²² , C ²³ , C ²⁴ , C ²⁵ or C ²⁶ . In certain embodiments, the fatty acid, fatty aldehyde, or fatty alcohol is a C6, C8, C ¹⁰ , C ¹² , C ¹³ , C ¹⁴ , C ¹⁵ , C ¹⁶ , C ^17, or C ^{18 fatty acid, fatty aldehyde, or fatty alcohol.} .
[0092] The R group of a fatty acid, fatty aldehyde or fatty alcohol can be a straight chain or a branched chain.
[0093] Branched chains can have more than one branch point and can include cyclic branches. In some embodiments, the branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is a branched fatty acid, branched fatty aldehyde, or branched C6, C ⁷ , C8, C ⁹ , C ¹⁰ , C ¹¹ , C ¹² , C ^{13 fatty alcohol} , C ¹⁴ , C ¹⁵ , C ¹⁶ ,
[0094] C ¹⁷ , C ¹⁸ , C ¹⁹ , C ²⁰ , C ²¹ , C ²² , C ²³ , C ²⁴ , C ²⁵ or C ²⁶ . In particular embodiments, the branched fatty acid, branched fatty aldehyde or branched fatty alcohol is a branched fatty acid, branched fatty aldehyde or branched C6, C8, C ¹⁰ , C ¹² , C ¹³ , C ¹⁴ , C ¹⁵ , C ^{16 fatty alcohol} , C ¹⁷ or C ¹⁸ . In certain branched fatty realms, branched fatty aldehyde or branched fatty alcohol is in the primary position (C ¹ ).
[0096] In certain embodiments, the branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is an iso-fatty acid, iso-fatty aldehyde, or iso-fatty alcohol, or an anteiso-fatty acid, an anteiso-fatty aldehyde, or anteiso-fatty alcohol. In exemplary embodiments, the branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is selected from branched fatty acid, branched fatty aldehyde or branched fatty alcohol iso-C70, iso-C ^{8: 0} , iso-C9: 0, iso- C ^{10: 0} , iso- ^{11: 0} , iso-C ^{12: 0} , iso-C13: 0, iso-C14: 0, iso-15: 0, iso-C ^{16: 0} , iso-C17: 0, iso -C ^{18: 0} , iso-C19: 0, anteiso-C7: 0, anteiso-C ^{8: 0} , anteiso-C9: 0, anteiso-Cm ⁰ , anteiso-Cn ^{: 0} , anteiso-C ^{12: 0} , anteiso -C13: 0, anteiso-C14: 0, anteiso-C15: 0, anteiso-C ^{16: 0} , anteiso-C17: 0, anteiso-C ^{18: 0} and anteiso-C19: 0.
[0098] The R group of a branched or unbranched fatty acid, branched or unbranched fatty aldehyde, or branched or unbranched fatty alcohol may be saturated or unsaturated. If unsaturated, the R group may have one or more than one point of unsaturation. In some embodiments, the unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol is a monounsaturated fatty acid, monounsaturated fatty aldehyde, or monounsaturated fatty alcohol.
[0099] In certain embodiments, the unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol is an unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol C6: 1, C7: 1, C8: 1, C9: 1, C10: 1, C11 : 1, C12: 1, C13: 1, C14: 1, C15: 1, C16: 1, C17: 1, C18: 1, C19: 1, C20: 1, C21: 1, C22: 1, C23: 1 , C24: 1, C25: 1 or C26: 1. In certain preferred embodiments, the unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol is C10: 1, C12: 1, C14: 1, C16: 1, or C18: 1. In still other embodiments, the unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol is unsaturated at the omega-7 position. In certain embodiments, the unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol comprises a cis double bond.
[0100] As used herein, the term "alkane" means saturated hydrocarbons or compounds consisting only of carbon (C) and hydrogen (H), wherein these atoms are linked together by single bonds (i.e., they are saturated compounds ).
[0102] The terms "olefin" and "alkene" are used interchangeably herein and refer to hydrocarbons that contain at least one carbon-carbon double bond (ie, they are unsaturated compounds).
[0104] The terms "terminal olefin", "a-olefin", "terminal alkene" and "1-alkene" are used interchangeably herein with reference to a-olefins or alkenes with a chemical formula CxH2x, they are distinguished from other olefins with a similar molecular formula for the linearity of the hydrocarbon chain and the position of the double bond in the primary or alpha position.
[0106] As used herein, the term "fatty ester" can be used in reference to an ester. In a preferred embodiment, a fatty ester is any ester prepared from a fatty acid, for example a fatty acid ester. In some embodiments, a fatty ester contains an A side and a B side. As used herein, an "A side" of an ester refers to the oxygen-bonded carbon chain of the ester carboxylate. As used herein, a "B-side" of an ester refers to the carbon chain that comprises the parent carboxylate of the ester. In embodiments where the fatty ester is derived from the fatty acid biosynthetic pathway, the A side is provided by an alcohol and the B side is provided by a fatty acid.
[0108] Any alcohol can be used to form the A side of fatty esters. For example, alcohol can be derived from the fatty acid biosynthetic pathway. Alternatively, alcohol can be produced through non-fatty acid biosynthetic pathways. Also, alcohol can be provided exogenously. For example, alcohol can be supplied in the fermentation broth in cases where the fatty ester is produced by an organism. Alternatively, a carboxylic acid, such as a fatty acid or acetic acid, can be supplied exogenously in cases where the fatty ester is produced by an organism that can also produce alcohol.
[0110] The carbon chains comprising side A or side B can be of any length. In one embodiment, the A side of the ester is at least about 1,2,3,4,5,6,7,8,10,12,14,16, or 18 carbons in length. When the fatty ester is a fatty acid methyl ester, the A side of the ester is 1 carbon in length. When the fatty ester is a fatty acid ethyl ester, the A side of the ester is 2 carbons long. The B side of the ester can be at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 carbons in length. Side A and / or side B can be straight or branched chain. Branched chains can have one or more branch points. Furthermore, branched chains can include cyclic branches. Also, side A and / or side B can be saturated or unsaturated. If unsaturated, side A and / or side B may have one or more points of unsaturation.
[0111] In some embodiments, the fatty acid ester is a fatty acid methyl ester (FAME) or a fatty acid ethyl ester (FAEE). In certain embodiments, the FAME is a beta-hydroxy (B-OH) FAME. In one embodiment, the fatty ester is biosynthetically produced. In this embodiment, the fatty acid is "activated" first. Non-limiting examples of "activated" fatty acids are acyl-CoA, acyl ACP, and acyl phosphate. Acyl-CoA can be a direct product of fatty acid biosynthesis or degradation. Furthermore, acyl-CoA can be synthesized from a free fatty acid, a CoA, and an adenosine nucleotide triphosphate (ATP). An example of an enzyme that produces acyl-CoA is acyl-CoA synthase.
[0113] After a fatty acid is activated, it can easily be transferred to a receptor nucleophile. Examples of nucleophiles are alcohols, thiols or phosphates.
[0115] In one embodiment, the fatty ester is a wax. The wax can be derived from a long chain alcohol and a long chain fatty acid. In another embodiment, the fatty ester is a fatty acid thioester, for example, fatty acyl Coenzyme A (CoA). In other embodiments, the fatty ester is a fatty acyl pantothenate, an acyl carrier protein (ACP), or a fatty phosphate ester.
[0117] As used herein "acyl CoA" refers to an acyl thioester formed between the carbonyl carbon atom of the alkyl chain and the sulfhydryl group of the 4'-phosphopantethionyl moiety of coenzyme A (CoA), having the formula RC (O) S-CoA, where R is any alkyl group having at least 4 carbon atoms. In some cases, an acyl CoA will be an intermediate in the synthesis of fully saturated acyl CoA, including, but not limited to, 3-keto-acyl CoA, a 3-hydroxy acyl CoA, a delta-2-trans-enoyl-CoA, or an alkyl acyl CoA. In some embodiments, the carbon chain will have approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 carbon atoms. In other embodiments the acyl CoA will be branched. In one embodiment, the branched acyl CoA is an isoacyl CoA, in another, it is an anti-isoacyl CoA. Each of these "acyl CoAs" are substrates for enzymes that convert them to fatty acid derivatives such as those described herein.
[0118] The terms "altered expression level" and "modified expression level" are used interchangeably, and mean that a polynucleotide, polypeptide, or hydrocarbon is present at a different concentration in a modified host cell compared to its concentration in a wild-type cell. corresponding in the same terms.
[0120] "Polynudeotide" refers to a polymer of DNA or RNA, which can be single-stranded or double-stranded and which can contain unnatural or modified nucleotides. The terms "polynudeotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule and therefore include double and single stranded DNA, and double and single stranded RNA. The terms include, as equivalents, RNA or DNA analogs made from nucleotide analogs and modified polynucleotides such as, but not limited to, methylated and / or end-capped polynucleotides. The polynudeotide can be in any form, including, but not limited to, plasmid, viral, chromosomal, EST, cDNA, mRNA, and rRNA.
[0122] The term "nucleotide", as used herein, refers to a monomeric unit of a polynudeotide consisting of a heterocyclic base, a sugar, and one or more phosphate groups. Natural bases (guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) are normally derived from purine or pyrimidine, although it should be understood that base analogs are also included. natural and unnatural. The natural sugar is pentose (five-carbon sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), although it should be understood that analogs of natural and unnatural sugars are also included. Nucleic acids are normally linked by phosphate linkages to form nucleic acids or polynucleotides, although many other linkages are known in the art (eg, phosphorothioates, boranophosphates, and the like).
[0124] The polynucleotides described herein may comprise degenerate nucleotides that are defined according to the IUPAC code for nucleotide degeneracy where B is C, G, or T; D is A, G, or T; H is A, C, or T; K is G or T; M is A or C; N is A, C, G, or T; R is A or G; S is C or G; V is A, C, or G; W is A or T; and Y is C or T.
[0126] The terms "polypeptide" and "protein" refer to a polymer of amino acid residues. The term "recombinant polypeptide" refers to a polypeptide that is produced by recombinant DNA techniques, where generally the DNA encoding the expressed protein or RNA is inserted into a suitable expression vector which in turn is used to transform a host cell to produce the polypeptide or RNA.
[0128] In some embodiments, the polypeptide, polynudeotide, or hydrocarbon that has an altered or modified expression level is "overexpressed" or has an "increased expression level." As used herein, "overexpressing" and "increasing the level of expression" mean expressing or causing a polynudeotide, polypeptide, or hydrocarbon to be expressed in a cell at a concentration greater than that normally expressed in a cell of type corresponding wild under the same conditions. For example, a polypeptide can be "overexpressed" in an engineered host cell when the polypeptide is present at a higher concentration in the engineered host cell compared to its concentration in a non-engineered host cell of the same species under the same conditions.
[0130] In other embodiments, the polypeptide, polynudeotide, or hydrocarbon that has an altered expression level is "attenuated" or has a "decreased expression level." As used herein, "attenuate" and "decrease the level of expression" mean to express or cause a polynudeotide, polypeptide, or hydrocarbon to be expressed in a cell at a concentration lower than that normally expressed in a cell of type corresponding wild under the same conditions.
[0132] The degree of overexpression or attenuation can be 1.5 times or more, for example, 2 times or more, 3 times or more, 5 times or more, 10 times or more, or 15 times or more. Alternatively, or in addition, the degree of overexpression or attenuation may be 500 times or less, for example, 100 times or less, 50 times or less, 25 times or less, or 20 times or less. Thus, the degree of overexpression or attenuation may be limited by any two of the above endpoints. For example, the degree of overexpression or attenuation can be 1.5-500 times, 2-50 times, 10-25 times, or 15-20 times.
[0134] In some embodiments, a polypeptide described herein has "an increased level of activity." By "increased activity level" is meant that a polypeptide has a higher level of biochemical or biological function (eg, DNA binding or enzymatic activity) in an engineered host cell compared to its level of biochemical function and / or biological in a corresponding wild-type host cell under the same conditions. The degree of enhanced activity can be about 10% or more, about 20% or more, about 50% or more, about 75% or more, about 100% or more, about 200% or more, about 500% or more, about 1000% or more, or any range included therein.
[0135] A polynucleotide or polypeptide can be attenuated using methods known in the art. In some embodiments, the expression of a gene or polypeptide encoded by the gene is attenuated by mutation of the regulatory polynucleotide sequences that control the expression of the gene. In other embodiments, the expression of a gene or polypeptide encoded by the gene is attenuated by overexpression of a repressor protein or by providing an exogenous regulatory element that activates a repressor protein. In still other embodiments, DNA or RNA-based gene silencing methods are used to attenuate the expression of a gene or polynucleotide. In some embodiments, the expression of a gene or polypeptide is totally attenuated, for example, by deleting all or a portion of the polynucleotide sequence of a gene.
[0137] A polynucleotide or polypeptide can be overexpressed using methods known in the art. In some aspects, overexpression of a polypeptide is achieved through the use of an exogenous regulatory element. The term "exogenous regulatory element" generally refers to a regulatory element that originates outside the host cell. However, in certain aspects, the term "exogenous regulatory element" can refer to a host cell-derived regulatory element whose function is replicated or usurped in order to control the expression of an endogenous polypeptide. For example, if the host cell is an E. coli cell and the FadR polypeptide is encoded by an endogenous fadR gene, then the expression of the endogenous fadR can be controlled by a promoter derived from another E. coli gene.
[0139] In some aspects, the exogenous regulatory element is a chemical compound, such as a small molecule. As used herein, the term "small molecule" refers to a substance or compound that has a molecular weight of less than about 1,000 g / mol.
[0141] In some aspects, the exogenous regulatory element that controls the expression of an endogenous fadR gene is an expression control sequence that is operably linked to the endogenous fadR gene by recombinant integration into the genome of the host cell. In certain aspects, the expression control sequence is integrated into a host cell chromosome by homologous recombination, using methods known in the art (eg, Datsenko et al., Proc. Natl. Acad. Sci. USA, 97 (12): 6640-6645 (2000)).
[0143] Expression control sequences are known in the art and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), ribosome binding sites (RBS), and the like, which provide for expression of the polynucleotide sequence in a host cell. Expression control sequences specifically interact with cellular proteins that participate in transcription (Maniatis et al., Science, 236: 1237-1245 (1987)). Examples of expression control sequences are described in, for example, Goeddel, "Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990). In the methods of the invention the sequence Control of expression is a heterologous promoter and / or ribosome binding site.
[0145] In the methods of the invention, an expression control sequence is operably linked to a polynucleotide sequence. By "operably linked" it is meant that a polynucleotide sequence and an expression control sequence or sequences are linked in a manner that allows gene expression when appropriate molecules (eg, transcription activator proteins) are linked to the sequence or sequences. expression control sequences. The operably linked promoters are upstream of the selected polynucleotide sequence in terms of the direction of transcription and translation. Operably linked enhancers can be located 5 ', within, or 3' of the selected polynucleotide.
[0147] In some embodiments, the polynucleotide sequence is provided to the host cell by means of a recombinant vector, comprising a promoter operably linked to the polynucleotide sequence. In certain embodiments, the promoter is a developmentally regulated, organelle-specific, tissue-specific, inducible, constitutive, or cell-specific promoter.
[0149] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid, ie, a polynucleotide sequence, to which it has been linked. One type of useful vector is an episome (ie, a nucleic acid capable of extrachromosomal replication). Useful vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are attached. Vectors capable of directing the expression of the genes to which they are operably linked are referred to herein as "expression vectors." In general, expression vectors useful in recombinant DNA techniques are typically in the form of "plasmids" which generally refer to circular double-stranded DNA loops that, in their vector form, are not attached to the chromosome. The terms "plasmid" and "vector" are used interchangeably herein, as the plasmid is the most commonly used form of vector. However, other forms of expression vectors that have equivalent functions and are disclosed in the art below are also included.
[0151] The term "regulatory sequences", as used herein, typically refers to a sequence of bases in DNA, operably linked to DNA sequences encoding a protein that ultimately controls the expression of the protein. Examples of regulatory sequences include, but are not limited to, RNA promoter sequences, transcription factor binding sequences, transcription termination sequences, transcription modulators (such as enhancer elements), nucleotide sequences that affect stability RNA and translation regulatory sequences (such as ribosome binding sites (eg, Shine-Dalgarno sequences in prokaryotes or Kozak sequences in eukaryotes), start codons, stop codons).
[0152] As used herein, the term "the expression of said nucleotide sequence is modified relative to the wild-type nucleotide sequence" means an increase or decrease in the level of expression and / or activity of a endogenous nucleotide sequence or the expression and / or activity of a nucleotide sequence encoding a heterologous or non-native polypeptide.
[0154] As used herein, the term "express" with respect to a polynucleotide is to make it work. A polynucleotide encoding a polypeptide (or protein), when expressed, will be transcribed or translated to produce that polypeptide (or protein). As used herein, the term "overexpress" means to express or cause to be expressed a polynucleotide or polypeptide in a cell at a concentration higher than that normally expressed in a corresponding wild-type cell under the same conditions.
[0156] In some aspects, the recombinant vector comprises at least one sequence selected from the group consisting of (a) an expression control sequence operably coupled to the polynucleotide sequence; (b) a selection marker operably coupled to the polynucleotide sequence; (c) a marker sequence operably coupled to the polynucleotide sequence; (d) a purification fraction operably coupled to the polynucleotide sequence; (e) a secretory sequence operably coupled to the polynucleotide sequence; and (f) a targeting sequence operably coupled to the polynucleotide sequence.
[0158] The expression vectors described herein include a polynucleotide sequence that is described herein in a form suitable for the expression of the polynucleotide sequence in a host cell. Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the level of expression of the desired polypeptide, and the like. The expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the polynucleotide sequences as described herein.
[0160] Expression of genes encoding polypeptides in prokaryotes, eg, E. coli, is most often done with vectors containing constitutive or inducible promoters that drive the expression of fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, generally to the amino or carboxyl terminus of the recombinant polypeptide. Such fusion vectors typically serve one or more of the following three purposes: (1) to increase expression of the recombinant polypeptide; (2) increase the solubility of the recombinant polypeptide; and (3) assist in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide. This allows the separation of the recombinant polypeptide from the fusion fraction after purification of the fusion polypeptide. Examples of such enzymes, and their cognate recognition sequences, include factor Xa, thrombin, and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech, Inc., Piscataway, NJ; Smith et al., Gene, 67: 31-40 (1988)), pMAL (New England Biolabs, Beverly, MA), and pRITS (Pharmacia Biotech, Inc., Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose-binding protein E, or protein A, respectively, to the target recombinant protein.
[0162] Suitable expression systems for prokaryotic and eukaryotic cells are well known in the art; see, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual", second edition, Cold Spring Harbor Laboratory (1989). Examples of inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene, 69: 301-315 (1988)) and PET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185 , Academic Press, San Diego, CA, pp. 60-89 (1990)). In certain embodiments, a polynucleotide sequence of the invention is operably linked to a promoter derived from bacteriophage T5. Examples of vectors for expression in yeast include pYepSec1 (Baldari et al., EMBO J., 6: 229-234 (1987)), pMFa (Kurjan et al., Cell, 30: 933-943 (1982)), pJRY88 (Schultz et al., Gene, 54: 113-123 (1987)), pYES2 (Invitrogen Corp., San Diego, CA) and picZ (Invitrogen Corp., San Diego, CA). Baculovirus vectors available for protein expression in cultured insect cells (eg, Sf9 cells) include, for example, the pAc series (Smith et al., Mol. Cell Biol., 3: 2156-2165 (1983)) and the pVL series (Lucklow et al., Virology, 170: 31-39 (1989)). Examples of mammalian expression vectors include pCDM8 (Seed, Nature, 329: 840 (1987)) and pMT2PC (Kaufinan et al., EMBO J, 6: 187-195 (1987)).
[0163] Vectors can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" refer to various art-recognized techniques for introducing foreign nucleic acid (eg, DNA) into a host cell, including co-precipitation with calcium phosphate or calcium chloride, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in, for example, Sambrook et al. (above).
[0165] For the stable transformation of bacterial cells, it is known that, depending on the expression vector and the transformation technique used, only a small fraction of cells will take up and replicate the expression vector. To identify and select these transformants, a gene encoding a selectable marker (eg, antibiotic resistance) can be introduced into host cells along with the gene of interest. The markers Selectors include those that confer resistance to drugs such as, but not limited to, ampicillin, kanamycin, chloramphenicol, or tetracycline. Nucleic acids encoding a selectable marker can be introduced into a host cell in the same vector as that encoding a polypeptide described herein or they can be introduced into a separate vector. Cells stably transformed with the introduced nucleic acid can be identified by growth in the presence of an appropriate selection drug.
[0166] In the same way, for stable transfection in mammalian cells, it is known that, depending on the expression vector and the transfection technique used, only a small fraction of the cells can integrate the foreign DNA into their genome. In order to identify and select for these integrants, a gene encoding a selectable marker (eg, antibiotic resistance) can be introduced into host cells along with the gene of interest. Preferred selectable markers include those that confer drug resistance, such as G418, hygromycin, and methotrexate. Nucleic acids encoding a selectable marker can be introduced into a host cell in the same vector as that encoding a polypeptide described herein or they can be introduced into a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by growth in the presence of an appropriate selection drug.
[0168] In some embodiments, the FadR polypeptide has the amino acid sequence of SEQ ID NO: 1.
[0170] In other embodiments, the FadR polypeptide is encoded by a fadR gene obtained from microorganisms of the genus Escherichia, Salmonella, Citrobacter, Enterobacter, Klebsiella, Cronobacter, Yersinia, Serratia, Erwinia, Pectobacterium, Photorhabdus, Edwardsiella, Shewanella, or Vibrio.
[0172] In other embodiments, the FadR polypeptide is a FadR homolog having an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO : one.
[0174] The identity of a FadR polypeptide having at least 80% identity to the amino acid sequence of SEQ ID NO: 1 is not particularly limited and one of ordinary skill in the art can easily identify homologues of FadR derived from E. coli. MG1655 using the methods described herein as well as methods known in the art.
[0176] As used herein, the terms "homologous" and "homologous" refer to a polynucleotide or a polypeptide that comprises a sequence that is at least 50% identical to the corresponding polynucleotide or polypeptide sequence. Preferably, the polynucleotides or homologous polypeptides have polynucleotide sequences or amino acid sequences that are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 2%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% homology to the corresponding amino acid sequence or polynucleotide sequence. As used herein, the terms sequence "homology" and sequence "identity" are used interchangeably.
[0178] In summary, "homology" calculations between two sequences can be performed as follows. Sequences are aligned for optimal comparative purposes (for example, gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment, and non-homologous sequences can be discarded for comparative purposes) . The amino acid or nucleotide residues of the corresponding amino acid positions or nucleotide positions of the first and second sequences are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or acid "identity" nucleic is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which is necessary to introduce for optimal alignment of the two sequences.
[0180] Sequence comparison and determination of percent homology between two sequences can be performed using a mathematical algorithm, such as BLAST (Altschul et al. J. Mol. Biol., 215 (3): 403-410 (1990)). The percent homology between two amino acid sequences can also be determined using the Needleman and Wunsch algorithm, which has been incorporated into the GAP program in the GCG software package, using a Blossum 62 matrix or a PAM250 matrix, and a gap weight. of 16, 14, 12, 10, 8, 6 or 4 and a weight of length of 1, 2, 3, 4, 5 or 6 (Needleman and Wunsch, J. Mol. Biol., 48: 444-453 (1970 )). The percent homology between two nucleotide sequences can also be determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. One skilled in the art can perform initial homology calculations and adjust the algorithm parameters accordingly. A preferred set of parameters (and the one that should be used if a practitioner is unsure about which parameters should be applied to determine whether a molecule is within a claims homology limitation) is a 62 Blossum scoring matrix with a penalty of Gap of 12, an extended gap penalty of 4, and a phase shift gap penalty of 5. They are known Additional sequence alignment methods in biotechnology techniques (see, eg, Rosenberg, BMC Bioinformatics, 6: 278 (2005); Altschul et al., FEBS J, 272 (20): 5101-5109 (2005)).
[0182] As used herein, the term "hybrid under conditions of low stringency, medium stringency, high stringency, or very high stringency" describes the conditions for hybridization and washing. Guidance for carrying out hybridization reactions can be found in "Current Protocols in Molecular Biology", John Wiley & Sons, N. Y. (1989), 6.3.1 - 6.3.6. In that reference, aqueous and non-aqueous methods are described, and either method can be used. The specific hybridization conditions cited herein are as follows: 1) Low stringency hybridization conditions in 6X sodium chloride / sodium citrate (SSC) at approximately 45 ° C, followed by two washes in 0.2X SSC, 0.1% SDS at least 50 ° C (wash temperature can be increased to 55 ° C for low stringency conditions); 2) medium stringency hybridization conditions in 6X SSC at approximately 45 ° C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60 ° C; 3) high stringency hybridization conditions in 6X SSC at approximately 45 ° C, followed by one or more washes in 0.2x SSC, 0.1% SDS at 65 ° C; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65 ° C, followed by one or more washes in 0.2X SSC, 1% SDS at 65 ° C. Very high stringency conditions (4) are the preferred conditions unless otherwise specified.
[0184] In some embodiments, the polypeptide is a fragment of any of the polypeptides described herein. The term "fragment" refers to a shorter part of a full-length polypeptide or protein ranging in size from four amino acid residues to the complete amino acid sequence minus one amino acid residue. In certain embodiments of the invention, a "fragment" refers to the entire amino acid sequence of a domain of a polypeptide or protein (eg, a substrate-binding domain or a catalytic domain).
[0186] An "endogenous" polypeptide refers to a polypeptide encoded by the genome of the parental microbial cell (also referred to as a "host cell") from which the recombinant cell is engineered (or "derived").
[0188] An "exogenous" polypeptide refers to a polypeptide that is not encoded by the genome of the parental microbial cell. A polypeptide variant (ie, mutant) is an example of an exogenous polypeptide.
[0190] The term "heterologous", as used herein, typically refers to a nucleotide sequence or protein that is not naturally present in an organism. For example, a polynucleotide sequence endogenous to a plant can be introduced into a host cell by recombinant methods and the plant polynucleotide is then a heterologous polynucleotide in a recombinant host cell.
[0192] In some embodiments, the polypeptide is a mutant or variant of any of the polypeptides described herein. The terms "mutant" and "variant", as used herein, refer to a polypeptide that has an amino acid sequence that differs from a wild-type polypeptide by at least one amino acid. For example, the mutant may comprise one or more of the following conservative amino acid substitutions: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine; replacement of a threonine with a serine; replacing an acid residue, such as aspartic acid and glutamic acid, with another acid residue; the replacement of a moiety bearing an amide group, such as asparagine and glutamine, with another moiety bearing an amide group; exchanging a basic residue, such as lysine and arginine, with another basic residue; and the replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue. In some embodiments, the mutant polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid substitutions, additions, insertions, or deletions.
[0194] As used herein, the term "mutagenesis" refers to a process by which the genetic information of an organism is stably changed. Mutagenesis of a nucleic acid sequence encoding a protein produces a mutant protein. Mutagenesis also refers to changes in noncoding nucleic acid sequences that result in modified protein activity.
[0196] As used herein, the term "gene" refers to nucleic acid sequences that encode an RNA product or a protein product, as well as operably linked nucleic acid sequences that affect the expression of the RNA or protein (for For example, such sequences include, but are not limited to, promoter or enhancer sequences) or operably linked nucleic acid sequences that encode sequences that affect RNA or protein expression (for example, such sequences include, but are not limited to, binding sites for ribosomes or translation control sequences).
[0198] In certain embodiments, the FadR polypeptide comprises a mutation in an amino acid residue corresponding to amino acid 219 of s Eq ID NO: 1. In certain embodiments, the mutation results in a substitution of the amino acid residue corresponding to amino acid 219 of the SEQ ID NO: 1 with an asparagine residue. The FadR (S219N) mutation has been described previously (Raman et al., J. Biol. Chem., 270: 1092-1097 (nineteen ninety five)).
[0200] Preferred or mutant fragments of a polypeptide retain some or all of the biological function (eg, enzymatic activity) of the corresponding wild-type polypeptide. In some embodiments, the fragment or mutant retains at least 75%, at least 80%, at least 90%, at least 95%, or at least 98% or more of the biological function of the corresponding wild-type polypeptide. . In other embodiments, the fragment or mutant retains approximately 100% of the biological function of the corresponding wild-type polypeptide. Guidance can be found to determine which amino acid residues can be substituted, inserted, or deleted without affecting biological activity using computer programs well known in the art, eg, LASERGENE ™ software (DNASTAR, Inc., Madison, WI).
[0202] In still other embodiments, a fragment or mutant exhibits increased biological function compared to the corresponding wild-type polypeptide. For example, a fragment or mutant may show at least 10%, at least 25%, at least 50%, at least 75%, or at least 90% improvement in enzyme activity compared to the polypeptide of corresponding wild type. In other embodiments, the fragment or mutant exhibits an improvement of at least 100% (eg, at least 200% or at least 500%) in enzyme activity compared to the corresponding wild-type polypeptide.
[0204] It is understood that the polypeptides described herein may have additional non-essential or conservative amino acid substitutions, which do not have a substantial effect on the function of the polypeptide. Whether or not a certain substitution will be tolerated (ie, whether it will not adversely affect the desired biological function, such as DNA binding or enzymatic activity) can be determined as described in Bowie et al. (Science, 247: 1306-1310 (1990)).
[0206] A "conservative amino acid substitution" is one in which the amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, thyroid, cysteine), nonpolar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (for example, threonine, valine, isoleucine), and side chains aromatic (eg tyrosine, phenylalanine, tryptophan, histidine).
[0208] Variants can be natural or created in vitro. In particular, such variants can be created using genetic engineering techniques, such as site-directed mutagenesis, random chemical mutagenesis, exonuclease III removal procedures, or standard cloning techniques. Alternatively, such variants, fragments, analogs, or derivatives can be created by chemical synthesis or modification procedures.
[0210] Methods for creating variants are well known in the art. These include procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids that encode polypeptides that have characteristics that enhance their value in industrial or laboratory applications. In such procedures, a large number of variant sequences are generated and characterized that have one or more nucleotide differences from the sequence obtained from the natural isolate. Typically, these nucleotide differences result in amino acid changes relative to polypeptides encoded by the nucleic acids of natural isolates.
[0212] For example, variants can be prepared using random and targeted mutagenesis (see, eg, Arnold, Curr. Opin. Biotech., 4: 450-455 (1993)). Random mutagenesis can be accomplished using error-prone PCR (see, for example, Leung et al., Technique, 1: 11-15 (1989); and Caldwell et al., PCR Methods Applic., 2: 28-33 ( 1992)). Site-directed mutagenesis can be performed using oligonucleotide-directed mutagenesis to generate site-specific mutations in any cloned DNA of interest (see, eg, Reidhaar-Olson et al., Science, 241: 53-57 (1988)). Other methods of generating variants include, for example, PCR assembly (see, for example, US Patent 5,965,408), sexual PCR mutagenesis (see, for example, Stemmer, Proc. Natl. Acad. Sci ., USA, 91: 10747-10751 (1994) and US Patents 5,965,408 and 5,939,250), recursive ensemble mutagenesis (see, for example, Arkin et al., Proc. Natl. Acad Sci., USA, 89: 7811-7815 (1992)) and exponential set mutagenesis (see, for example, Delegrave et al., Biotech. Res, 11: 1548-1552 (1993).
[0214] Variants can also be created by in vivo mutagenesis. In some embodiments, random mutations are generated in a nucleic acid sequence by propagating the sequence in a bacterial strain, such as an E. coli strain, that carries mutations in one or more of the DNA repair pathways. Such "mutator" strains have a higher random mutation rate than a wild-type strain. Propagation of a DNA sequence (eg, a polynucleotide sequence that encodes a PPTase) in one of these strains will eventually generate random mutations in the DNA. Mutant strains suitable for use in in vivo mutagenesis are described, for example, in International Patent Application Publication No. WO 1991/016427.
[0216] Variants can also be generated using cassette mutagenesis. In cassette mutagenesis, a small region of a double-stranded DNA molecule is replaced with a synthetic oligonucleotide "cassette" that differs from the native sequence. The oligonucleotide usually contains a complete and / or partially random native sequence.
[0217] As used herein, a "host cell" is a cell used to produce a product described herein (eg, a fatty aldehyde or a fatty alcohol). In any of the aspects of the disclosure described herein, the host cell may be selected from the group consisting of a mammalian cell, plant cell, insect cell, fungal cell (e.g., a filamentous fungal cell or a cell of yeast) and bacterial cell. In the present invention, the host cell is a bacterial cell. A host cell is referred to as a "genetically engineered host cell" or a "recombinant host cell" if the expression of one or more polynucleotides or polypeptides in the host cell is altered or modified compared to its expression in a corresponding wild-type host cell. Under the same conditions.
[0219] In some embodiments, the host cell is a gram-positive bacterial cell. In other embodiments, the host cell is a gram-negative bacterial cell.
[0221] In some aspects, the host cell is selected from the genus Escherichia, Bacillus, Lactobacillus, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Phachaemeurium, Penchaemeurium, Penchaemeurium, Penchaemeurium, Penchaemeurium, Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces, Yarrowia or Streptomyces.
[0223] In other embodiments, the host cell is a Bacillus lentus cell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, a Bacillus lichenformis cell, a Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillus thuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell, a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell.
[0225] In other aspects, the host cell is a Trichoderma koningii cell, a Trichoderma viride cell, a Trichoderma reesei cell, a Trichoderma longibrachiatum cell, an Aspergillus awamori cell, an Aspergillus fumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulans cell, an Aspergillus niger cell, an Aspergillus oryzae cell, a Humicola insolens cell, a Humicola lanuginose cell, a Rhodococcus opacus cell, a Rhizomucor miehei cell, or a Mucor michei cell.
[0226] In still other embodiments, the host cell is a Streptomyces lividans cell or a Streptomyces murinus cell.
[0228] In still other embodiments, the host cell is an Actinomycetes cell.
[0230] In some aspects, the host cell is a Saccharomyces cerevisiae cell. In some aspects, the host cell is a Saccharomyces cerevisiae cell.
[0232] In still other aspects, the host cell is a CHO cell, COS cell, VERO cell, BHK cell, HeLa cell, Cvl cell, MDCK cell, 293 cell, 3T3 cell, or PC12 cell.
[0234] In other respects, the host cell is a cell of a eukaryotic plant, alga, cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, purple non-sulfur bacteria, Extremophilic, yeast, fungus, an organism of genetically engineered from it or a synthetic organism. In some respects, the host cell is dependent on light or fixes carbon. In some aspects, the host cell has autotrophic activity. In some aspects, the host cell has photoautotrophic activity, such as in the presence of light. In some aspects, the host cell is heterotrophic or mixotrophic in the absence of light. In certain aspects, the host cell is an Avabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, Zea mays, Botryococcuse braunii, Chlamydomonas reinhardtii, Dunaliela salina, Synechococcus Sp. PCC 7002, ^{Synechococcus Sp. PCC 79803 Sp. Thermosynechococcus elongates} bP-1, ^Chlorobium tepidum, Chlorojlexus auranticus, Zromatiumm Vinosum, Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridium ljungdahlii, Clostridiuthermocellichia chromosomyconassymoresis, Penischari ssumvillium.
[0235] In certain preferred embodiments, the host cell is an E. coli cell. In some embodiments, the E. coli cell is an E. coli strain B, strain C, strain K, or strain W.
[0237] In other embodiments, the host cell is a Pantoea citrea cell.
[0239] As used herein, the term "permissive conditions for production" means any conditions that allow a host cell to produce a desired product, such as a fatty acid or a derivative of a fatty acid. In the same way, the expression "conditions in which the polynucleotide sequence of a vector is expressed" means any conditions that allow a host cell to synthesize a polypeptide. Suitable conditions include, for example, fermentation conditions. Fermentation conditions can comprise many parameters, such as temperature ranges, aeration levels, and media composition. Each of these conditions, individually and in combination, allows the host cell to grow. Illustrative culture media include broths or gels. Generally, the medium includes a carbon source that can be directly metabolized by a host cell. In addition, enzymes can be used in the medium to facilitate mobilization (eg, depolymerization of starch or cellulose to fermentable sugars) and subsequent metabolism of the carbon source.
[0241] As used herein, the phrase "carbon source" refers to a substrate or compound suitable for use as a carbon source for the growth of simple prokaryotic or eukaryotic cells. Carbon sources can be in a variety of forms, including, but not limited to, polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, and gases (eg, CO and CO ² ). Examples of carbon sources include, but are not limited to, monosaccharides, such as glucose, fructose, mannose, galactose, xylose, and arabinose; oligosaccharides, such as fructo-oligosaccharide and galacto-oligosaccharide; polysaccharides, such as starch, cellulose, pectin, and xylan; disaccharides, such as sucrose, maltose, and turanose; cellulosic material and variants such as sodium methyl cellulose and sodium carboxymethyl cellulose; saturated or unsaturated fatty acid esters, succinate, lactate and acetate; alcohols, such as ethanol, methanol, and glycerol, or mixtures thereof. Furthermore, the carbon source can be a product of photosynthesis, such as glucose. In certain preferred embodiments, the carbon source is biomass. In other preferred embodiments, the carbon source is glucose. In other preferred embodiments, the carbon source is sucrose.
[0243] As used herein, the term "biomass" refers to any biological material from which a carbon source is derived. In some embodiments, a biomass is processed into a carbon source, which is suitable for bioconversion. In other embodiments, the biomass does not require additional processing at a carbon source. The carbon source can be turned into a biofuel. An illustrative source of biomass is plant matter or vegetation, such as corn, sugar cane, or prairie grass. Another illustrative source of biomass is metabolic waste products, such as animal matter (eg, cow manure). Other illustrative sources of biomass include algae and other marine plants. Biomass also includes waste products from industry, agriculture, forestry, and households, including, but not limited to, fermentation residues, silage, straw, unprocessed wood, sewage, garbage, urban cellulosic waste, and food scraps. The term "biomass" can also refer to carbon sources, such as carbohydrates (eg, monosaccharides, disaccharides, or polysaccharides).
[0245] As used herein, the term "clone" typically refers to a cell or group of cells descended from and essentially genetically identical to a single common ancestor, for example, bacteria in a cloned bacterial colony arose from a single bacterial cell.
[0247] As used herein, the typical term "culture" refers to a liquid medium comprising viable cells. In one embodiment, a culture comprises cells that grow in a predetermined culture medium under controlled conditions, for example, a culture of recombinant host cells grown in liquid media comprising a source of selected carbon and nitrogen.
[0249] By "culture" or "culture" is meant the cultivation of a population of recombinant host cells under suitable conditions in a liquid or solid medium. In particular embodiments, cultivating refers to the fermentative bioconversion of a substrate until a final product is obtained. Culture media are well known, and the individual components of such culture media are available from commercial sources, for example, under the Difco ™ and BBL ™ trademarks. In a non-limiting example, the aqueous nutrient medium is a "rich medium" comprising complex sources of nitrogen, salts and carbon, such as YP medium, comprising 10 g / L peptone and 10 g / L extract of yeast from said medium.
[0251] To determine whether the conditions are sufficient to allow the production of a product or the expression of a polypeptide, a host cell can be cultured, for example, for about 4, 8, 12, 24, 36, 48, 72 or more hours. During and / or after cultivation, samples can be obtained and analyzed to determine if conditions allow production or expression. For example, host cells in the sample or the medium in which the host cells were cultured can be assayed for the presence of a desired product. When assaying for the presence of a fatty acid or fatty acid derivative, assays such as, but not limited to, EM, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), chromatography can be used. liquid (LC), CG coupled to a flame ionization detector (DIL), CG-MS and LC-MS. When assayed for expression of a polypeptide, techniques such as, but not limited to, Western blotting and dot blotting can be used.
[0253] In the methods of the invention, the production and isolation of fatty acids and fatty acid derivatives can be enhanced by optimizing the fermentation conditions. In some embodiments, the fermentation conditions are optimized to increase the percentage of the carbon source that is converted to hydrocarbon products. During normal cellular life cycles, carbon is used in cellular functions, such as the production of lipids, saccharides, proteins, organic acids, and nucleic acids. Reducing the amount of carbon needed for growth-related activities it can increase the efficiency of the conversion of the carbon source into product. This can be achieved by, for example, first growing the host cells to a desired density (eg, a density reached at the peak of the log phase of growth). At that point, replication checkpoint genes can be harnessed to stop cells from growing. Specifically, quorum sensing mechanisms can be used (reviewed in Camilli et al., Science 311: 1113 (2006); Venturi, FEMS Microbiol. Rev., 30: 274-291 (2006); and Reading et al., FEMS Microbiol Lett, 254: 11 (2006)) to activate checkpoint genes, such as p53, p21 or other checkpoint genes.
[0255] Genes that can be turned on to stop E. coli cell replication and growth include the umuDC genes. Overexpression of umuDC genes arrests progression from stationary phase to exponential growth (Murli et al., J. Bacteriol., 182: 1127-1135 (2000)). UmuC is a DNA polymerase that can perform translesional synthesis on noncoding lesions that are usually the result of ultraviolet (UV) and chemical mutagenesis. The umuDC gene products are involved in the translesional synthesis process and also serve as a checkpoint for DNA sequence damage. The umuDC gene products include UmuC, UmuD, umuD ', UmuD' ² C, UmuD ' ^2, and UmuD ² . Simultaneously, the product-producing genes can be activated, so that the need to use replication and maintenance pathways is minimized while preparing a fatty aldehyde or fatty alcohol. Host cells can also be engineered to express E. coli umuC and umuD in pBAD24 under the prpBCDE promoter system through de novo synthesis of this gene with the appropriate end-product production genes.
[0257] The host cell can be further engineered to express a recombinant cellulosome, which can allow the host cell to use cellulosic material as a carbon source. Exemplary cellulosomes suitable for use in the methods of the invention include, for example, the cellulosomes described in International Patent Application Publication WO 2008/100251. The host cell can also be engineered to efficiently assimilate carbon and use cellulosic materials as carbon sources according to the methods described in US Patents 5,000,000; 5,028,539; 5,424,202; 5,482,846; and 5,602,030. Furthermore, the host cell can be genetically engineered to express an invertase so that sucrose can be used as a carbon source.
[0259] In some embodiments of the fermentation methods of the invention, the fermentation chamber encloses a fermentation that undergoes continuous reduction, thereby creating a stable reducing environment. Electron balance can be maintained by releasing carbon dioxide (in gaseous form). Efforts to increase the balance of NAD / H and NADP / H can also facilitate stabilization of the electron balance. The availability of intracellular NADPH can also be enhanced by genetic engineering of the host cell to express a NADH: NADPH transhydrogenase. The expression of one or more transhydrogenases NADH: NADPH converts the NADH produced in glycolysis to NADPH, which can enhance the production of fatty aldehydes and fatty alcohols.
[0261] For small-scale production, the engineered host cells can be grown in batches of, for example, about 100 ml, 500 ml, 1 L, 2 L, 5 L, or 10 L; ferment; and induced to express a desired polynucleotide sequence, such as a polynucleotide sequence encoding a PPTase. For large-scale production, the engineered host cells can be grown in batches of approximately 10 µl, 100 µl, 1000 µl, 10,000 µl, 100,000 µl, 1,000,000 µl or larger; ferment; and induced to express a desired polynucleotide sequence.
[0263] Fatty acids and derivatives thereof produced by the methods of the invention are generally isolated from the host cell. The term "isolated", as used herein with respect to products, such as fatty acids and derivatives thereof, refers to products that are separated from cellular components, cell culture media, or chemical precursors. or synthetics. Fatty acids and derivatives thereof produced by the methods described herein can be relatively immiscible in the fermentation broth, as well as in the cytoplasm. Therefore, fatty acids and their derivatives can accumulate in an organic phase, either intracellularly or extracellularly. Collecting the products in the organic phase can reduce the impact of the fatty acid or fatty acid derivative on cell function and can allow the host cell to produce more product.
[0265] In some embodiments, the fatty acids and fatty acid derivatives produced by the methods of the invention are purified. As used herein, the term "purify", "purified" or "purification" means the removal or isolation of a molecule from its environment by, for example, isolation or separation. "Essentially purified" molecules are at least about 60% free (eg, at least about 70% free, at least about 75% free, at least about 85% free, at least about 90% free, at least about 95% free, at least about 97% free, at least about 99% free) of other components with which they are associated. As used herein, these terms also refer to the removal of contaminants from a sample. For example, removal of contaminants can result in an increase in the percentage of a fatty aldehyde or a fatty alcohol in a sample. For example, when a fatty aldehyde or fatty alcohol is produced in a host cell, the fatty aldehyde or fatty alcohol can be purified by the removal of proteins from the host cell. After purification, the percentage of a fatty acid or derivative thereof in the sample is increased.
[0267] As used herein, the terms "purify", "purified" and "purification" are relative terms that do not require absolute purity. Thus, for example, when a fatty acid or derivative thereof is produced in host cells, a purified fatty acid or derivative thereof is a fatty acid or derivative thereof that is substantially separated from other cellular components (e.g. , nucleic acids, polypeptides, lipids, carbohydrates or other hydrocarbons).
[0269] Additionally, a purified fatty acid preparation or a purified fatty acid derivative preparation is a fatty acid preparation or a fatty acid derivative preparation in which the fatty acid or derivative thereof is substantially free of contaminants, such as that could be after fermentation. In some embodiments, a fatty acid or derivative thereof is purified when at least 50% by weight of a sample is composed of the fatty acid or fatty acid derivative. In other embodiments, a fatty acid or derivative thereof is purified when at least about 60%, for example, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 92% or more by weight of a sample is composed of the fatty acid or derivative thereof. Alternatively, or in addition, a fatty acid or derivative thereof is purified when less than about 100%, for example, less than about 99%, less than about 98%, less than about 95%, less than about 90% or less of about 80% by weight of a sample is composed of the fatty acid or derivative thereof. Thus, a purified fatty acid or derivative thereof may have a purity level limited by either of the two endpoints above. For example, a fatty acid or derivative thereof can be purified when at least about 80% -95%, at least about 85% -99%, or at least about 90% -98% of a sample is composed of the acid. fatty or fatty acid derivative.
[0271] The fatty acid or derivative thereof may be present in the extracellular environment, or it may be isolated from the extracellular environment of the host cell. In certain embodiments, a fatty acid or derivative thereof is secreted by the host cell. In other embodiments, a fatty acid or derivative thereof is transported to the extracellular environment. In still other embodiments, the fatty acid or derivative thereof is passively transported to the extracellular medium. A fatty acid or derivative thereof can be isolated from a host cell using methods known in the art, such as those disclosed in International Patent Application Publications WO 2010/042664 and WO 2010/062480.
[0273] The methods described herein can result in the production of homogeneous compounds wherein at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least approximately 95%, of the fatty acids or fatty acid derivatives produced will have carbon chain lengths varying by less than 6 carbons, less than 5 carbons, less than 4 carbons, less than 3 carbons, or less than about 2 carbons. Alternatively, or in addition, the methods described herein may result in the production of homogeneous compounds wherein less than about 98%, less than about 95%, less than about 90%, less than about 80% or less of about 70% of the fatty acids or fatty acid derivatives produced will have carbon chain lengths that vary by less than 6 carbons, less than 5 carbons, less than 4 carbons, less than 3 carbons or less of about 2 carbons. Thus, fatty acids or fatty acid derivatives can have a degree of homogeneity limited by either of the two endpoints above. For example, the fatty acid or fatty acid derivative may have a degree of homogeneity where about 70% -95%, about 80% -98%, or about 90% -95% of the fatty acids or acid derivatives fats produced will have carbon chain lengths that vary by less than 6 carbons, less than 5 carbons, less than 4 carbons, less than 3 carbons, or less than about 2 carbons. These compounds can also be produced with a relatively uniform degree of saturation.
[0275] As a result of the methods of the present invention, one or more of the titer, yield, or productivity of the fatty acid or derivative thereof produced by the engineered host cell having an altered expression level of a FadR polypeptide increases relative to that of the corresponding wild-type host cell.
[0277] The term "titer" refers to the amount of fatty acid or fatty acid derivative produced per unit volume of host cell culture. In any aspect of the compositions and methods described herein, a fatty acid or fatty acid derivative such as a terminal olefin, a fatty aldehyde, a fatty alcohol, an alkane, a fatty ester, a ketone, or a internal olefin is produced at a titer of about 25mg / l, about 50mg / l, about 75mg / l, about 100mg / l, about 125mg / l, about 150mg / l, about 175mg / l, about 200mg / l, about 225mg / l, about 250mg / l, about 275mg / l, about 300mg / l, about 325mg / l, about 350mg / l, about 375mg / l, about 400 mg / l, about 425 mg / l, about 450 mg / l, about 475 mg / l, about 500 mg / l, about 525 mg / l, about 550mg / l, about 575mg / l, about 600mg / l, about 625mg / l, about 650mg / l, about 675mg / l, about 700mg / l, about 725mg / l, about 750 mg / l, about 775 mg / l, about 800 mg / l, about 825 mg / l, about 850 mg / l, about 875 mg / l, about 900 mg / l, about 925 mg / l, about 950 mg / l l, about 975 mg / l, about 1000 g / l, about 1050 mg / l, about 1075 mg / l, about 1100 mg / l, about 1125 mg / l, about 1150 mg / l, about 1175 mg / l, about 1200 mg / l, about 1225 mg / l, about 1250 mg / l, about 1275 mg / l, about 1300 mg / l, about 1325 mg / l, about 1350 mg / l, about 1375 mg / l, about 1400 mg / l, about 1425 mg / l, about 1450 mg / l, about 1475 mg / l, about 1500 mg / l, about 1525 mg / l, about 1550 mg / l, about 1575 mg / l, about 1600 mg / l, about 1625 mg / l, about 1650 mg / l, about 1675 mg / l, about 1700 mg / l l, about 1725 mg / l, about 1750 mg / l, about 1775 mg / l, about 1800 mg / l, about 1825 mg / l, about 1850 mg / l, about 1875 mg / l, about 1900 mg / l, about 1925 mg / l, about 1950 mg / l, about 1975 mg / l, about 2000 mg / l or a range limited by either of the two above values. In other embodiments, a fatty acid or fatty acid derivative is produced at a titer of more than 2000 mg / l, more than 5000 mg / l, more than 10,000 mg / l or more, such as 50 g / l, 70 g / l, 100 g / l, 120 g / l, 150 g / l or 200 g / l.
[0279] As used herein, "yield of fatty acid derivative produced by a host cell" refers to the efficiency with which an input carbon source is converted to product (ie, fatty alcohol or fatty aldehyde). in a host cell. Host cells engineered to produce fatty acid derivatives according to the methods of the invention have a yield of at least 3%, at least 4%, at least 5%, at least 6%, when at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least one 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23% , at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29% or at least 30% or a range limited by any of the two previous values. In other embodiments, a fatty acid derivative or derivatives are produced in a yield greater than 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Alternatively, or in addition, the yield is about 30% or less, about 27% or less, about 25% or less, or about 22% or less. Therefore, performance may be limited by any two of the above endpoints. For example, the yield of a fatty acid derivative or derivatives produced by the recombinant host cell according to the methods of the invention can be 5% to 15%, 10% to 25%, 10% to 22%, 15% to 27%, 18% to 22%, 20% to 28%, or 20% to 30%. Yield can refer to a particular fatty acid derivative or to a combination of fatty acid derivatives produced by a given recombinant host cell culture.
[0281] In one approach, the term "productivity of the fatty acid or derivative thereof produced by a host cell" refers to the amount of fatty acid or fatty acid derivative produced per unit of host cell culture volume per unit of culture density. host cell. In any aspect of the compositions and methods described herein, the productivity of a fatty acid or a fatty acid derivative such as an olefin, a fatty aldehyde, a fatty alcohol, an alkane, a fatty ester, or a ketone produced by an engineered host cell is at least about 3 mg / L / OD ⁶⁰⁰ , at least about 6 mg / L / OD ⁶⁰⁰ , at least about 9 mg / L / OD ⁶⁰⁰ , at least about 12 mg / l / OD ⁶⁰⁰ or at least about 15 mg / l / OD ⁶⁰⁰ . Alternatively, or in addition, the productivity is about 50 mg / L / OD ⁶⁰⁰ or less, about 40 mg / L / OD ⁶⁰⁰ or less, about 30 mg / L / OD ⁶⁰⁰ or less, or about 20 mg / L / OD ⁶⁰⁰ or less. Therefore, productivity can be limited by any two of the above endpoints. For example, the productivity can be from about 3 to about 30 mg / L / OD ⁶⁰⁰ , from about 6 to about 20 mg / L / OD ^600, or from about 15 to about 30 mg / L / OD ⁶⁰⁰ .
[0283] In another approach, the term "productivity" refers to the amount of a fatty acid derivative or derivatives produced per unit volume of host cell culture per unit time. In any aspect of the compositions and methods described herein, the productivity of a fatty acid derivative or derivatives produced by a recombinant host cell is at least 100 mg / L / hour, at least 200 mg / L / hour0, at least 300 mg / l / hour, at least 400 mg / l / hour, at least 500 mg / l / hour, at least 600 mg / l / hour, at least 700 mg / l / hour, at least 800 mg / l / hour, at least 900 mg / l / hour, at least 1000 mg / l / hour, at least 1100 mg / l / hour, at least 1200 mg / l / hour, at least 1300 mg / l / hour , at least 1400 mg / l / hour, at least 1500 mg / l / hour, at least 1600 mg / l / hour, at least 1700 mg / l / hour, at least 1800 mg / l / hour, at least 1900 mg / l / hour, at least 2000 mg / l / hour, at least 2100 mg / l / hour, at least 2200 mg / l / hour, at least 2300 mg / l / hour, at least 2400 mg / l / hour or at least 2500 mg / l / hour. As an alternative, or in addition, the productivity is 2500 mg / l / hour or less, 2000 mg / l / OD600 or less, 1500 mg / l / OD600 or less, 120 mg / l / hour or less, 1000 mg / l / hour or less, 800 mg / l / hour or less, or 600 mg / l / hour or less. Therefore, productivity can be limited by any two of the above endpoints. For example, productivity can be 3 to 30 mg / l / hour, 6 to 20 mg / l / hour, or 15 to 30 mg / l / hour. For example, the productivity of a fatty acid derivative or derivatives produced by a recombinant host cell according to the methods can be 500 mg / L / hour to 2500 mg / L / hour, or 700 mg / L / hour at 2000 mg / l / hour. Productivity can refer to a particular fatty acid derivative or a combination of fatty acid derivatives produced by a given recombinant host cell culture.
[0285] In the methods of the invention, the production and isolation of a desired fatty acid or derivative thereof (eg, acyl-CoA, fatty acids, terminal olefins, fatty aldehydes, fatty alcohols, alkanes, alkenes, wax esters, ketones and internal olefins) can be enhanced by altering the expression of one or more genes involved in the regulation of the production, degradation and / or secretion of fatty acid, fatty ester, alkane, alkene, olefin, fatty alcohol, in the engineered host cell genetics.
[0287] FadR is known to modulate the expression and / or activity of numerous genes, including fabA, fabB, iclR, fadA, fadB, fadD, fadE, fadI, fadJ, fadl, fadM, uspA, aceA, aceB, and aceK. In some embodiments of the methods described herein, the engineered host cell further comprises an altered level of expression of one or more genes selected from the group consisting of fabA, fabB, iclR, fadA, fadB, fadD, fadE, fadI, fadJ, fadl, fadM, uspA, aceA, aceB, and aceK compared to the expression level of the selected gene (s) in a corresponding wild-type host cell. Example accession numbers for polypeptides encoded by the FadR target genes include fabA (NP_415474), fabB (BAA16180), (NP_418442), fadA (YP_026272.1), fadB (NP_418288.1), fadD (AP_002424), fadE (NP_414756.2), fadI (NP_416844.1), fadJ (NP_416843.1), fadl (AAC75404), fadM (NP_414977.1), uspA (AAC76520), aceA (AAC76985.1), aceB (AAC76984.1) and aceK (AAC76986.1).
[0289] In FIG. 1 exemplary enzymes and polypeptides for use in the practice of the invention are listed. One of ordinary skill in the art will understand that depending on the purpose (eg, the desired fatty acid or fatty acid derivative product), the specific genes (or combinations of genes) listed in FIG. 1 can be overexpressed, modified, attenuated, or suppressed in a host cell having an altered expression level of a FadR polypeptide.
[0291] In some embodiments, the method comprises modifying the expression of a gene encoding one or more of a thioesterase (eg, TesA), a decarboxylase, a carboxylic acid reductase (CAR; eg, CarB), an alcohol dehydrogenase (aldehyde reductase); an aldehyde decarbonylase, a fatty alcohol forming acyl-CoA reductase (FAR), an acyl ACP reductase (AAR), an ester synthase, an acyl-CoA reductase (ACR1), OleA, OleCD and OleBCD.
[0293] In certain embodiments of the invention, the engineered host cell having an altered expression level of a FadR polypeptide can be engineered to further comprise a polynucleotide sequence encoding a polypeptide: (1) having thioesterase activity ( EC 3.1.2.14), wherein the genetically engineered host cell synthesizes fatty acids; (2) having decarboxylase activity, wherein the engineered host cell synthesizes terminal olefins; (3) having carboxylic acid reductase activity, wherein the engineered host cell synthesizes fatty aldehydes; (4) having carboxylic acid reductase and alcohol dehydrogenase activity (EC 1.1.1.1), wherein the genetically engineered host cell synthesizes fatty alcohols; (5) having carboxylic acid reductase and aldehyde decarbonylase activity (EC 4.1.99.5), wherein the engineered host cell synthesizes alkanes; (6) having acyl-CoA reductase activity (EC 1.2.1.50), where the microorganism synthesizes fatty aldehydes; (7) having acyl-CoA reductase activity (EC 1.2.1.50) and alcohol dehydrogenase activity (EC 1.1.1.1), wherein the genetically engineered host cell synthesizes fatty alcohols; (8) having acyl-CoA reductase activity (EC 1.2.1.50) and aldehyde decarbonylase activity (EC 4.1.99.5), wherein the engineered host cell synthesizes alkanes; (9) having alcohol-forming acyl CoA reductase activity wherein the engineered host cell synthesizes fatty aldehydes and fatty alcohols; (10) having carboxylic acid reductase activity, wherein the engineered host cell synthesizes fatty aldehydes; (11) having acyl ACP reductase activity, wherein the engineered host cell synthesizes fatty aldehydes; (12) having acyl ACP reductase activity and alcohol dehydrogenase activity (EC 1.1.1.1), wherein the genetically engineered host cell synthesizes fatty alcohols; (13) having acyl ACP reductase activity and aldehyde decarbonylase activity (EC 4.1.99.5), wherein the engineered host cell synthesizes alkanes; (14) having ester synthase activity (EC 3.1.1.67), wherein the engineered host cell synthesizes fatty esters; (15) having ester synthase activity (EC 3.1.1.67) and (a) carboxylic acid reductase activity, (b) acyl-CoA reductase activity, (c) acyl ACP reductase activity or (d) alcohol dehydrogenase activity (EC 1.1. 1.1), wherein the engineered host cell synthesizes wax esters; (16) having OleA activity, wherein the engineered host cell synthesizes 2-alkyl-3-keto-acyl CoA and ketones; or (17) having OleCD or OleBCD activity, wherein the engineered host cell synthesizes internal olefins.
[0295] In some embodiments, the method further comprises modifying the expression of a gene encoding a fatty acid synthase in the host cell. As used herein, "fatty acid synthase" means any enzyme involved in fatty acid biosynthesis. In certain embodiments, modifying the expression of a gene encoding a fatty acid synthase includes expressing a gene encoding a fatty acid synthase in the host cell and / or increasing the expression or activity of a fatty acid synthase. endogenous to the host cell. In alternative embodiments, modifying the expression of a gene encoding a fatty acid synthase includes attenuating a gene encoding a fatty acid synthase in the host cell and / or decreasing the expression or activity of a fatty acid synthase. endogenous to the host cell. In some embodiments, the fatty acid synthase is a thioesterase (EC 3.1.1.5 or EC 3.1.2.14). In particular embodiments, the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
[0296] In other embodiments, the host cell is genetically modified to express an attenuated level of a fatty acid degrading enzyme relative to a wild-type host cell. As used herein, the term "fatty acid degrading enzyme" means an enzyme involved in the decomposition or conversion of a fatty acid or fatty acid derivative into another product, such as, but not limited to, an acyl -CoA synthase. In some embodiments, the host cell is genetically modified to express an attenuated level of an acyl-CoA synthase relative to a wild-type host cell. In particular embodiments, the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfl, PJI-4354, EAV15023, fadDI, fadD2, RPC_4074, fadDD35, fadDD22, faa3p, or the gene encoding the YP_002028218 protein. In certain embodiments, the genetically modified host cell comprises an inactivation of one or more genes encoding a fatty acid degrading enzyme, such as the acyl-CoA synthase genes mentioned above.
[0298] The fatty acid biosynthetic pathway in host cells uses the precursors acetyl-CoA and malonyl-CoA. Steps in this pathway are catalyzed by enzymes from the fatty acid biosynthesis (fab) and acetyl-CoA carboxylase (acc) gene families (see, for example, Heath et al., Prog. Lipid Res. 40 (6) : 467-97 (2001)). Acetyl-CoA is carboxylated by acetyl-CoA carboxylase (EC 6.4.1.2), which is a multi-subunit enzyme encoded by four separate genes (accA, accB, accC, and accD) in most prokaryotes, to form malonil -CoA. In some bacteria, such as Corynebacterium glutamicus, acetyl-CoA carboxylase consists of two subunits, ^{AccDA [YP_225123.1] and AccBC} [Yp_224991], ^{encoded by accDA and accBC, respectively. Depending on the} desired fatty acid or fatty acid derivative product, specific fab and / or acc genes (or combinations thereof) can be overexpressed, modified, attenuated, or deleted in an engineered host cell.
[0300] In some embodiments, an acetyl-CoA carboxylase complex is overexpressed in the engineered host cell. In certain embodiments, acetyl-CoA carboxylase subunit genes are derived from one or more of Corynebacterium glutamicum, Escherichia coli, Lactococcus lactis, Kineococcus radiotolerans, Desulfovibrio desulfuricans, Erwinia amylovora, Rhodostenriocystrophrumii, Rhodostenriocystrophrumii. sp. PCC6803 and Synechococcus elongatus.
[0302] Biotin protein ligase (EC 6.3.4.15) is an enzyme that catalyzes the covalent binding of biotin to the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. In some embodiments of the present invention, a biotin protein ligase is expressed or overexpressed in the engineered host cell. In certain embodiments, the biotin protein ligase is Corynebacterium glutamicum birA (YP_225000) or Saccharomyces cerevisiae bpll (NP_010140).
[0304] The production of fatty acid esters such as FAME or FAEE in a host cell can be facilitated by the expression or overexpression of an ester synthase (EC 2.3.1.75 or EC 3.1.1.67) in a genetically engineered host cell. In some embodiments, the ester synthase is ES9 from Marinobacter hydrocarbonoclasticus (SEQ ID NO: 2), ES8 from Marinobacterhydrocar-bonoclasticus (SEQ ID NO: 3), AtfA1 from Alcanivorax borkumensis SK2 (SEQ ID NO: 4), AtfA2 from Alcanivorax borkumens SK2 (SEQ ID NO: 5), diacylglycerol O-acyltransferase from Marinobacter aquaeolei VT8 (SEQ ID NO: 6 or SEQ ID NO: 7), a wax synthase or a wax ester bifunctional synthase / acyl-CoA: diacylglycerol acyltransferase (wax- dgaT).
[0306] In certain embodiments, a gene encoding a fatty aldehyde biosynthetic polypeptide is expressed or overexpressed in the host cell. Exemplary fatty aldehyde biosynthetic polypeptides suitable for use in the methods of the invention are disclosed, for example, in International Patent Application Publication WO 2010/042664. In preferred embodiments, the fatty aldehyde biosynthetic polypeptide has carboxylic acid reductase activity (EC 6.2.1.3 or EC 1.2.1.42), eg, fatty acid reductase activity.
[0307] In the methods of the invention, the polypeptide having carboxylic acid reductase activity is not particularly limited. Exemplary polypeptides having carboxylic acid reductase activity that are suitable for use in the methods of the present invention are disclosed, for example, in ^{International Patent Application Publications WO 2010/062480 and} wO ^{2010/042664. In some embodiments, the polypeptide having} carboxylic acid reductase activity is CarB from M. smegmatis (YP_889972) (SEQ ID NO: 8). In other embodiments, the polypeptide having carboxylic acid reductase activity is CarA [ABK75684] from M. smegmatis, FadD9 [AAK46980] from M. tuberculosis, CAR [AAR91681] from Nocardia sp. NRRL 5646, CAR [YP-001070587] from Mycobacterium sp. JLS or CAR [YP-118225] from Streptomyces griseus. The expressions "carboxylic acid reductase", "CAR", and "polypeptide fatty aldehyde biosynthetic "are used interchangeably herein.
[0309] In certain embodiments, a thioesterase and a carboxylic acid reductase are expressed or overexpressed in the engineered host cell.
[0311] In some embodiments, a gene encoding a fatty alcohol biosynthetic polypeptide is expressed or overexpressed in the host cell. Exemplary fatty alcohol biosynthetic polypeptides suitable for use in the methods of the invention are disclosed, for example, in International Patent Application Publication WO 2010/062480. In certain embodiments, the fatty alcohol biosynthetic polypeptide has aldehyde reductase or alcohol dehydrogenase activity (EC 1.1.1.1). Examples of fatty alcohol biosynthetic polypeptides include, but are not limited to, AlrA from Acenitobacter sp. M-1 (SEQ ID NO: 9) or homologs of AlrA and alcohol dehydrogenases from endogenous E. coli such as YjgB, (AAC77226) (SEQ ID NO: 10), DkgA (NP_417485), DkgB (NP_414743), YdjL (AAC74846 ), YdjJ (NP_416288), AdhP (NP_415995), YhdH (NP_417719), YahK (NP_414859), YphC (AAC75598), YqhD (446856) and YbbO [AAC73595.1].
[0313] As used herein, the term "alcohol dehydrogenase" is a peptide capable of catalyzing the conversion of a fatty aldehyde to alcohol (eg, fatty alcohol). One of ordinary skill in the art will appreciate that certain alcohol dehydrogenases are capable of catalyzing other reactions as well. For example, certain alcohol dehydrogenases will accept substrates other than fatty aldehydes, and these nonspecific alcohol dehydrogenases are also included in the term "alcohol dehydrogenase." Exemplary alcohol dehydrogenases suitable for use in the methods of the invention are disclosed, for example, in International Patent Application Publication WO 2010/062480.
[0315] In some embodiments, a thioesterase, a carboxylic acid reductase, and an alcohol dehydrogenase are expressed or overexpressed in the engineered host cell. In certain embodiments, the thioesterase is tesA (SEQ ID NO: 11), the carboxylic acid reductase is carB (SEQ ID NO: 8), and the alcohol dehydrogenase is YjgB (SEQ ID NO: 10) or AlrAadp1 (SEQ ID NO: 9 ).
[0317] Phosphopantetheine transferases (PPTases) (EC 2.7.8.7) catalyze the transfer of 4'-phosphopantetheine from CoA to a substrate. CAR from Nocardia and several of its homologues contain a putative binding site for 4-phospho-pantethein (PPT) (He et al., Appl. Environ. Microbiol., 70 (3): 1874-1881 (2004)). In some embodiments of the invention, a PPTase is expressed or overexpressed in an engineered host cell. In certain embodiments, the PPTase is EntD from E. coli MG1655 (SEQ ID NO: 12).
[0319] In some embodiments, a thioesterase, a carboxylic acid reductase, a PPTase, and an alcohol dehydrogenase are expressed or overexpressed in the engineered host cell. In certain embodiments, the thioesterase is tesA (SEQ ID NO: 11), the carboxylic acid reductase is carB (SEQ ID NO: 8), the PPTase is entD (SEQ ID NO: 12), and the alcohol dehydrogenase is yjgB (SEQ ID NO: 10) or alrAadp1 (SEQ ID NO: 9).
[0320] The disclosure also provides a fatty acid or a fatty derivative produced by any of the methods described herein. A fatty acid or derivative thereof produced by any of the methods described herein can be used directly as fuel, fuel additives, starting materials for the production of other chemical compounds (e.g. polymers, surfactants, plastics, fabrics, solvents, adhesives, etc.) or personal care additives. These compounds can also be used as feedstock for subsequent reactions, eg, hydrogenation, catalytic cracking (eg, via hydrogenation, pyrolysis, or both), to prepare other products.
[0322] In some aspects, the disclosure provides a biofuel composition comprising the fatty acid or derivative thereof produced by the methods described herein. As used herein, the term "biofuel" refers to any fuel derived from biomass. Biofuels can replace petroleum-based fuels. For example, biofuels include transportation fuels (eg gasoline, diesel, jet fuel, etc.), heating fuels, and electricity generation fuels. Biofuels are a renewable energy source. As used herein, the term "biodiesel" means a biofuel that can be a substitute for diesel, which is derived from petroleum. Biodiesel can be used in internal combustion diesel engines, either in pure form, which is called "pure" biodiesel, or as a blend in any concentration with petroleum-based diesel. Biodiesel can include esters or hydrocarbons, such as alcohols. In certain aspects, the biofuel is selected from the group consisting of a biodiesel, a fatty alcohol, a fatty ester, a triacylglyceride, a gasoline, or a jet fuel.
[0324] To boost the performance of a fuel or engine, fuel additives are used. For example, fuel additives can be used to alter the freezing / gelling point, cloud point, lubricity, viscosity, oxidative stability, ignition quality, octane level, and / or flash point of a fuel. fuel. In the United States, all fuel additives must be registered with the Environmental Protection Agency (EPA). The names of the fuel additives and the companies that sell the fuel additives are available to the public by contacting the EPA or visiting the EPA website.
[0325] One of ordinary skill in the art will appreciate that a biofuel produced according to the methods described herein can be mixed with one or more fuel additives to convey a desired quality.
[0327] The disclosure also provides a surfactant composition or a detergent composition comprising a fatty alcohol produced by any of the methods described herein. One of ordinary skill in the art will appreciate that, depending on the intended purpose of the surfactant or detergent composition, different fatty alcohols can be produced and used. For example, when the fatty alcohols described herein are used as a raw material for the production of surfactants or detergents, one of ordinary skill in the art will appreciate that the characteristics of the fatty alcohol raw material will affect the characteristics of the surfactant or detergent composition produced. Thus, the characteristics of the surfactant or detergent composition can be selected by producing particular fatty alcohols for use as a raw material.
[0329] A fatty alcohol-based surfactant and / or detergent composition described herein may be mixed with other surfactants and / or detergents well known in the art. In some aspects, the blend may include at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60% or a range limited by either of the above two values, by weight of the fatty alcohol. In other examples, a surfactant or detergent composition may be prepared that includes at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 40%. less about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or a range limited by either of the two previous values, by weight of a fatty alcohol that includes a carbon chain that has 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 carbons in length. Such surfactant or detergent compositions may also include at least one additive, such as a microemulsion or a surfactant or detergent from non-microbial sources such as vegetable oils or petroleum, which may be present in an amount of at least about 5%, at least about 5%. less about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or a range limited by either of the above two values, by weight of the fatty alcohol.
[0331] Bioproducts (eg, fatty acids, acyl-CoA, hydrocarbons, fatty aldehydes, fatty alcohols, fatty esters, surfactant compositions, and biofuel compositions) produced according to the methods of the invention can be distinguished from organic compounds derived from carbon. petrochemical based on double carbon isotopic footprint or 14C dating. Additionally, the specific source of biologically derived carbon (eg, glucose versus glycerol) can be determined by double carbon isotopic footprinting (see, eg, US Patent 7,169,588).
[0333] The ability to distinguish bioproducts from petroleum-based organic compounds is beneficial in tracking these materials in trade. For example, organic compounds or chemicals that comprise both biological and petroleum-derived carbon isotope profiles can be distinguished from organic compounds and chemicals made only from petroleum-based materials. Thus, materials prepared according to the inventive methods can be followed commercially based on their unique carbon isotope profile.
[0335] Bioproducts can be distinguished from petroleum-based organic compounds by comparing the stable carbon isotope ratio (13C / 12C) in each fuel. The 13C / 12C ratio in a given bioproduct is a consequence of the 13C / 12C ratio in atmospheric carbon dioxide at the time the carbon dioxide is fixed. It also reflects the exact metabolic pathway. There are also regional variations. Oil, C ³ plants (broadleaf), C ⁴ plants (grasses) and marine carbonates show significant differences in 13C / 12C and the corresponding 813C values. Furthermore, the lipid matter of C ³ and C ⁴ plants is analyzed differently from that of materials derived from the carbohydrate components of the same plants as a consequence of the metabolic pathway.
[0337] The 13C measurement scale was originally defined by a zero setting with Pee Dee Belemnite limestone (PDB, Pee Dee Belemnite), where values are given in parts per thousand deviations from this material. Values for "813C" are expressed in parts per thousand (per thousand), abbreviated,% or and are calculated as follows:
[0339] 813C (% o) = [(13C / 12C) sample - (13C / 12C) standard] / (13C / 12C) standard X 1000
[0341] In some aspects, a bioproduct produced in accordance with the methods of the invention has an 813C of about -30 or more, about -28 or more, about -27 or more, about -20 or plus, about -18 or more, about -15 or more, about -13 or more, or about -10 or more. Alternatively, or in addition, a bioproduct has an 813C of about -4 or less, about -5 or less, about -8 or less, about -10 or less, about -13 or less, about -15 or less, about - 18 or less or about -20 or less. Thus, the byproduct may have an 813C limited by any two of the above endpoints. For example, the bioproduct may have an 813C of from about -30 to about -15, from about -27 to about -19, from about -25 to about -21, from about -15 to about -5, from about -13 to about -7, or about -13 to about -10. In some embodiments, the byproduct can have an 813C of about -10, -11, -12, or -12.3. In other aspects, the byproduct has an 813C of about -15.4 or more. In still other aspects, the byproduct has an 813C of about -15.4 to about -10.9, or an 813C of about -13.92 to about -13.84.
[0343] Bioproducts can also be distinguished from petroleum-based organic compounds by comparing the amount of 14C in each compound. Because 14C has a nuclear half-life of 5730 years, "older" carbon-containing petroleum fuels can be distinguished from "newer" carbon-containing byproducts (see, for example, Currie, "Source Apportionment of Atmospheric Partiols "Characterization of Environmental Partióles, J. Buffle and HP van Leeuwen, Eds., Vol. I of the IUPAC Environmental Analytical Chemistry Series, Lewis Publishing, Inc., pp. 3-74 (1992)).
[0345] 14C can be measured by accelerator mass spectrometry (EMA), with results given in units of "modern carbon fraction" (fM). The fM is defined by the National Institute of Standards and Technology (NIST, or f Standards and Technology) Standard Reference Materials (MRP) 4990B and 4990C. As used herein, "modern carbon fraction" or fM has the same meaning as defined by the National Institute of Standards and Technology (NISt) Standard Reference Materials (SRM) 4990B and 4990C. ), known as oxalic acid standards HOxI and HOxII, respectively. The fundamental definition refers to 0.95 times the 14C / 12C isotope ratio of HOxI (referred to as AD 1950). This is roughly equivalent to wood from before the industrial revolution corrected for disintegration. For the current living biosphere (plant matter), fM is approximately 1.1.
[0347] In some aspects, a bioproduct produced in accordance with the methods of the invention has an fM14C of at least about 1, for example, at least about 1.003, at least about 1.01, at least about 1.04, at least about 1.111. , at least about 1.18 or at least about 1.124. Alternatively, or in addition, the byproduct has a fM14C of about 1.130 or less, eg, about 1.124 or less, about 1.18 or less, about 1.111 or less, or about 1.04 or less. Thus, the byproduct may have an fM14C limited by any two of the above endpoints. For example, the byproduct may have an fM14C of about 1.003 to 1.124, an fM14C of about 1.04 to about 1.18, or an fM14C of about 1.111 to about 1.124.
[0349] Another measure of 14C is known as the modern carbon percentage, that is, pMC. For an archaeologist or geologist using 14C dating, AD 1950 equals "zero years." This also represents a pMC of 100. The "carbon bomb" in the atmosphere reached almost double the normal level in 1963 at the peak of thermo-nuclear weapons tests. Its distribution within the atmosphere has approximated since its appearance, showing values above 100 pMC for plants and animals living since AD 1950. It has gradually decreased over time and the current value is close to a pMC of 107.5. This means that a new biomass material, such as corn, would provide a distinctive 14C of pMC close to 107.5. Petroleum based compounds will have a pMC value of zero. The combination of fossil carbon with current carbon will lead to a dilution of the current pMC content. Assuming that a pMC of 107.5 represents the 14C content of current biomass materials and a pMC of 0 represents the 14C content of petroleum products, the measured value of pMC for that material will reflect the proportions of the two component types. For example, a material derived 100% from today's soybeans would have a radiocarbon distinctive with a pMC value close to 107.5. If that material were diluted 50% with petroleum-based products, the resulting mixture would have a distinctive radiocarbon of about 54 pMC.
[0351] A biobased carbon content is derived by assigning "100%" equal to 107.5 pMC and "0%" equal to 0 pMC. For example, a sample that measures a pMC of 99 will provide an equivalent biobased carbon content of 93%. This value is known as the mean biobased carbon result, and it assumes that all components within the analyzed material originated from either the current biological material or the petroleum-based material.
[0353] In some aspects, a bioproduct produced in accordance with the methods of the invention has a pMC of at least about 50, at least about 60, at least about 70, at least about 75, at least about 80, at least about 85, at least about less about 90, at least about 95, at least about 96, at least about 97, or at least about 98. Alternatively, or in addition, the bioproduct has a pMC of about 100 or less, about 99 or less, about 98 or less, about 96 or less, about 95 or less, about 90 or less, about 85 or less, or about 80 or less. Thus, the bioproduct may have a pMC limited by any two of the above endpoints. For example, a byproduct can have a pMC of from about 50 to about 100; from about 60 to about 100; from about 70 to about 100; from about 80 to about 100; from about 85 to about 100; from about 87 to about 98; or from about 90 to about 95. In other aspects, a byproduct described herein has a pMC of about 90, about 91, about 92, about 93, about 94, or about 94.2.
[0355] The following examples further illustrate the invention.
[0357] EXAMPLE 1
[0359] This example demonstrates a method to identify genetically engineered host cells that show enhanced production of fatty acids and derivatives thereof.
[0361] ALC310 is a previously characterized E. coli strain having the genotype MG1655 (AfadE :: FRT AfhuA :: FRT fabB [A329V] AentD :: PT5-entD) carrying plasmid ALC310 (pCL1920_PTRc_carBopt_13G04_alrA_sthA: 13) (SEQ ID ID) and produces fatty acids and derivatives thereof. To identify strains showing improved titer or yield of fatty acids or derivatives thereof, transposon mutagenesis of ALC310 was performed, followed by high-throughput screening.
[0363] Transposon DNA was prepared by cloning a DNA fragment into the plasmid EZ-Tn5 ™ pMOD ™ <R6K ori / MCS> (Epicenter Biotechnologies, Madison, WI). The DNA fragment contains a T5 promoter and a chloramphenicol resistance gene (cat) flanked by loxP sites. The resulting plasmid was named p100.38 (SEQ ID NO: 14). Plasmid p100.38 was optionally digested with restriction enzyme PshAI, incubated with EZ-Tn5 ™ Transposase enzyme (Epicenter Biotechnologies, Madison, WI), and electroporated into electrocompetent ALC310 cells according to manufacturer's instructions. The resulting colonies contained the transposon DNA randomly inserted into the ALC31 0 chromosome.
[0365] The transposon clones were then subjected to high throughput screening to measure the production of fatty alcohols. Briefly, colonies were cultured by picking in deep well plates containing Luria-Bertani (LB) medium. After overnight growth, each culture was inoculated into fresh LB. After 3 hours of growth, each culture was inoculated into fresh FA-2 medium. Spectinomycin (100 pg / ml) was included in all media to maintain selection for plasmid 7P36. FA-2 medium is M9 medium with 3% glucose supplemented with antibiotics, 10 pg / l iron citrate, 1 pg / l thiamine, 0.1 M Bis-Tris buffer (pH 7.0) and a dilution of 1: 1000 of the trace mineral solution described in Table 1.
[0372] After 20 hours of growth in FA-2, the cultures were extracted with butyl acetate. The crude extract was derivatized with BSTFA (N, O-bis [trimethylsilyl] trifluoroacetamide) and the total fatty species (eg, fatty alcohols, fatty aldehydes and fatty acids) were measured with CG-DIL as described in the Publication of ^{International Patent} Application Wo ^2008/119082.
[0374] Clones that produced 15% more total fat species than ALC310 were subjected to further verification using shake flask fermentation. Briefly, clones were grown in 2 ml of LB medium supplemented with spectinomycin (100 mg / l) at 37 ° C. After overnight growth, 100 µl of culture was transferred to 2 ml of fresh LB supplemented with antibiotics. After 3 hours of growth, 2 ml ^{of culture was transferred to a 125 ml flask containing 18 ml of f} A-2 ^{medium supplemented with spectinomycin (100 mg / l).} When the OD ⁶⁰⁰ of the culture reached 2.5, 1 mM IPTG was added to each flask. After 20 hours of growth at 37 ° C, a 400 µl sample was removed from each flask and the total fatty species were extracted with 400 µl of butyl acetate. The crude extracts were analyzed directly with CG-DIL as described above.
[0375] A transposon clone (named D288) was identified that presented increased titers of the fatty species total compared to parental ALC310 strain (FIG. 2).
[0376] The results of this example demonstrate a method of identifying engineered host cells exhibiting enhanced production of fatty acids and derivatives thereof compared to a corresponding wild-type host cell.
[0377] EXAMPLE 2
[0378] This example demonstrates that an engineered host cell with a transposon insertion in the nhaB gene exhibits enhanced production of fatty acids and derivatives thereof compared to the corresponding wild-type host cell.
[0379] Sequence analysis was performed to identify the location of the transposon insertion in the D288 strain identified in Example 1. To do this, genomic DNA was purified from a 3 ml LB overnight culture of D288 cells using the ZR kit. of MiniPrep ™ fungal / bacterial DNA (Zymo Research) according to manufacturer's instructions. The purified genomic DNA was sequenced out of the transposon using primers DG150 (GCAGTTATTGGTGCCCTTAAACGCCTGTGTTGCTACGCCTG) (SEQ ID NO: 15) and DG153 (CCCAGGCTTCCCGGTATCAACAGGACACCAGG) (SEQ ID NO: transposon 16) (SEQ ID NO: transposon 16).
[0380] Strain D288 was determined to have a transposon insert in the nhaB gene (FIG. 3).
[0381] The results of this example demonstrate that an engineered E. coli host cell with a transposon insertion in the nhaB gene exhibits enhanced production of fatty acids and derivatives thereof compared to an E. coli host cell from corresponding wild type.
[0382] EXAMPLE 3
[0383] This example demonstrates that engineered host cells having an altered level of FadR production exhibit enhanced production of fatty acids and derivatives thereof.
[0384] The nhaB gene is close to the gene encoding the fatty acid degradation regulator, FadR (FIG. 3). To determine whether the alteration of FadR expression affects the production of fatty acids or derivatives thereof in host cells, a FadR expression library was cloned and screened.
[0385] To clone the expression library, the wild-type fadR gene was amplified from genomic DNA of E. coli MG1655 by PCR using primers DG191 (SEQ ID NO: 17) and DG192 (SEQ ID NO: 18). A mutant fadR gene containing the S219N amino acid change was also amplified from E. coli MG1655 fadR [S219N] genomic DNA using the DG191 and DG192 primer set. The primers used in this example are listed in Table 2.
[0387] Primer Identifier Sequence Sequence
[0389] NNNATGGTCATTAAGGCGCAAAGCCCGG
[0390] A gene cassette encoding kanamycin resistance (kan) was amplified by PCR from plasmid pKD13 using primers SL03 (SEQ ID NO: 19) and SL23 (SEQ ID NO: 20). Each fadR cassette (i.e. wild type and S219N mutant) was separately ligated with the kanamycin resistance cassette using overlap extension (SOE) PCR splicing using primers SL23 and DG193 (SEQ ID NO : twenty-one). Primer DG193 contained degenerate nucleotides for the generation of expression variants.
[0391] Plasmid p100.487 (pCL1920_PTRC_carBopt_13G04_alrA_fabB [A329G]) (SEQ ID NO: 22) was linearized via restriction digestion with SwaI and XhoI enzymes. Each of the two SOE PCR fadR-kan products was separately cloned into linearized plasmid p100.487 using the INFUSION ™ system (Clontech, Mountain View, CA) and the plasmids were then transformed into NEB TURBO ™ chemically competent cells (New England Biolabs, Ipswich, MA). Transformants were plated on LB agar containing 50 µg / ml kanamycin.
[0393] Thousands of colonies were obtained for fadR and fadR [S219N]. Colonies were scraped from the plates and plasmids were isolated by miniprep according to standard protocols. The resulting set of plasmids was transformed into an E. coli strain EG149 with a genotype of MG1655 (AfadE :: FRT AfhuA :: FRT fabB [A329V] AentD :: PT5-entD)) and selected on LB plates containing spectinomycin 100 µg / ml.
[0395] The transformants were then screened for the production of total fatty species (eg, fatty acids, fatty aldehydes, and fatty alcohols) using the deep-well procedure described in Example 1. Numerous strains were identified that exhibited enhanced production of fatty species. total compared to control strain ALC487 (strain EG149 carrying plasmid p100.487) (FIG. 4). Strains expressing wild-type fadR or [S219N] fadR exhibited enhanced production of total fat species compared to strain ALC487, although the highest titers were observed in strains expressing wild-type FadR (FIG. 4).
[0397] Several of the major producer strains expressing wild-type FadR identified in the initial screening were assigned strain IDs and validated in shake flask fermentation. Briefly, each strain was streaked to determine individual colonies and three separate colonies from each strain were grown in three separate flasks according to the shake flask fermentation protocol described in Example 1. Total fat species were measured using CG-DIL as described in Example 1. All strains expressing wild-type FadR had higher total fat species titers compared to the control ALC487 strain (FIG. 5).
[0399] Several of the major producer strains expressing wild-type FadR were then characterized in order to determine the yield of fat species. For this, a shake flask fermentation was carried out as described above, except that (i) the temperature was maintained at 32 ° C, (ii) additional glucose was added after 18 and 43 hours and (iii) the extraction it was done at 68.5 hours. The total fat species produced was divided by the total glucose consumed to calculate the yield of fat species. All strains expressing wild-type FadR showed a higher yield of total fat species compared to the control strain ALC487 (FIG. 6).
[0401] The D512 strain was further characterized by evaluating the total fat species titer and the yield after fermentation in a 5 L bioreactor. At a glucose-glucose feed rate of 10 g / l / h, strain D512 produced higher titers of fatty acids and fatty alcohols compared to control strain ALC487 (FIG. 7). Furthermore, the total yield of all fatty species increased in strain D512 compared to strain ALC487 (FIG. 7). At a higher glucose feed rate of 15 g / l / h, strain D512 produced approximately 68.5 g / l of total fat species with a yield of approximately 20% (FIG. 7). Strain D512 produced a higher total fat species titer and a yield of 15 g / l / h compared to 10 g / l / h (FIG. 7).
[0403] Plasmid DNA was isolated from strain D512 and sequenced according to standard protocols. The plasmid obtained from strain D512, designated pDG109, was determined to have the sequence corresponding to SEQ ID NO: 23.
[0405] The results of this example demonstrate that engineered host cells having an altered expression level of FadR produce higher titers and yields of fatty acids and derivatives thereof compared to corresponding wild-type host cells.
[0407] EXAMPLE 4
[0409] This example demonstrates a method for producing high fatty acid titers in engineered host cells that have an altered level of FadR expression.
[0411] The E. coli strain EG149 used in Example 3 overexpresses the entD gene, which encodes a phosphopantetheine transferase (PPTase) involved in the activation of the CarB enzyme that catalyzes the reduction of fatty acids to aldehydes and fatty alcohols.
[0413] To evaluate the effect of entD expression on fatty acid and fatty alcohol production in strain D512, a D512 variant containing a deletion of the entD gene (D512 AentD) was generated. Shake flask fermentations were performed with strain D512 and strain D512 AentD as described in Example 1. Strain D512 produced high titers of fatty alcohols and comparatively lower titers and yields of fatty acids (FIG.
[0414] 7). In contrast, the D512 AentD strain produced high titers and yields of fatty acids and relatively low titers of fatty alcohols (FIG. 8). Total fat species titers were similar between strain D512 and strain D512 AentD (FIG. 8).
[0416] The results of this example demonstrate that genetically engineered host cells that have an altered level of FadR expression and produce high fatty acid titers when the entD gene is deleted.
[0418] EXAMPLE 5
[0420] This example demonstrates a method to identify genetically engineered host cells that show enhanced production of fatty acids and derivatives thereof.
[0422] To further evaluate the effect of altered expression of FadR on the production of fatty acids and derivatives thereof, libraries of wild-type FadR (S219N) and FadR ribosome binding sites (RBS) were prepared and screened in cells. host of E. coli.
[0424] An RBS library was inserted upstream of the fadR (S219N) gene into pDS57 as follows. The genomic DNA of a strain containing the allele of fadR (S219N) Moniker stEP005; id: s26z7 was amplified by PCR using primer set DG191 (SEQ ID No: 17) and fadR (S219N) _pme319rc (SEQ ID NO: 24). The primers used in this example are listed in Table 3.
[0427] Primer Sequence Sequence identifier DG191 ATGGTCATTAAGGCGCAAAGCCCGG SEQ ID NO: 17 fadR (S219N) _ C AA A AC AGCC A AGCT GGAGACCGTTTTT ATCGCC SEQ ID NO: 24 pme31 9rc
[0428] CCTGAATGGCTAAATCACC
[0429] 377-rbs-fadR SEQ ID NO: 25 (S219N) f GCCCGAACCCGCAAGTAANHHARNDDHDDNWAG
[0430] GG ^NH246 AAAAACGGTCTCCAGCTTGGCTGTTTTGGCGGAT SEQ ID NO: 26 GAGAGAAGATTTTC
[0431] 377-3r TT ACTT GCGGGTTCGGGCGC SEQ ID NO: 27
[0432] After the fadR (S219N) template was prepared, RBS was added by PCR using primer set 377-rbs-fadR (S219N) f (SEQ ID NO: 25) and fadR (S219N) -pme319rc (SEQ ID NO: 24). The 377-rbsfadR (S219N) f primer contained degenerate nucleotides to introduce variability into the RBS library. RBS-fadR (S219N) was ligated with a vector backbone pDS57 (described in Example 5), using the CLONEZ ™ kit commercially available from Genscript (Piscataway, NJ) with primer set NH246 (SEQ ID NO : 26) and 377-3r (SEQ ID NO: 27).
[0434] An RBS library was also inserted upstream of the wild-type fadR gene into pDS57 using a similar protocol, except that the wild-type fadR gene was PCR amplified using E. coli DV2 genomic DNA.
[0436] The ligated constructs of pDS57-rbs-fadR (S219N) and pDS57-rbs-fadR were separately transformed into an E. coli strain DAM1 by electroporation. The DAM1 strain was produced as a derivative of the DV2 strain (MG1655 AfadE, AfhuA), where the l acIq-PTrc-tesA-fadD genes were integrated into the chromosome using the Tn7-based delivery system present in the plasmid pGRG25 (described in McKenzie et al., BMC Microbiology 6:39 (2006)). After transformation, cells were harvested for 1 hour at 37 ° C, followed by culturing on LB agar plate containing spectinomycin. After incubation overnight at 37 ° C, individual colonies were picked for screening in 96 deep well plates containing 300 µl / well of LB with spectinomycin. The plates were incubated on a 32 ° C shaker with 80% humidity and shaken at 250 RPM for approximately 5 hours. After 5 hours of growth, 30 µl / well of LB culture was transferred to FA2 medium 300 µl / well (2 g / l nitrogen) containing spectinomycin. The plates were incubated again on a 32 ° C shaker with 80% humidity and shaken at 250 RPM overnight. 30 µl / well of the culture was inoculated overnight in FA2300 µl / well medium (1 g / l nitrogen) containing spectinomycin, IPTg ImM and 2% methanol. One replica plate was incubated on a shaker at 32 ° C and another was incubated on a shaker at 37 ° C with 80% humidity and shaking at 250 RPM overnight. The recipe for FA2 medium is listed in Table 4.
[0437] Reagent ml of Reagent per 1000 ml of FA2 5X 200 saline solution
[0438] Thiamine (10 mg / ml) _0.1
[0439] MgSO41M 1
[0440] CaCb 1M _0.1
[0441] Glucose 50% 60
[0442] TM2 (trace minerals without iron) 1
[0443] Ferric citrate 10 g / l 1
[0444] Buffer Bis-Tris 2M 50
[0445] NH4Cl * 10
[0446] Water q.s. for 1000 ml
[0447] * removed for FA2 medium (nitrogen 1 g / l)
[0449] After approximately 24 hours of incubation, the plates were removed by adding 40 µl / well 1M HCl and 300 µl / well butyl acetate. The plates were shaken for 15 minutes at 2000 RPM and then centrifuged for 10 minutes at 4500 RPM at room temperature. 50 µl of the organic layer was transferred per well to a shallow 96-well plate containing 50 µl / well BSTFA (Sigma Aldrich, St. Louis, MO) and the extracts were analyzed by CG-DL.
[0451] Several E. coli DAM1 clones transformed with pDS57-rbs-fadR were identified as producing substantially higher titers of FAME and free fatty acids compared to control E. coli DAM1 transformed with pDS57 alone. In general, the FadR variants produced low C14 to C16 ratios and had higher overall titers at 32 ° C compared to 37 ° C.
[0453] Numerous E. coli DAM1 clones transformed with pDS57-rbs-FadR (S219N) were also identified as producing substantially higher titers of FAME and free fatty acids compared to control E. coli DV2 or E DAM1. coli transformed with pDS57 alone.
[0455] Two of the pDS57-rbs-FadR (S219N) transformed clones identified in the initial screen (designated P1A4 and P1G7) were further characterized in shake flask fermentations. Briefly, each colony was inoculated into 5 ml of LB containing spectinomycin and incubated at 37 ° C with shaking at approximately 200 RPM for approximately 5 hours. 1.5 ml of the LB culture was transferred to 13.5 ml of FA2 medium (2 g / l nitrogen) containing 0.05% Triton X-100 and spectinomycin in a 125 ml baffled flask. The flask cultures were incubated overnight at 32 ° C, 80% humidity and 250 RPM. 1.5 ml of the overnight culture was transferred to a new 125 ml baffled flask containing 13.5 ml of FA2 medium (1 g / l nitrogen) containing 0.05% Triton X-100, IPTG ImM, 2% methanol, and spectinomycin. The flask cultures were then incubated at 32 ° C, 80% humidity and 250 RPM. After 56 hours of incubation, 500 µl samples were taken from each flask. 100 µl of each sample was diluted with 900 µl of water to measure the OD of the culture, 100 µl of each sample was diluted with 900 µl of water to measure the remaining glucose, and 300 µl of each sample was extracted and analyzed using GC- DIL as described above.
[0457] Both FadR variants (ie, P1A4 and P1G7) produced higher titers and yields of total fat species compared to the control strain transformed with pDS57 alone in shake flask fermentation (FIG. 9).
[0459] The production of fatty species by P1A4 and P1G7 was also measured in large-scale fermentations. For this, cells from a frozen stock solution were cultured in LB medium for a few hours and then transferred to a defined medium consisting of ^{3 g / l KH 2} PO ⁴ , Na2HPO dihydrate46 g / l, 2 g / l NH4Cl, MgSO4 x 7 H ² O 0.24 g / l, glucose 20 g / l, bis-Tris buffer 200 mM (pH 7.2), trace metal solution 1.0 ml / l and thiamine 1.0 mg / l, and were grown overnight. The trace metal solution was composed of FeCb x 6 H ² O 27 g / l, ZnCb x ⁴ H ² O 2 g / l, CaCb x 6 H ² O 2 g / l, Na2MoO4 x ² H ² O 2 g / l, CuSO4 x 5 H ² O 1.9 g / l, H ³ BO ³ 0.5 g / l and concentrated HCl 40 ml / l.
[0461] 50 ml of each culture were inoculated overnight in 1 liter of production medium in a fermenter with temperature, pH, agitation, aeration and dissolved oxygen control. The composition of the medium was as follows: KH ² PO ⁴ 1 g / l, (NH4) 2SO4 0.5 g / l, MgSO4 x 7 H ² O 0.5 g / l, Bacto casaminoacids 5 g / l , ferric citrate 0.034 g / l, 1M HCl 0.12 ml / l, ZnCb x 4 H ² O 0.02 g / l, CaCb x ² H ² O 0.02 g / l, Na2MoO4 x ² H ² O 0 , 02 g / l, CuSO4 x 5 H ² O 0.019 g / l, H ³ BO ³ 0.005 g / l and 1.25 ml / l of a vitamin solution. The vitamin solution contained riboflavin 0.06 g / l, pantothenic acid 5.40 g / l, niacin 6.0 g / l, pyridoxine 1.4 g / l, and folic acid 0.01 g / l.
[0463] Fermentations were carried out at 32 ° C, pH 6.8 and dissolved oxygen (DO) equal to 25% of saturation. The pH is

权利要求:
Claims (15)
[1]
1. A method of producing a fatty acid or derivative thereof, comprising;
(a) providing an engineered bacterial host cell comprising a heterologous promoter and / or ribosome binding site operably linked to a polynucleotide sequence encoding a FadR polypeptide, causing said promoter and / or ribosome binding site the overexpression of the FadR polypeptide in said cell,
(b) cultivating the genetically engineered bacterial host cell in culture medium under conditions that allow the production of a fatty acid or a derivative thereof, and
(c) isolating the fatty acid or derivative thereof from the genetically engineered bacterial host cell,
wherein the fatty acid or derivative thereof is a fatty acid, an acyl-ACP, an acyl-CoA, a fatty aldehyde, a short-chain alcohol, a long-chain alcohol, a fatty alcohol, a hydrocarbon, or an ester .
[2]
2. The method of claim 1, wherein the FadR polypeptide is encoded by a FadR gene obtained from Escherichia, Salmonella, Citrobacter, Enterobacter, Klebsiella, Cronobacter, Yersinia, Serratia, Erwinia, Pectobacterium, Photorhabdus, Edwardsiella, Shewanella or Vibrio.
[3]
The method of claim 1 or 2, wherein the FadR polypeptide is a wild-type FadR polypeptide or a mutant FadR polypeptide.
[4]
4. The method of claim 3, wherein the FadR polypeptide comprises a mutation in an amino acid residue corresponding to amino acid 219 of SEQ ID NO: 1, preferably wherein the mutation is a substitution of the amino acid residue corresponding to the amino acid 219 of SEQ ID NO: 1 with an asparagine residue.
[5]
The method of any one of claims 1-4, wherein the FadR polypeptide comprises an amino acid sequence that has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
[6]
6. The method of any one of claims 1-5, wherein the expression of one or more genes selected from the group consisting of fabA, fabB, iclR, fadA, fadB, fadD, fadE, fadl, fadJ, fadl, fadM, uspA, aceA, aceB, and aceK is attenuated in the engineered bacterial host cell.
[7]
7. The method of any one of claims 1-6, wherein the fatty acid or derivative thereof is an ester, and the ester is a wax, a fatty ester, or a fatty acid ester.
[8]
The method of claim 7, wherein the fatty acid or derivative thereof is a fatty acid ester, and the fatty acid ester is a fatty acid methyl ester (FAME) or a fatty acid ethyl ester (FAEE ).
[9]
The method of claim 7 or 8, wherein an ester synthase is overexpressed in the engineered host cell, preferably wherein the ester synthase is ES9 from Marinobacter hydrocarbonoclasticus (SEQ ID NO: 2), ES8 from Marinobacter hydrocarbonoclasticus (SEQ ID NO: 3), AtfAl from Alcanivorax borkumensis SK2 (SEQ ID NO: 4), AtfA2 from Alcanivoraxborkumensis SK2 (s Eq ID NO: 5), O-acylglycerol diacetyltransferase from Marinobacteraquaeolei VT8 (SEQ ID NO: 6 NO: 7), a wax synthase or a bifunctional wax ester synthase / acyl-CoA: diacylglycerol acyltransferase (wax-dgaT).
[10]
The method of any one of claims 7-9, wherein an acetyl-CoA carboxylase complex is overexpressed in the genetically engineered host cell, preferably wherein the acetyl-CoA carboxylase complex is encoded by two or more genes of the acetyl-CoA carboxylase subunit obtained from one or more of Corynebacterium glutamicum, Escherichia coli, Lactococcus lactis, Kineococcus radiotolerans, Desulfovibrio desulfuricans, Erwinia amylovora, Rhodospirillum rubrum, Vibrio furniliaphissii, Stenotrocyst sp. PCC6803 or Synechococcus elongatus.
[11]
The method of claim 10, wherein a biotin protein ligase is overexpressed in the engineered host cell.
[12]
12. The method of claim 7, wherein the fatty acid or derivative thereof is a fatty aldehyde or a fatty alcohol and wherein a carboxylic acid reductase and a thioesterase are overexpressed in the genetically engineered host cell, preferably wherein An alcohol dehydrogenase is overexpressed in the engineered host cell.
[13]
The method of claim 12, wherein the carboxylic acid reductase is carB (SEQ ID NO: 8), the thioesterase is tesA (SEQ ID NO: 11) and the alcohol dehydrogenase is YjgB (SEQ ID NO: 10) or AlrAadpl (SEQ ID NO: 9).
[14]
The method of claim 12 or 13, wherein a phosphopantetheinyl transferase (PPTase) is overexpressed in the engineered host cell, preferably wherein the PPTase is EntD from E. coli MG1655 (SEQ ID NO: 12).
[15]
The method of claim 14, wherein the host cell is selected from the group consisting of an E. coli cell, a Pantoea citrea cell, a Bacillus lentus cell, a Bacillus brevis cell, a Bacillus brevis cell. Bacillus stearothermophilus, a Bacillus lichen formis cell, a Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillus thuringiensis cell, a Bacillus clausii cell, a Bacillus clausii cell megaterium, a Bacillus subtilis cell, a Bacillus amyloliquefaciens cell, a Trichoderma koningii cell, a Trichoderma viride cell, a Trichoderma reesei cell, a Trichoderma longibrachiatum cell, an Aspergillus awamori cell, an Aspergillus fumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulans cell, an Aspergillus niger cell, an Aspergillus oryzae cell, a Humicola insolens, a Humicola lanuginose cell, a Rhodococcus opacus cell, a Rhizomucor miehei cell, a Mucor miehei cell, a Streptomyces lividans cell, a Streptomyces murinus cell, an Actinomycetes cell, an Avabidopsis thaliana cell, a Panicum virgatum cell, a Miscanthus giganteus cell, a Zea mays cell, a Botryococcuse braunii cell, a Chlamydomonas reinhardtii cell, a Dunaliela salina cell, a Synechococcus Sp. PCC 7002 cell, a Synechococcus Sp. PCC cell 7942, a Synechocystis Sp. PCC 6803 cell, a Thermosynechococcus elongates BP-1 cell, a Chlorobium tepidum cell, a Chlorojlexus auranticus cell, a Cromatiumm vinoum cell, a Rhodospirillum rubrum cell, a Rhodobacter capsulatus cell, a Rhodopseudomonas palusris cell, a Clostridium ljungdahlii cell, a Clostridiuthermocellum cell, a Penicillium chrysogenum cell, a that of Pichia pastoris, a Saccharomyces cerevisiae cell, a Schizosaccharomyces pombe cell, a Pseudomonasjluorescens cell and a Zymomonas mobilis cell.

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US201161470989P| true| 2011-04-01|2011-04-01|

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PCT/US2012/031881|WO2012154329A1|2011-04-01|2012-04-02|Methods and compositions for improved production of fatty acids and derivatives thereof|

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