WO2021123811A1 - Bio-based production of toxic chemicals - Google Patents

Bio-based production of toxic chemicals Download PDF

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Publication number
WO2021123811A1
WO2021123811A1 PCT/GB2020/053292 GB2020053292W WO2021123811A1 WO 2021123811 A1 WO2021123811 A1 WO 2021123811A1 GB 2020053292 W GB2020053292 W GB 2020053292W WO 2021123811 A1 WO2021123811 A1 WO 2021123811A1
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seq
enzyme
octanol
microorganism
thioesterase
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PCT/GB2020/053292
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French (fr)
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Patrik Raymond Jones
Pachara SATTAYAWAT
Ian Sofian YUNUS
Jonathan Emmanuel MULLER
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Imperial College Innovations Ltd
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12N9/1033—Chloramphenicol O-acetyltransferase (2.3.1.28)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1062—Sucrose synthase (2.4.1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01028—Chloramphenicol O-acetyltransferase (2.3.1.28)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01013—Sucrose synthase (2.4.1.13)
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    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/02—Thioester hydrolases (3.1.2)
    • C12Y301/02014—Oleoyl-[acyl-carrier-protein] hydrolase (3.1.2.14), i.e. ACP-thioesterase

Definitions

  • the present invention relates to a method of producing derivatives of medium or short chain length compounds, such as medium or short chain length alcohols.
  • the present invention relates to a method of producing a derivative of 1-octanol, as well as microorganisms and expression vectors for use in said method.
  • the present invention also relates to novel thioesterase enzymes.
  • the thioesterase enzymes may be particularly useful in the method of the invention. Accordingly, the present invention also relates to the use of the thioesterase enzymes in the method of the invention.
  • Microbial biotechnology offers an attractive method for renewable production of chemicals that replace those currently sourced from fossil fuel feedstocks (1) (e.g. monomers for plastic or textile polymer synthesis) or nature (2, 3) (e.g. vanillin).
  • a critical factor determining success with such a process is the compatibility between the engineered metabolism and its microbial host (4). If the target molecule, or its metabolic intermediates, are toxic to the host organism, the maximum potential for cost-effective production is likely not achieved. And, for lower value chemicals, economics really matter in the face of competition from fossil fuels (5). Nonetheless, when strategies to enhance product tolerance have been implemented, this has been found to improve productivity (6).
  • the present invention provides methods for the production of chemicals, as well as microorganisms for use in the methods.
  • the invention provides a method of producing a chemical comprising expressing an enzyme in a microorganism, wherein the microorganism produces a first chemical and the enzyme converts the first chemical into a second chemical.
  • the conversion may be through the formation of a hydrophobic (e.g. esterification) or hydrophilic (e.g. glycosylation) derivative.
  • the second chemical may then be removed from the microorganism cell, and optionally converted back into the first chemical or, alternatively, optionally converted into a third chemical.
  • the removal may, for example, be through secretion from the cell, followed by harvesting of the secreted chemical, or by extraction of the second chemical from stores in the cell.
  • the removal may involve the use of a solvent overlay, for example to capture volatile products.
  • the enzyme is artificially expressed in the cell, i.e. the enzyme would not be expressed at appropriate levels in the cell in the absence of steps taken in order to carry out the invention.
  • expression of the enzyme in the cell will be due to the presence of a nucleic acid (for example heterologous nucleic acid) sequence encoding the enzyme, for example on a plasmid construct.
  • the enzyme may be a glycosyltransferase or an alcohol acetyltransferase.
  • the first and second chemicals are organic compounds, and preferably of medium or short chain length (C4 to C10).
  • the first chemical has a functional group that is modified by the enzyme, for example a hydroxyl group, OH-.
  • the bioderivatization of the first chemical i.e. the modification of the functional group by the enzyme, may reduce the toxicity of the first chemical, such that the second chemical is less toxic to the cell than the first chemical, and/or it may change the chemical properties of the first chemical, e.g. increase or decrease the water solubility such that the second chemical is more or less water soluble than the first chemical, and/or it may protect the molecule from further conversion, e.g. by oxidation of the functional group so that the second chemical is less reactive than the first chemical.
  • the present invention provides a method of producing a derivative of a medium or short chain length compound, comprising: providing a first enzyme to a microorganism that produces a medium or short chain length compound, wherein the first enzyme modifies the hydroxyl group of the medium or short chain length compound to form a derivative of the medium or short chain length compound; and harvesting the derivative of the medium or short chain length compound from the microorganism.
  • the medium or short chain length compound is a medium or short chain length alcohol.
  • the alcohol may comprise 4, 5, 6, 7, 8, 9, or 10 carbons.
  • the short chain length alcohol is n-butanol.
  • This embodiment may give rise to a particularly preferred aspect of the invention which provides a method of producing a derivative of n-butanol, comprising: providing a first enzyme to a microorganism that produces n-butanol, wherein the first enzyme modifies the hydroxyl group of the n-butanol to form a derivative of n-butanol; and harvesting the derivative of n-butanol from the microorganism.
  • the medium chain length alcohol is 1-octanol.
  • This embodiment may give rise to a particularly preferred aspect of the invention which provides a method of producing a derivative of 1-octanol, comprising: providing a first enzyme to a microorganism that produces 1-octanol, wherein the first enzyme modifies the hydroxyl group of the 1-octanol to form a derivative of 1-octanol; and harvesting the derivative of 1-octanol from the microorganism.
  • the medium chain length alcohol is 1-decanol.
  • This embodiment may give rise to a particularly preferred aspect of the invention which provides a method of producing a derivative of 1-decanol, comprising: providing a first enzyme to a microorganism that produces 1-decanol, wherein the first enzyme modifies the hydroxyl group of the 1-decanol to form a derivative of 1-decanol; and harvesting the derivative of 1-decanol from the microorganism.
  • the microorganism is a bacterium.
  • the bacterium may be a heterotrophic bacterium.
  • the bacterium may be selected from the group consisting of Escherichia, Halomonas and Cyanobacterium.
  • the Escherichia may be Escherichia coli (E. coli), for example B strain C43 (DE3) or K-12 strain BW25113.
  • Escherichia coli E. coli
  • B strain C43 DE3
  • K-12 strain BW25113 BW25113.
  • the cyanobacterium may be Synechocystis sp (for example PCC 6803).
  • two or more first enzymes are provided.
  • the first enzyme may be heterologous or homologous.
  • the first heterologous enzyme may be selected from the group consisting of an alcohol acetyltransferase and a glycosyltransferase.
  • the alcohol acetyltransferase may be selected from the group consisting of CAT (SEQ ID NO: 1), SAAT (SEQ ID NO: 2) and ATF1 (SEQ ID NO: 3), or a variant thereof.
  • the alcohol acetyltransferase may be selected from the group consisting of ATF1 (SEQ ID NO: 3), VAAT (SEQ ID NO:103) or UAAT4 (SEQ ID NO:82).
  • the alcohol may be n-butanol.
  • the alcohol acetyltransferase is UAAT4 (SEQ ID NO:82).
  • the glycosyltransferase may be selected from the group consisting of AdGT4 (SEQ ID NO: 4), VvGT1 (SEQ ID NO: 5), MtGT1 (SEQ ID NO: 6), AtGT1 (SEQ ID NO: 7) and MtH2 (SEQ ID NO: 8), or a variant thereof.
  • the glycosyltransferase is MtH2 (SEQ ID NO: 8), or a variant thereof.
  • the derivative of 1-octanol may be octyl acetate and/or octyl glucoside.
  • the derivative of n-butanol may be butyl acetate and/or butyl glucoside.
  • the derivative of 1-decanol may be decyl acetate and/or decyl glucoside.
  • the method may further comprise providing a second enzyme to the microorganism.
  • the second enzyme may be heterologous.
  • the second heterologous enzyme may be selected from the group consisting of thioesterase, carboxylic acid reductase, and phosphopantetheinyl.
  • the thioesterase may be a C8-preferring thioesterase.
  • the C8-preferring thioesterase may be selected from the group consisting of CpFatBI (SEQ ID NO: 9), CaFatB3 (SEQ ID NO:10), Tes3 (SEQ ID NO: 11), or a variant thereof.
  • the variant may be CpFatB1-4 (SEQ ID NO: 12) or CaFatB3-5 (SEQ ID NO: 13), or a variant thereof.
  • the thioesterase is CaFatB3-5 (SEQ ID NO: 13) or variant thereof.
  • the thioesterase may be a C10-preferring thioesterase.
  • the thioesterase enzyme comprises or consists of a truncated SEQ ID NO: 15 or SEQ ID NO: 16, or a variant thereof.
  • such a thioesterase may be a C8- preferring thioesterase or a C 10- preferring thioesterase.
  • the thioesterase enzyme comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO: 90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, and SEQ ID NO:102, or a variant thereof.
  • the thioesterase enzyme comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22, and SEQ ID NO:23.
  • a thioesterase may be a C8-preferring thioesterase or a C 10- preferring thioesterase.
  • a C8-preferring thioesterase is SEQ ID NO:23 or 25, or variants thereof.
  • a C10-preferring thioesterase is selected from SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22, SEQ ID NO:25 or a variant thereof.
  • the microorganisms is a cyanobacterium and the thioesterase is a C10- preferring thioesterase.
  • the C10-preferring thioesterase is selected from SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22 or a variant thereof.
  • the thioesterase may be a C4-preferring thioesterase.
  • the C4-preferring thioesterase may be CAH09236 from B.fragilis (SEQ ID NO: 102).
  • the second enzyme may be homologous.
  • the second homologous enzyme may be aldehyde reductase.
  • the method may further comprise providing a third enzyme to the microorganism.
  • the third enzyme may be a heterologous enzyme.
  • the third heterologous enzyme is sucrose synthase.
  • the sucrose synthase may be Arabidopsis thaliana sucrose synthase. In a suitable embodiment, the sucrose synthase may be Arabidopsis thaliana sucrose synthase is according to SEQ ID NO: 14, or a fragment or variant thereof.
  • the third enzyme may be homologous.
  • the first, second, and/or third enzyme may be provided to the microorganism directly or indirectly.
  • the first, second, and/or third enzyme may be provided to the microorganism indirectly by an expression vector comprising a nucleic acid encoding the enzyme.
  • the method may further comprise providing a fourth protein to the microorganism.
  • the fourth protein is an acyl-carrier protein (ACP).
  • ACP acyl-carrier protein
  • the fourth protein may be homologous or heterologous, suitably it is heterologous.
  • the acyl-carrier protein is selected from EcACP (SEQ ID NO:83) , 6803ACP (SEQ ID NO:84), and CIACP2 (SEQ ID NO:85).
  • the acyl-carrier protein comprises or consists of a sequence selected from SEQ ID NO:83, SEQ ID NO:84, and SEQ ID NO:85.
  • the acyl-carrier protein is overexpressed in the microorganism.
  • the provision of overexpressed ACP increases metabolic flux through the enzymatic pathway.
  • the fourth protein may be provided to the microorganism indirectly by an expression vector comprising a nucleic acid encoding the protein.
  • the method may further comprise supplying the microorganism with a precursor of the medium or short chain length alcohol.
  • the method may further comprise supplying the microorganism with a precursor of 1-octanol, n-butanol or 1-decanol.
  • the precursor may be an internal or external substrate.
  • the precursor may be selected from the group consisting of octanoic acid, butanoic acid, decanoic acid, glucose, octanoyl-ACP, butanoyl-ACP, decanoyl-ACP, octanal, butanal and decanal.
  • the invention provides a vector for use in a method of the invention.
  • the expression vector may comprise a nucleic acid encoding a first enzyme, and optionally a nucleic acid encoding a second and/or third enzyme, and optionally a fourth protein, wherein the first enzyme modifies the hydroxyl group of a medium or short chain length compound to form a derivative of the medium or short chain length compound.
  • the expression vector may comprise a nucleic acid encoding a first enzyme, and optionally a nucleic acid encoding a second and/or third enzyme, and optionally a fourth protein, wherein the first enzyme modifies the hydroxyl group of the 1-octanol to form a derivative of 1-octanol.
  • the invention provides a microorganism for use in a method of the invention.
  • an expression vector comprising a nucleic acid encoding a first enzyme, and optionally a nucleic acid encoding a second and/or third enzyme, and optionally a fourth protein, wherein the first enzyme modifies the hydroxyl group of the medium or short chain length compound to form a derivative of the medium or short chain length compound;
  • an expression vector comprising a nucleic acid encoding a first enzyme, and optionally a nucleic acid encoding a second and/or third enzyme, and optionally a fourth protein, wherein the first enzyme modifies the hydroxyl group of the 1-octanol to form a derivative of 1-octanol;
  • the invention provides a thioesterase enzyme, wherein the thioesterase comprises or consists of a truncated SEQ ID NO: 15 or SEQ ID NO: 16, or a variant thereof.
  • the thioesterase enzyme may comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21 , SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO: 90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, or a variant thereof.
  • the present invention provides use of the thioesterase enzyme of the invention in the method of the invention. Except for where the context requires otherwise, the considerations set out in this disclosure should be considered to be applicable to methods, vectors and microorganisms of the invention.
  • the present invention is based upon the purposeful in vivo transformation of chemicals into chemical derivatives by modification of functional groups, defined as bioderivatization. Often, these functional groups (for example hydroxyl groups, OH-) are central to rendering a chemical toxic towards cells, including cells that produce the chemical.
  • functional groups for example hydroxyl groups, OH-
  • bioderivatization may also change the chemical properties (e.g. water solubility) of the target chemical, and/or protect the molecule from further conversion (e.g. oxidation). Bioderivatization could also open new opportunities for strategic product: process separation that is more cost-efficient.
  • the method of the invention may comprise the step of converting a harvested derivative of a medium or short chain length compound (such as a derivative of 1-octanol) to the corresponding medium or short chain length compound (for example 1-octanol).
  • this step may be performed ex vivo.
  • FIG. 1 A general bioderivatization concept according to the invention is illustrated in Figure 1.
  • the present invention is based on the inventors’ development of an in vivo method for the transformation of chemicals into chemical derivatives that have more desirable properties, for example that are less toxic or more soluble.
  • the methods of the invention allow the transformation of 1-octanol into less toxic derivatives of 1-octanol, such as octyl acetate.
  • the inventors have surprisingly found that whilst E. coli cells are unable to grow when the concentration of 1-octanol is above 0.75mM, the cells are able to grow in the presence of 1- octanol derivatives even when those are present at a much higher concentration which speaks to the reduced toxicity of the derivatives.
  • the cells are able to grow in the presence of octyl acetate at a concentration as high as 50mM, or the in the presence of octyl glucoside at a concentration as high as 2.5mM. It will be appreciated that as a result of the cells’ ability to grow under these increased concentrations of derivatives, the cells are able to produce more of the derivatives.
  • the derivatives may be then converted ex vivo back into 1- octanol, allowing for increased amounts of 1-octanol to be ultimately produced.
  • the present invention provides a method of producing a derivative of a medium or short chain length compound, comprising: providing a first enzyme to a microorganism that produces a medium or short chain length compound, wherein the first enzyme modifies the hydroxyl group of the medium or short chain length compound to form a derivative of the medium or short chain length compound; and harvesting the derivative of the medium or short chain length compound from the microorganism.
  • the term “medium chain length compound” as used herein refers to a compound having a medium length carbon chain.
  • a medium length carbon chain may have 6, 7, 8, 9, 10, 11, or 12 carbons.
  • the term “short chain length compound” as used herein refers to a compound having a short length carbon chain.
  • a short length carbon chain may have 1, 2, 3, 4 or 5 carbons.
  • the compound may be an organic compound.
  • the compound may be an alcohol.
  • the alcohol may be butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol or dodecanol.
  • the alcohol may be octanol.
  • the alcohol may be butanol.
  • the alcohol may be decanol.
  • the alcohol may be a terpene alcohol, for example linalool, menthol, eugenol or cinnamoyl alcohol.
  • the present invention provides a method of producing a derivative of 1-octanol, n-butanol or 1-decanol.
  • the method comprises providing a first enzyme to a microorganism that produces 1-octanol, n-butanol or 1-decanol, wherein the first enzyme modifies the hydroxyl group of the 1-octanol, n-butanol or 1-decanol to form a derivative of 1- octanol, n-butanol or 1-decanol.
  • the method may further comprise the step of harvesting the derivative of 1-octanol from the microorganism.
  • the term “derivative of the medium or short chain length compound” or “derivative”, refers to a compound obtained from the medium or short chain length compound, wherein the compound has a modified hydroxyl group as compared to the corresponding compound that it is derived from.
  • the term “derivative of 1-octanol” refers to a compound obtained from 1-octanol, wherein the compound has a modified hydroxyl group as compared to 1-octanol.
  • the derivative of the medium or short chain length compound such as for example the derivative of 1-octanol, n-butanol or 1- decanol, may be referred to as “a target molecule”.
  • the derivative may be hydrophobic or hydrophilic.
  • a hydrophobic derivative may be obtained, for example, by esterification of the hydroxyl group.
  • a hydrophobic derivative may be obtained, for example, by esterification of the hydroxyl group of 1-octanol, resulting in the production of octyl acetate.
  • a hydrophilic derivative may be obtained, for example, by glycosylation of the hydroxyl group.
  • a hydrophilic derivative may be obtained, for example, by glycosylation of the hydroxyl group of 1-octanol, resulting in the production of octyl glucoside.
  • the derivative of 1-octanol may be selected from the group consisting of octyl acetate, octyl glucoside, glycosides with other glycosyl groups and esters with other acyl groups.
  • the derivative of n-butanol may be selected from the group consisting of butyl acetate, butyl glucoside, glycosides with other glycosyl groups and esters with other acyl groups.
  • the derivative of 1-decanol may be selected from the group consisting of decyl acetate, decyl glucoside, glycosides with other glycosyl groups and esters with other acyl groups.
  • the derivative of the terpene alcohols may be selected from the group consisting of menthyl acetate, linalyl acetate, linalyl butyrate, eugenyl acetate, and cinnamyl acetate.
  • the method of the invention may be particularly useful in the context of producing compounds that are toxic to the microorganisms which produce said compounds.
  • the derivative such as derivative of 1-octanol
  • the derivative may be less toxic than the compound from which the derivative is produced from (for example 1-octanol).
  • Toxicity may be determined, for example, by determining the compounds effect on cell growth, proliferation and/or genetic stability. Method for determining a compounds effect on cell growth, proliferation and/or genetic stability will be known to those skilled in the art.
  • Unstable compounds may be reactive in the environment and/or during normal use. Compounds may be unstable as a result of being reactive.
  • the method may comprise the use of a solvent overlay.
  • the solvent overlay may be provided by adding the solvent to the culture medium for culturing the microorganism.
  • the use of a solvent overlay may increase the amount of derivative produced and/or the amount of derivative harvested.
  • the solvent may be hexadecane and/or pentadecane, but other suitable solvents will be known to those skilled in the art. It will also be appreciated by those skilled in the art that the choice of a solvent for use in a solvent overlay may depend upon the type of derivative produced.
  • first enzyme refers to an enzyme which converts a medium or short chain length compound to a derivative of the medium or short chain length compound by modifying the hydroxyl group of the medium or short chain length compound.
  • the first enzyme may convert 1-octanol, n-butanol or 1-decanol respectively to a derivative of 1- octanol, n-butanol or 1-decanol respectively by modifying the hydroxyl group of 1-octanol, n- butanol or 1-decanol respectively.
  • the first enzyme may be heterologous.
  • the first heterologous enzyme such an enzyme may be referred to as “the first heterologous enzyme”.
  • a heterologous enzyme is one which does not naturally exist in the microorganism to which the enzyme is provided.
  • the first enzyme may be homologous.
  • the first homologous enzyme such an enzyme may be referred to as “the first homologous enzyme”.
  • a homologous enzyme is one which naturally exists in the microorganism to which the enzyme is provided.
  • a heterologous enzyme may be provided to produce a derivative of 1-octanol that might naturally be not produced by the microorganism.
  • a homologous enzyme may be provided to increase the amount of an enzyme that naturally exists in the microorganism. Increasing the amount of an enzyme may be useful to increase the amount of the derivative (for example derivative of 1-octanol) being produced, or when the amount of the naturally existing enzyme is insufficient to produce the derivative (for example derivative of 1-octanol).
  • the microorganism may be provided with one or more first enzyme.
  • the microorganism may be provided with one, two, three, four, five, six, or more first enzymes.
  • some or all of the first enzymes may be heterologous or homologous.
  • the enzymes may produce different derivatives, for example derivatives of 1-octanol, such as acetyltransferase and glycosyltransferase. Alternatively, they may produce the same derivative, such as acetyltransferase or glycosyltransferase.
  • the first heterologous enzyme may be an alcohol acetyltransferase or glycosyltransferase.
  • the microorganism is provided with more than one first heterologous enzyme it may be provided, for example, with one or more alcohol acetyltransferase and one or more glycosyltransferase, or two or more different alcohol acetyltransferase, or two or more different glycosyltransferases.
  • a suitable alcohol acetyltransferase may be selected from the group consisting of CAT (SEQ ID NO: 1), SAAT (SEQ ID NO: 2) and ATF1 (SEQ ID NO: 3).
  • a suitable glycosyltransferase may be selected from the group consisting of AdGT4 (SEQ ID NO: 4), VvGT1 (SEQ ID NO: 5), MtGT1 (SEQ ID NO: 6), AtGT1 (SEQ ID NO: 7) and MtH2 (SEQ ID NO: 8).
  • the alcohol acetyltransferase may be selected from the group consisting of ATF1 (SEQ ID NO: 3), VAAT (SEQ ID NO:103) or UAAT4 (SEQ ID NO:82).
  • the first enzyme provided to the microorganism may dictate what derivative, for example what derivative of 1-octanol, is produced by the microorganism.
  • the derivative produced may be octyl acetate, whereas when the first enzyme is a glycosyltransferase the derivative produced may be octyl glucoside. This is the same for any other alcohol described herein.
  • providing refers to any techniques by which the microorganism will receive a first, second and/or third enzyme.
  • the enzyme may be provided to the microorganism either indirectly or directly.
  • the enzyme in an embodiment where the enzyme is provided to the microorganism indirectly, it may be provided in the form of a nucleic acid encoding such an enzyme.
  • a nucleic acid encoding the enzyme may be provided to the microorganism, for example, through the use of an expression vector comprising a nucleic acid sequence encoding such an enzyme.
  • the enzyme in an embodiment where the enzyme is provided to the microorganism directly, it may be provided in the form of the protein itself.
  • the microorganism may be cultured in the presence of the enzyme.
  • the microorganism may be cultured in the presence of an agent which may facilitate the transport of the enzyme into the microorganism.
  • expression vector refers to an isolated DNA molecule which upon transfection into the microorganism provides for expression of the enzyme within the microorganism.
  • the enzyme may be heterologous or homologous.
  • the expression vector may comprise regulatory DNA sequences that are required for an efficient transcription of the DNA coding sequence into mRNA and for an efficient translation of the mRNAs into enzymes in the microorganism.
  • the expression vector may comprise a DNA sequence that encodes the first, second and/or third enzyme.
  • the expression vector may be viral or non-viral. More suitably, the expression vector may be viral.
  • a suitable viral expression vector may be derived from a virus selected from the group consisting of paramyxovirus, retrovirus, adenovirus, lentivirus, pox virus, alphavirus, and herpes virus.
  • Methods of delivering expression vectors to a cell are well known in the art. Merely by way of example, such methods include viral transfection, electroporation and sonoporation.
  • Suitable non-viral expression vectors may be selected from the group consisting of inorganic particle expression vectors (such as calcium phosphate, silica, and gold), lipid based particle expression vectors (for example cationic lipids, lipid nano emulsions, and solid lipid nanoparticles) and polymer based particle expression vectors (for example peptides, polyethylenimine, chitosan, and dendimers).
  • inorganic particle expression vectors such as calcium phosphate, silica, and gold
  • lipid based particle expression vectors for example cationic lipids, lipid nano emulsions, and solid lipid nanoparticles
  • polymer based particle expression vectors for example peptides, polyethylenimine, chitosan, and dendimers.
  • suitable non-viral expression vectors will be known to those skilled in the art.
  • microorganism refers to any prokaryotic or eukaryotic organism, such as a bacterium, protozoa, a virus or any kind of higher organism, such as a fungus (for example yeast), a plant, or an animal, which can be maintained in the form of a cell suspension or cell culture, and which produces a medium or short chain length compound, for example 1- octanol, n-butanol or 1-decanol.
  • the microorganism is a bacterium.
  • the bacterium may be heterotrophic.
  • the bacterium may be selected from the group consisting of Escherichia, Halomonas, Cyanobacterium.
  • the Escherichia may be Escherichia coli (E. coli).
  • the E. coli may be B strain C43 (DE3) or K-12 strain BW25113.
  • the cyanobacterium may be Synechocystis sp (for example PCC 6803). It will be appreciated that these are merely exemplary microorganisms, and other suitable microorganisms, such as other suitable bacteria will be known to those skilled in the art.
  • the microorganism may be capable of producing the medium or short chain length compound, such as 1-octanol, n-butanol or 1-decanol on its own (i.e. without the need of being supplied with a precursor of 1-octanol, n-butanol or 1-decanol), or alternatively or additionally, the microorganism may be capable of producing the medium or short chain length compound (such as 1-octanol, n-butanol or 1-decanol) from a precursor of the medium or short chain length compound (such as a precursor of 1-octanol n-butanol or 1-decanol) that has been supplied to the microorganism.
  • the medium or short chain length compound such as 1-octanol, n-butanol or 1-decanol
  • the microorganism may comprise a first enzyme, and optionally a second and/or third enzyme, or an expression vector comprising a nucleic acid encoding a first enzyme, and optionally a second and/or third enzyme.
  • the first, second and/or third enzyme is heterologous.
  • precursor of medium or short chain length compound refers to a compound that can be converted into a medium or short chain length compound.
  • the precursor may be converted into a medium or short chain compound via a single step reaction or via a multistep reaction.
  • the precursor may be selected from the group consisting of a lignocellulosic substrate, sucrose, starch, and amylose.
  • the term “precursor of 1-octanol” as used herein refers to a compound that can be converted into 1-octanol.
  • the precursor may be converted into 1-octanol via a single step reaction or via a multistep reaction.
  • the precursor may be selected from the group consisting of a lignocellulosic substrate, sucrose, starch, and amylose.
  • the precursor may be an internal or external substrate.
  • the precursor may be selected from the group consisting of octanoic acid, CO2, formate, glucose, octanoyl-ACP, octanoyl- CoA, octanal, and hexose.
  • the precursor may be one that naturally exists in the microorganism or one that doesn’t naturally exist in the microorganism.
  • Providing the microorganism with the precursor may allow the production of medium or short chain length compound, for example 1-octanol, n-butanol or 1-decanol, (if the precursor does not naturally exist in the microorganism) or may increase the production of medium or short chain length compound, for example 1-octanol, n-butanol or 1-decanol (if the precursor naturally exists in the microorganism).
  • a method of the invention may comprise the step of supplying the microorganism with a precursor medium or short chain length compound.
  • the precursor may be supplied to the microorganism by adding the precursor to the medium in which the microorganism is cultured.
  • a method of the invention may comprise the step of supplying the microorganism with a precursor of 1-octanol, n-butanol or 1-decanol (for example octanoic acid).
  • the precursor may be supplied to the microorganism by adding the precursor to the medium in which the microorganism is cultured.
  • the method of the invention may further comprise providing the microorganism with a second enzyme, wherein the enzyme will convert the precursor into medium or short chain length compound or into a different precursor of medium or short chain length compound and/or the medium chain length compound.
  • the precursor might not comprise a hydroxyl group and/or the precursor may for example comprise a carboxylic acid group.
  • the method of the invention may further comprise providing the microorganism with a second enzyme.
  • the second enzyme may convert the precursor into the medium or short chain length compound.
  • the enzyme may convert the precursor into 1-octanol, n-butanol or 1-decanol, or into a different precursor of 1-octanol, n-butanol or 1-decanol.
  • the second enzyme maybe heterologous or homologous.
  • Providing the second enzyme may be particularly desirable when the method of the invention comprises the step of supplying the microorganism with a precursor of 1-octanol, n-butanol or 1-decanol (for example octanoic acid, butanoic acid, or decanoic acid respectively).
  • the second enzyme may convert the precursor into 1-octanol, n-butanol or 1-decanol, or into a different precursor of 1-octanol, n-butanol or 1-decanol.
  • the method of the invention may comprise providing the microorganism with one or more second enzyme.
  • the method may comprise providing the microorganism with one, two, three, or more second enzymes.
  • some or all of the second enzymes may be heterologous or homologous.
  • the second homologous enzyme may be aldehyde reductase.
  • the second heterologous enzyme may be selected from the group consisting of thioesterase, carboxylic acid reductase, and phosphopantetheinyl.
  • the thioesterase may be a C8-preferring thioesterase.
  • the thioesterase may be a C4- preferring thioesterase.
  • the thioesterase may be a C 10- preferring thioesterase.
  • the C8-preferring thioesterase may be selected from the group consisting of ‘CpFatBI (SEQ ID NO: 9), ‘CaFatB3 (SEQ ID NO: 10), Tes3 (SEQ ID NO: 11), or variants thereof.
  • a suitable variant of ‘CpFatBI is ‘CpFatB1-4 (SEQ ID NO:12).
  • a suitable variant of ‘CaFatB3 is ‘CaFatB3-5 (SEQ ID NO: 13).
  • Other suitable exemplary variants are disclosed in US2019284588, which is herein incorporated by reference.
  • the thioesterase enzyme comprises or consists of a truncated SEQ ID NO: 15 or SEQ ID NO: 16, or a variant or derivative thereof.
  • a thioesterase may be a C8-preferring thioesterase.
  • the thioesterase enzyme comprises a truncated SEQ ID NO: 15 or SEQ ID NO: 16
  • the enzyme does not comprise or consist of the full-length sequence according to SEQ ID NO: 15 or SEQ ID NO: 16.
  • the thioesterase enzyme that comprises or consists of a truncated SEQ ID NO: 15 or SEQ ID NO: 16 may comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21 , SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO: 90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, or a variant or derivative thereof.
  • the thioesterase enzyme that comprises or consists of a truncated SEQ ID NO: 15 or SEQ ID NO: 16 may comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21 , SEQ ID NO: 22, and SEQ ID NO:23.
  • Such thioesterases may be C8-preferring thioesterases, or C 10- preferring thioesterases.
  • thioesterase enzymes that comprises or consists of a truncated SEQ ID NO: 15 or SEQ ID NO: 16, such as thioesterase enzymes that comprise or consist of SEQ ID NO: 17, SEQ ID: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21 , SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO: 90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 or a variant or derivative thereof may increase the production of fatty acids in the microorganisms, and/or fatty acid derivatives as compared to the wild-type enzymes according
  • the fatty acid may be a C6, C8, C10, C12, C14, C16 or C18 fatty acid. More suitably a C8 or C10 fatty acid.
  • the fatty acid derivative may be an alcohol.
  • the alcohol has a 6, 8, 10, 12, 14, 16 or 18 carbon backbone. More suitably the alcohol is an alcohol that has an 8 or 10 carbon backbone.
  • the microorganisms is a cyanobacterium and the thioesterase is a C10- preferring thioesterase.
  • the C10-preferring thioesterase is selected from SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22 or a variant thereof.
  • the thioesterase may be a C4-preferring thioesterase.
  • the C4-preferring thioesterase may be CAH09236 from B.fragilis (SEQ ID NO: 102).
  • Thioesterase enzymes described herein that comprise or consist of a truncated SEQ ID NO: 15 or SEQ ID NO: 16 have been developed by the present inventors, and surprisingly found to increase the production of fatty acids or fatty acid derivatives, such as alcohols. This finding gives rises to a further aspect of the invention, directed to the novel thioesterase enzymes themselves.
  • the invention provides a thioesterase enzyme, wherein the thioesterase comprises or consists of a truncated SEQ ID NO: 15 or SEQ ID NO: 16, or a variant thereof.
  • the thioesterase enzyme comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO: 90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, or a variant or derivative thereof.
  • a thioesterase enzyme that is particularly useful in the context of increased fatty acid production or fatty acid derivative production may comprise or consist of SEQ ID NO: 23 or SEQ ID NO: 25.
  • variant refers to an amino acid sequence in which one or more amino acids have been replaced by different amino acids as compared to the corresponding amino acid sequence.
  • the variant may be at least 70%, at least 75%, at least 80%, be at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to the corresponding sequence.
  • a cysteine or proline is substituted for, or by, any other residue
  • a residue having an electropositive side chain e.g., Arg, His or Lys
  • an electronegative residue e.g., Glu or Asp
  • a residue having a bulky side chain e.g., Phe or Trp
  • one having a smaller side chain e.g., Ala, Ser
  • no side chain e.g., Gly
  • the present invention provides use of the thioesterase enzyme of the invention in the method of the invention.
  • the method of the invention may further comprise the step of providing a third enzyme to the microorganism.
  • the third enzyme may generate a substrate involved in producing a derivative of the medium or short chain length compound, such as a derivative of 1-octanol, n-butanol or 1-decanol.
  • the third enzyme may be heterologous or homologous (herein also referred to as the third heterologous enzyme or third homologous enzyme, respectively).
  • the method of the invention may comprise providing the microorganism with one or more third enzyme.
  • the method may comprise providing the microorganism with one, two, three, or more second enzymes.
  • some or all of the third enzymes may be heterologous or homologous.
  • the third heterologous enzyme may be sucrose synthase, for example Arabidopsis thaliana sucrose synthase (AtSUSI).
  • Arabidopsis thaliana sucrose synthase may be according to SEQ ID NO: 14 or a fragment or variant thereof.
  • fragment refers to an amino acid that consists of a truncation in the corresponding amino acid sequence (for example SEQ ID NO: 14).
  • sucrose synthase for example AtSUSI
  • sucrose synthase for example AtSUSI
  • octyl glucoside is the produced derivative of 1-octanol.
  • the substrate may be UDP-glucose.
  • UDP-glucose in produced by sucrose synthase from UDP and sucrose, rendering sucrose synthase particularly useful in the context of producing octyl glucoside.
  • harvesting refers to removing and/or isolating the derivative of medium or short chain length alcohol, for example derivative of 1-octanol, from the microorganism or culture medium in which the microorganism is grown.
  • Methods of harvesting the derivative, such as derivative of 1-octanol will be known to those skilled in the art. Merely by way of example harvesting may be by distillation. The harvesting may involve the use of a solvent overlay, for example to capture volatile products.
  • the method may further comprise providing a fourth protein to the microorganism.
  • the fourth protein is an acyl-carrier protein (ACP).
  • ACP acyl-carrier protein
  • the fourth protein may be homologous or heterologous, suitably it is heterologous.
  • the acyl-carrier protein is selected from EcACP (SEQ ID NO:83), 6803ACP (SEQ ID NO:84) , and CIACP2 (SEQ ID NO:85) .
  • the acyl-carrier protein is overexpressed in the microorganism.
  • the provision of overexpressed ACP increases metabolic flux through the enzymatic pathway.
  • the fourth protein may be provided to the microorganism indirectly by an expression vector comprising a nucleic acid encoding the protein.
  • the method may further comprise supplying the microorganism with a precursor of the medium or short chain length alcohol.
  • the derivative for example derivative of 1-octanol may be reconverted to 1-octanol.
  • a derivative of 1-octanol may be reconverted into 1-octanol by hydrolysis.
  • the derivative may be converted into a different compound, for example by oxidation.
  • the derivative of 1-octanol may be converted to a different compound, such as its corresponding aldehyde, octanal.
  • 1-octanol may be converted to octanol by oxidation.
  • the method of the invention is carried out in a cyanobacteria
  • the cyanobacteria comprises a thioesterase and a glycosyltransferase, wherein the thioesterase is CaFatB3-5, and the glycosyltransferase is MtH2.
  • the cyanobacteria is Synechocystis sp (for example PCC 6803).
  • the cyanobacteria may further comprise an acyl-carrier protein, suitably an overexpressed acyl-carrier protein.
  • the method of the invention is carried out in E.coli, and the E.coli comprises a thioesterase and an alcohol acetyltransferase, wherein the thioesterase is CaFatB3-5, and the alcohol acetyltransferase is ATF1.
  • the alcohol is n-butanol and the microorganism comprises a thioesterase and an alcohol acetyltransferase, wherein the thioesterase is a C4-preferring thioesterase and the alcohol acetyltransferase is selected from the group consisting of ATF1 (SEQ ID NO: 3), VAAT (SEQ ID NO: 103) or UAAT4 (SEQ ID NO:82).
  • the C4-preferring thioesterase is CAH09236 from B.fragilis (SEQ ID NO: 102).
  • the method of the invention is carried out in E.coli, and the E.coli comprises a thioesterase, wherein the thioesterase is a C8-preferring thioesterase selected from SEQ ID NO:23 or 25, or variants thereof.
  • the method of the invention is carried out in a cyanobacteria
  • the cyanobacteria comprises a thioesterase, wherein the thioesterase is a C 10- preferring thioesterase selected from SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22 or a variant thereof.
  • the cyanobacteria is Synechocystis sp (for example PCC 6803).
  • Figure 1 shows an overview of the bioderivatization process.
  • A Concept scheme. Inside the cell, the target molecule is enzymatically converted into a derivative (e.g. ester) (1), with potential consequences for both toxicity and purification. After the product has been excreted from the cell (2), the original target molecule can be recovered by enzymatic or chemical processes that remove the conjugate group (3).
  • the yellow box illustrates a cell or a production system.
  • the filled circle represents the conjugate, e.g. acid. 'Met' represents the target chemical in question.
  • B Non-stoichiometric overview of the two derivatization processes considered in this study, (1) esterification and (2) O-glucosylation.
  • FAS fatty acid synthesis
  • GT glycosyltransferase
  • AAT alcohol acetyltransferase
  • Figure 2 shows toxic effect comparison of 1-octanol, octyl acetate and octyl glucoside based on specific growth rates.
  • E. coli C43 (DE3) was cultivated in M9 minimal media and different concentrations of compounds of interest (0-50 mM) were added at the beginning of the cultivation.
  • the specific growth rate was determined by calculating the slopes of three biological replicates from average growth curves and only considered data in the range of 1- 4 hours.
  • the empty symbols represent the mean average from 3 biological replicates and each filled circle represents data from independent biological replicates.
  • Figure 3 shows comparison of toxicity of externally added octyl acetate or 1-octanol to Synechocystis sp.
  • PCC 6803 Twenty-five millilitre of Synechocystis sp. PCC 6803 lacking acyl-ACP synthetase was cultivated in a 100-ml Erlenmeyer flask with a starting OD 730 0.2 at 30°C, 180 rpm (60 mhioI photons/m 2 . s, 1% CO2) in AlgaeTron AG 230 (Photon Systems
  • Figure 4 shows pathway engineering for the synthesis of octyl glucoside and octyl acetate.
  • A Metabolic scheme depicting a novel synthetic pathway for in vivo production of octyl glucoside and octyl acetate extended from ACP-dependent 1-octanol pathway (TPC3) (17).
  • FIG. 5 shows identification of limiting substrates in 1-octanol and octyl acetate production by substrate feeding cultivation and effect of bioderivatization on E. coli growth and glucose consumption without overlay use.
  • E. coli C43 (DE3)
  • A Strain No. 2 (Tes3-Sfp-CAR) (Table 1)
  • B Strain No. 4 (Tes3-Sfp-CAR + SAAT) (Table 1) were cultivated in M9 media with 2% (w/v) glucose, 0.5 mM IPTG induction, 10% (v/v) hexadecane overlay and different concentrations of octanoic acid were added to the cultures.
  • Figure 6 shows average growth curves of E. coli C43 (DE3) cultivated in M9 media with varying concentrations of (A) 1-octanol (0 - 50 mM), (B) octyl acetate (0 - 50 mM), and (C) octyl glucoside (0 - 100 mM). All cells were cultivated in 96-well microtiter plates at 37°C, and 432 rpm. Data are the average ⁇ standard deviation from 3 replicates.
  • Figure 7 shows amino acid sequence alignments of CpFatB1_wild type (SEQ ID NO: 100), ‘CpFatBI (SEQ ID NO: 9), OpFatB1-4 (SEQ ID NO: 12), CaFatB3_ wild type (SEQ ID NO: 101), ‘CaFatB3 (SEQ ID NO: 10) and QaFatB3-5 (SEQ ID NO: 13).
  • Figure 8 shows selection of thioesterases and IPTG optimization for 1-octanol production and effect of bioderivatization on E. coli growth and metabolism in the absence of solvent overlay. Yield (mmol/mol glucose) from in vivo production of 1-octanol from E. coli BW25113 Strain No. 7 to 11 (Table 1) at 24 h and 48 h when cultivated in M9 media overlaid with 10% (v/v) hexadecane overlay with different IPTG used to induce at (A) 0.02 mM IPTG, (B) 0.05 mM IPTG, (C) 0.2 mM IPTG, and (D) 0.5 mM IPTG.
  • E Average growth curves of E. coli BW25113 Strain No. 12 (‘CpFatB1-4-Sfp-CAR; indicated as open black circles) and 14 (‘CpFatBI -4-Sfp-CAR + ATF1; indicated as open red squares) (Table 1) and (F) glucose consumptions when cultivated in M9 minimal media and 0.05 mM IPTG in the absence of solvent overlay.
  • the empty symbols represent the mean average from 3 biological replicates and each filled circle represents data from independent biological replicates.
  • Figure 9 shows toxic effect of octyl acetate on growth.
  • E. coli C43 E. coli C43 (DE3) was cultivated in M9 minimal media with 2% (w/v) glucose and different concentrations (50-200 mM) of octyl acetate were added at the beginning of the cultivation.
  • the specific growth rate was calculated using slopes from average growth curves and only considered data in the range of 1-4 h. Data are the average ⁇ standard deviation from 3 replicates.
  • FIG. 10 shows growth from octyl acetate producing strains and 1-octanol producing strains.
  • E. coli Strain No. 12 (‘CpFatBI -4-Sfp-CAR), 14 (‘CpFatB1-4-Sfp-CAR + ATF1), 13 (‘CaFatB3- 5-Sfp-CAR) and, 15 (‘CaFatB3-5-Sfp-CAR + ATF1) (Table 1) were cultivated in M9 minimal media with 2% (w/v) glucose and 10% (v/v) hexadecane overlay. Different concentrations of IPTG were used to induce the cultures (A) 0.05, (B) 0.2 and, (C) 0.5 mM.
  • FIG 11 shows glucose consumption from octyl acetate producing strains and 1-octanol producing strains.
  • E. coli Strain No. 12 (‘CpFatBI -4-Sfp-CAR), 14 (‘CpFatBI -4-Sfp-CAR + ATF1), 13 (CaFatB3-5-Sfp-CAR) and, 15 (‘CaFatB3-5-Sfp-CAR + ATF1) (Table 1) were cultivated in M9 minimal media with 2% (w/v) glucose and 10% (v/v) hexadecane overlay. Different concentrations of IPTG were used to induce the cultures (A) 0.05, (B) 0.2 and, (C) 0.5 mM.
  • Figure 12 shows comparison of in vivo 1-octanol and octyl acetate production from E. coli BW25113 in the presence of solvent overlay.
  • the 1-octanol and octyl acetate production from E. coli Strain No. 13 ‘CaFatB3-5-Sfp-CAR) and No.
  • Figure 13 shows GC-MS chromatogram of (A) 1-octanol and (B) octyl acetate peak.
  • One millimolar of 1-octanol or octyl acetate was spiked into 25 ml of M9 liquid media containing 2% (w/v) glucose in the presence of 10% (v/v) hexadecane overlay. Flasks were incubated at 30°C, 150 rpm for 24 h. The hexadecane overlay was sampled and analyzed by GC-MS, whilst the aqueous phase was extracted by 10% (v/v) hexadecane before the analysis.
  • Figure 14 shows evaluation of 1-octanol production from 2 E. coli strain backgrounds BW25113 and C43 (DE3).
  • the strains harboring pET-PA1lacO-1-TPC3 were cultivated in M9 minimal media with 2% (w/v) glucose, induced with 0.5 mM IPTG and overlaid with 10% (v/v) hexadecane overlay for 48 h. Data are the average from 3 biological replicates.
  • Figure 15 shows cellular health of E. coli strains harboring different thioesterases. Average growth of (A) Strain No. 10 (CpFatB1-4) and (B) 11 (CaFatB3-5) (Table 1) when cultivated in M9 media with 2% (w/v) glucose overlaid with 10% (v/v) hexadecane overlay with different IPTG used to induce the cultures (0.02, 0.05, 0.2 and 0.5 mM). Data are the average ⁇ standard deviation from 3 replicates.
  • FIG 16 shows growth from 1-octanol producing and octyl acetate producing strains in 24- well plate with solvent overlay.
  • E. coli Strain No. 12 CpFatB1-4-Sfp-CAR
  • 14 ‘CpFatB1-4- Sfp-CAR + ATF1
  • 13 CaFatB3-5-Sfp-CAR
  • 15 ‘CaFatB3-5-Sfp-CAR + ATF1
  • Table 1 were cultivated in M9 minimal media with 2% (w/v) glucose in 24-well microtiter plate at 30°C and 432 rpm.
  • IPTG was used to induce the strains at 0.2 mM and 10% (v/v) hexadecane overlay was applied at the beginning of the incubation. Data are the average ⁇ standard deviation from 4 replicates.
  • Figure 17 shows conversion of 1-octanol into octyl acetate resulted in enhanced titer, growth, and yield.
  • A Growth profile of 1-octanol producing strain (Table 2, Strain No. 3, Aaas-PnrsB- Sfp-CAR-Pcoa-‘CpFatB1-4) vs octyl acetate producing strain (Table 2, Strain No. 4, Aaas- PnrsB-Sfp-CAR-Pcoa-‘CpFatB1-4-SAAT) in the absence of overlay for 20 days. All strains were induced to express recombinant proteins on day 2 with 625 nM cobalt and 15 uM nickel.
  • Figure 18 shows engineering a synthetic pathway for octyl glucoside biosynthesis with pathway enhancement by introducing AtSUSI for in-situ UDP-glucose regeneration and pathway flux comparison.
  • A Schematic diagram of plasmids used to generate Strain No. 13,
  • Figure 19 shows comparison of in vivo 1-octanol and octyl acetate production from E. coli BW25113 (titers).
  • E. coli Strain No. 12 ‘CpFatB1-4-Sfp-CAR), 14 (‘CpFatB1-4-Sfp-CAR + ATF1) at (A) 24 h, (B) 48 h, (C) 72 h and Strain No.
  • Figure 20 shows the ratios between product yields from octyl acetate producing strains and 1-octanol producing strains.
  • Figure 21 shows the ratios between product titers from octyl acetate producing strains and 1- octanol producing strains.
  • FIG 22 shows localization of octyl glucoside in the absence of solvent overlay.
  • E. coli BW25113 Strain No. 13 (‘CaFatB3-5-Sfp-CAR) and 19 (‘CaFatB3-5-Sfp-CAR + MtH2) were cultivated in M9 media with 2% (w/v) glucose with 0.5 mM IPTG. The cultures were harvested at centrifuged (17,000 x g, 10 min) to separate culture media and cells. The supernatant was analysed with HPLC. The cell fraction was suspended and lyzed with glass beads (Sigma Aldrich) at 30 hertz for 6 min on Tissuelyser II (QIAGEN). After that, the liquid fraction was analysed with HPLC.
  • FIG. 23 shows comparison of in vivo 1-octanol and octyl glucoside production from E. coli BW25113 (titers).
  • E. coli Strain No. 13 (‘CaFatB3-5-Sfp-CAR), 21 (‘CaFatB3-5-Sfp-CAR + AtSUSI), 19 (‘CaFatB3-5-Sfp-CAR + MtH2) and 22 (‘CaFatB3-5-Sfp-CAR + MtH2 + AtSUSI) were cultivated in M9 media with 2% (w/v) glucose supplemented with 15 mM sucrose at (A) 24 h (B) 48 h.
  • the asterisk indicates significance by Student’s t-test (**, P£0.01 ;***, P£0.005). All data are shown as the average from 3 biological replicates and circles represent data from independent biological replicates.
  • Figure 24 shows growth from octyl glucoside producing strains and 1-octanol producing strains in the presence of solvent overlay.
  • E. coli Strain No. 13 (‘CaFatB3-5-Sfp-CAR), No. 19 (‘CaFatB3-5-Sfp-CAR + MtH2), No. 21 (‘CaFatB3-5-Sfp-CAR + AtSUSI), and No. 22 (‘CaFatB3-5-Sfp-CAR + MtH2 + AtSUSI) (Table 1) were cultivated in M9 minimal media with 2% (w/v) glucose, 0.5 mM and 10% (v/v) hexadecane overlay.
  • Figure 25 shows alcohol yield generated by wild type and novel truncated thioesterase enzymes.
  • A alcohol yield by truncated CvFatBI thioesterases.
  • B Fatty acid and fatty alcohol yield by truncated ChoFatB2 thioesterases.
  • Figure 26 shows (A) Photosynthetic growth in shake flasks of Synechocystis sp.
  • PCC 6803 strains engineered to contain either (i) the 1-octanol pathway ('CaFatB3-5') or (ii) the 1-octanol pathway + glucosyltransferase ('CaFatB3-5-MtH2'); (B) molar product titer of engineered Synechocystis sp.
  • PCC 6803 strains in the absence of solvent overlay left panel shows the weight of the biomass whilst the right panel indicates the visual density of the cultures.
  • Figure 27 shows different concentrations of alcohol or ester that were used to spike Escherichia coli cultures grown in 24-well plates with LB media. After 15h the OD600 of each culture was measured. In each panel, the white bar represent the alcohol whilst the grey bar(s) represent the corresponding acetyl ester, or in the case of linalool, also the butyryl ester (light grey).
  • Figure 29 shows (A) Chain-length specificity of CvFatBI truncations in E. coli after cultivation in minimal M9 media for 48 hours. All E. coli strains also express the CAR enzyme and helper Spf protein, resulting in conversion of released "free"' fatty acids into corresponding alcohols; (B) Chain-length specificity of CvFatBI truncations in Synechocystis sp. PCC 6803 after cultivation in BG11 media for 10 days. All Synechocystis strains also express the CAR enzyme and helper Spf protein, resulting in conversion of released "free"' fatty acids into corresponding alcohols.
  • ACPs acyl-carrier proteins
  • bioderivatization has been implemented without a rationale (e.g. vanillin glucoside biosynthesis as opposed to synthesizing plain vanillin (2)) or happened by chance through interactions with native metabolism in the biotechnological host (e.g. geraniol synthesis results in geranyl acetate formation through the action of native AATs (13)).
  • a rationale e.g. vanillin glucoside biosynthesis as opposed to synthesizing plain vanillin (2)
  • geraniol synthesis results in geranyl acetate formation through the action of native AATs (13)
  • solvent overlay offers an opportunity to reduce toxicity, most likely simply by facilitating product removal.
  • solvent overlay is undesirable, for example when attempting to separate a volatile product in the off-gas or when the energetic cost of solvent- product separation is excessive. The presence or absence of solvent overlay was therefore also evaluated.
  • E. coli DH5a Thermo Fisher Scientific was used to propagate all the plasmids used in this work.
  • Two strains of E. coli E. coli C43 (DE3) (Lucigen) and BW25113 (Keio collection)
  • one species of cyanobacteria Synechocystis sp. PCC 6803 were used as hosts for 1-octanol and octyl acetate production. All E. coli and cyanobacterial strains used in this study are listed in Table 1 and Table 2, respectively.
  • Tes3, Sfp, and CAR were obtained from plasmid pET-TPC3 (17), whereas the chloramphenicol acyltransferase (cat) was amplified from plasmid pACYC-petF-fpr (18).
  • a strawberry alcohol acetyltransferase gene (.
  • AdGT4 Actinidia deliciosa A0A077EMP8 (Yauk et al., 2014)
  • VvGTl Vitis vinifera P51094 (Christopher M. Ford et al., 1998)
  • AtGTl Arabadopsis thaliana 0.9 M 156 (Lim et al., 2002)
  • AtSUSl Arabidopsis thaliana P49040 (Zheng et al., 2011)
  • E. coli strain C43 (DE3) was transformed with plasmid pET-TPC3 (Akhtar et al., 2015) harboring tes3, sfp, and car genes. Plasmid pACYC-petF-fpr plasmid (Kallio et al., 2014) was used as a template to amplify the cat gene.
  • the strawberry alcohol acetyltransferase gene ( saat ) from Fragaria x ananassa (Aharoni et al., 2000) (UniProtKB: Q9FVF1) and the alcohol O-acetyltransferase gene ( atf1 ) from Saccharomyces cerevisiae (UniProtKB: P40353) were chemically synthesized from Integrated DNA Technologies (IDT) and codon optimized for E. coli.
  • the saat and atf1 genes were individually stored in a blunt-ended pJET1.2 plasmid (Thermo Fisher Scientific), resulting in pJET-SAAT and pJET-ATF1 plasmids, respectively.
  • pCDF-GFP plasmid harboring T7 promoter was constructed as described in Table 8.
  • the cat, saat, and atf1 genes were then sub-cloned into pCDF-GFP plasmid using Ssal restriction site which was introduced by PCR using the oligonucleotide primers listed in Table 4.
  • the resulting plasmid was transformed into the E. coli C43 (DE3) strain carrying the pET-TPC3 plasmid, generating Strain No. 3 to 5 in Table 1.
  • BASIC method was used to construct plasmids in Strain No. 7 to 15 in Table 1.
  • plasmid pET-PA1lacO-1-GFP and pCDF-PA1lacO-1-GFP harboring PA1lacO-1 promoter were constructed as shown in Table 7 No. 1 and 2, respectively.
  • the tes3 and sfp-car genes were amplified from plasmid pET-TPC3 (Akhtar et al., 2015) using primers listed in Table 4 and cloned into a blunt-ended plasmid pJET1.2 (Thermo Fisher Scientific).
  • genes encoding thieosterases ‘CpFatBI, ‘CaFatB3,’CpFatB1-4, and ‘CaFatB3-5 genes were chemically synthesized as gBIocks from Integrated DNA Technologies (IDT) and cloned into pJET1.2 blunt. The resulting plasmids were used to create plasmids used in Strain No. 7 to 15 in Table 1. The linkers and plasmids used for BASIC method are listed in Table 7 No. 3 to 9.
  • E. coli strain BW25113 was transformed with pET-‘CaFatB3-5- Sfp-CAR.
  • IDTT Integrated DNA Technologies
  • GFP gene was amplified from pJET-GFP (Yunus and Jones, 2018) using oligonucleotides with Ncol and Avrll cutting sites for the construction of pCDF-GFP plasmid backbone. The amplified GFP was then cloned into pCDF-Ahr s (Akhtar et al., 2015) by replacing Ahr s with GFP.
  • cat was amplified from pACYC-petF-fpr with Bsal prefix and suffix, while SAAT and ATF1 were order as a gblock from IDT technology. All 3 genes were
  • pCDF-GFP backbone cloned into pCDF-GFP backbone.
  • pET-TPC4 was digested with Ncol and Avrll and pCDF-GFP was digested with Bsal. Both backbones were recirculated using Quick BluntingTM and Quick LigationTM Kits from NEB after restriction digestion.
  • PB29 PB30 plY98 pJET::Sp R Spectinomycin resistance cassette was ordered as a gBIock. Genetic Part 6 - Terminator plY67 pJET::termB15 Plasmid storage containing rrnB T1 terminator and T7Te terminator.
  • Plasmid backbone for plasmid propagation in E. coli with a plY23 pColE1 ::AmpR carbenicillin selection marker cassette (requested from Dr. Geoff Baldwin)
  • 1MP plY442 phaABJJHR 1S plY453 is the plasmid carrying the upstream genetic parts for the
  • 1P plY98 Sp ,R> 2S plY454 is the plasmid carrying the downstream genetic parts for the plY454
  • Strain 6803- Aaas was constructed as described in (Yunus and Jones, 2018). To create strain 6803-Aaas-PnrsB-Sfp-CAR, a suicide plasmid targeting phaA and phaB sites (encoded by slr1993 and slr1994) was first constructed. All the genetic parts used for this plasmid construction are listed in Table 9. Next, the plasmids carrying the upstream (plY453) and downstream (plY454) genetic parts were assembled using the linkers shown in Table 10. Next, the suicide plasmid (plY475) carrying a gfp dropout gene were created as shown in Table 11.
  • sfp and car genes were then amplified using primers listed in Table 4 and sub-cloned into pJET1.2 blunt plasmid to give plasmid plY485. Finally, the sfp and car genes were cloned into plasmid plY475 using the linkers shown in Table 12. The expression of Sfp and CAR were under controlled of a nickel-inducible promoter PnrsB. Plasmid plY706 (pMB1- Amp-phaAup-PnrsB-Sfp-CAR-termB15-Sp-phaBdown) was then naturally transformed into 6803 -Aaas strain.
  • 6803- Aaas strain was inoculated in 25 ml of BG11-Co liquid medium at 30°C with continuous illumination at 60 mhioI photons/m 2 . s and 1% (v/v) CO2 in the Algaetron AG 230 (Photon Systems Instruments).
  • the OD730 reached 0.3-0.4, the cells were harvested and resuspended in 500 mI_ fresh BG11-Co medium.
  • One hundred microliters of concentrated liquid culture were mixed with four to seven micrograms of plasmid plY706 and incubated at at 60 pmol photons/m 2 .
  • a fully segregated mutant was confirmed by PCR using primers IY293 (5’-GGCAAAGCTTTATTTGCCAATGCG-3’ (SEQ ID NO: 78)) and IY292 (5’-CCGATGACACTAATCTCAAGGCGG-3’ (SEQ ID NO: 79)) and used in the subsequent experiments.
  • PnrsB Amp R 2MS plY475 is a suicide vector targeting phaAB
  • LRBS1-4P plY485 Amp R 1S plY706 is a suicide vector carrying sfp and car genes targeting plY706
  • plasmid plY849 and plY887 was first transformed individually into E. coli cargo HB101 strain carrying pRL623 plasmid (Elhai et al. , 1997).
  • E. coli cargo HB101 strain carrying pRL623 plasmid Elhai et al. , 1997.
  • conjugal strain carrying plasmid pRL443 Elhai et al., 1997)
  • the 6803-Aaas-PnrsB-Sfp-CAR strain were mixed and incubated for 2 h (30°C, 60 mhioI photons/m 2 . s).
  • all strains were washed with LB medium (for E.
  • E. coli strain C43 (DE3) was cultivated in 10 ml of Luria-Bertani (LB) liquid media (LB broth, Sigma Aldrich) overnight at 37°C, 180 rpm. The overnight culture was washed twice with fresh M9 minimal media (47.8 mM Na 2 HP0 4 , 22 mM KH 2 P0 4 , 8.55 mM NaCI, 18.69 mM NH 4 CI, 2 mM MgS0 4 , 0.1 mM CaCI 2 and 2% (w/v) glucose) and resuspended in the same media to an initial O ⁇ boo of ⁇ 0.1.
  • M9 minimal media 47.8 mM Na 2 HP0 4 , 22 mM KH 2 P0 4 , 8.55 mM NaCI, 18.69 mM NH 4 CI, 2 mM MgS0 4 , 0.1 mM CaCI 2 and 2% (w/v) glucose
  • the liquid cultures were spiked with different concentrations of 1-octanol (0-50 mM) and octyl acetate (0-200 mM) and transferred (200 pi) into a well in 96-well microtiter plate.
  • the plate was incubated in Tecan Infinite M200 Pro Spectrophotometer (Tecan AG) at 37°C with continuous shaking at 432 rpm and O ⁇ boo was measured every hour for 15 hours. The specific growth rates were calculated for each treatment.
  • octyl glucoside production the cultures were cultivated at 37°C, 180 rpm for 4 h and induced with 0.5 mM isopropyl b-D-l-thiogalactopyranoside (IPTG) before continuing the incubation at 30°C, 150 rpm for 48 h. Samples were centrifuged and the supernatants were analyzed using HPLC. Sucrose (15 or 100 mM) was supplemented to the culture media at the time of induction when the function of AtSUSI was assessed. Solvent overlay was used when the production of 1-octanol and octyl glucoside was compared.
  • IPTG isopropyl b-D-l-thiogalactopyranoside
  • BG11-Co cyanobacterial strains were cultivated in BG11 medium without cobalt (hereafter BG11-Co) as cobalt was used as an inducer.
  • the preculture was grown in 5 ml BG11-Co containing appropriate antibiotic(s) (final concentration: kanamycin 10 pg/ml, spectinomycin 10 pg/ml, and erythromycin 20 pg/ml) in a 6-well plate at 30°C, 180 rpm, with continuous illumination (warm-white LED) at 60 pmol photons/m 2 .
  • s and 1% (v/v) C0 2 i n an Algaetron AG 230 Photon Systems Instruments (PSI)).
  • the liquid preculture was transferred into an autoclaved 100-ml Erlenmeyer flask covered with aluminium foil.
  • the OD730 was adjusted to 0.2 by adding BG11-Co to a final volume of 25 ml and antibiotics were added accordingly.
  • the liquid culture was induced on day 2 with 15 pM nickel and 625 nM cobalt, and the OD730 was monitored for 20 days in the presence or absence of a 30% (v/v) hexadecane solvent overlay. On day 20, the liquid culture was transferred into fresh BG11-Co media containing antibiotics with initial OD730 -0.2.
  • the standard 1X BG11-Co liquid medium was prepared by mixing 10 ml 100X BG11-Co, 1 ml 1000X ferric ammonium citrate, 1 ml 1000X Na 2 C0 3 , and 1 ml 1000X K2HPO4 in 1 L of ultrapure water (PURELAB flex 2).
  • the concentrated BG11-Co stock solution (100X BG11- Co) containing 149.6 g NaN03, 7.49 g MgS04.7H20, 3.6 g CaCI2.2H20, 0.89 g Na- citrate.2H20, 1.12 ml 0.25 M NaEDTA pH 8.0, and 100 ml 1000X trace mineral solution in 1 L ultrapure water was prepared.
  • the 1000X trace mineral solution was made by dissolving 2.86 g H3B03, 1.81 g MnCI2.4H20, 0.22 g ZnS04.7H20, 0.39 g Na2Mo04.2H20, and 0.079 g CuS04.5H20 in 1 L ultrapure water and stored at 4°C until being used.
  • the 1000X ferric ammonium citrate, 1000X Na 2 C0 3 , and 1000X K2HPO4 solutions were prepared by dissolving 0.6 g ferric ammonium citrate, 2 g Na 2 C0 3 , and 3.05 g K2HPO4 in 100 ml ultrapure water, respectively.
  • the 1 L standard 1X BG11-Co solution in a 1-L Duran bottle was sterilized by autoclaving for 15 min at 15 psi (121°C) and cooled down to room temperature prior to use.
  • Hexadecane solvent overlay was used to capture 1-octanol and octyl acetate from the liquid culture.
  • One hundred microliters of solvent overlay were transferred into an insert in a 2-ml screw top GC vial (Agilent Technologies).
  • Samples were analyzed using an Agilent 7890B GC with HP-5ms column, a 7693 autosampler and 5977B MSD.
  • One pi of samples was injected using pulsed split ratio 10:1 and split flow at 10 min/ml.
  • the GC method was programmed with an initial temperature of 70°C for 30 s, followed by a first ramp at 30°C/min to 250°C before ramping up to 300°C with a final hold for 2 min at 40°C/min.
  • Target products were identified by comparing mass spectra and retention times with external standards. Serial dilutions of 1-octanol (399%, Sigma Aldrich) and octyl acetate (399%, ACROS organics) standards were used to quantify the concentration of 1-octanol and octyl acetate in the sample.
  • E. coli strains were used to inoculate LC media in 24 well-plates and incubated in a Tecan microplate reader at 37°C, 432 rpm, over 24h.
  • a range of different concentrations (0-30 mM) of chemicals were added to E. coli culture with the same starting OD600 at the beginning of the cultivation. Where a fully soluble stock solution was not possible to prepare, each compound was spiked directly to each well. The specific growth rates were calculated for each treatment.
  • Octyl acetate is naturally found in wild strawberry ( Fragaria vesca) (23).
  • Fragaria vesca For use as a food flavor additive, it is also synthesized by a direct esterification reaction between acetic acid and octyl alcohol, catalyzed by either acids, ion exchange resins or ionic liquids (24).
  • acids, ion exchange resins or ionic liquids 24.
  • the TPC3 pathway was extended with an alcohol acetyltransferase (AAT) under the assumption that native acetyl-CoA was not limiting ( Figure 4A).
  • AAT alcohol acetyltransferase
  • Three AAT enzymes CAT (25), SAAT (19), and ATF1 (26) were selected based on the literature.
  • CAT has previously been used in E. coli for ester biosynthesis (27), whilst both SAAT and ATF1 have been reported to utilize 1-octanol as a substrate (19, 27, 28).
  • Tes3-Sfp-CAR showed noticeably lower cell density (Figure 5C, 5D, 5G, and 5H) and glucose consumption (Figure 5E and 5F), an effect that was largely alleviated by esterification (Strain No. 4, Tes3-Sfp-CAR + SAAT), thereby providing early insight towards answering the main hypothesis of the work.
  • the differences between the alcohol and ester-forming strains were reduced in the absence of externally added octanoic acid (Figure 5C, 5D, 5G, and 5H) supporting the idea that further pathway optimization was essential in order to evaluate the effect of bioderivatization under conditions that were more likely to be relevant for application.
  • the first generation 1-octanol pathway was also evaluated in two different E. coli strain backgrounds; the E. coli B strain C43 (DE3) and K-12 strain BW25113.
  • E. coli B strain C43 DE3
  • the new thioesterases were based on Cuphea palustris (CpFatBI) (29) and Cuphea avigera pulcherrima (CaFatB3) (30) with varying mutations and truncations (Figure 7), two of which were designed according to recent work by Lozada et al. (31).
  • All new thioesterases showed an improvement in 1-octanol production compared to Tes3 (15) but the performance varied greatly in response to changes in the inducer concentration (Figure 8).
  • glycosylation This requires changes in the metabolic engineering and has implications for both cellular efflux and product separation.
  • the simplest glycosylation to implement is O-glucosylation, thereby resulting in the formation of octyl glucoside, a non-ionic alkyl glucoside that is used as a surfactant (32).
  • octyl glucoside was also shown to be less toxic than 1-octanol ( Figure 2).
  • Glycosides are also highly hydrophilic which will require a different choice of downstream processing for separation, compared to esters. We are not aware of any prior reports of biotechnological production of octyl glucoside with a microbial host.
  • the ‘CaFatB3-5 thioesterase 1-octanol pathway was extended by overexpressing a glycosyltransferase (GT) ( Figure 4A). Five candidates were selected based on their reported activity towards longer-chain alcohols (33, 34).
  • GT glycosyltransferase
  • MtH2 glycosyltransferase from Medicago iruncaiula
  • Insufficient glucosylation may be due to a limitation in the supply of substrates, i.e. UDP- glucose or inhibition by UDP (36), one of the products of the glucosylation reaction.
  • UDP-glucose Another possible limiting factor is UDP-glucose availability.
  • UDP-glucose is involved in bacterial cell wall synthesis and is produced at a basal level in E. coli (37). Given that the UDP- glucose pool is relatively low, regeneration is important in order to archive a high production of glucosides.
  • Sucrose synthase SUS is an enzyme catalyzing a reversible reaction that converts sucrose and UDP to UDP-glucose which has been shown to enhance glucoside production (38).
  • sucrose synthase from Arabidopsis thaliana namely AtSUSI was co-expressed with GT and 1-octanol pathway (Figure 4A) to improve octyl glucoside production. It was shown that the overexpression of AtSUSI improved the production of octyl glucoside ( Figure 18C) and highly excessive amount of sucrose (100 mM) supplied resulted in more octyl glucoside produced ( Figure 18D).
  • SUS will not only help maintain the supply of UDP-glucose, but it is expected that it also will lower the intracellular concentration of UDP, thereby lowering any inhibitory effect that it may have on MtH2.
  • Bio-based production technologies may complement or even replace petroleum-based production of chemicals.
  • the development of such biotechnological production systems faces a number of technical challenges, incl. toxicity to the host caused by the chemical it has produced.
  • Many plants and microorganisms are naturally capable of biosynthesizing toxic molecules but they often convert them into derivatives before storage or excretion.
  • bioderivatization strategy for biotechnological chemicals production defined as purposeful biochemical derivatization of intended target molecules.
  • hydrophobic e.g. esterification
  • hydrophilic e.g.
  • bioderivatization affected (1) cellular health (i.e. growth and glucose consumption), independently of an effect on productivity, whilst in other designs (2) productivity was influenced without any apparent effect on cellular health. Overall, under most conditions, bioderivatization was found to benefit production of the toxic model bioproduct selected in this study and could be considered also for other products that are toxic to the production host.
  • Each thioesterase-encoding gene was incorporated into an operon containing also CAR and Sfp in a pET-plasmid under the control of the Plac promoter, resulting in a series of pET-Plac- Tes-Sfp-CAR-KanR plasmids.
  • pCDF-Plac-Yjgb-SpecR When co-expressed together with pCDF-Plac-Yjgb-SpecR, this will result in the biosynthesis and excretion of fatty alcohols into the media.
  • Both plasmids were used to transform Escerichia coli strain JW1994-1. Pre-cultures were grown overnight in 5 ml_ LB, in a 50 mL falcon tube, supplemented with the appropriate antibiotics, at 37°C and 180 rpm. Overnight cultures were washed with fresh M9 and then used to inoculate 25 mL M9 with appropriate antibiotics, 2% (v/v) glucose and 10% (v/v) isopropyl myristate overlay in a 100 mL Erlenmeyer flask, at a starting OD600 of 0.1. Cultures were then incubated at 37°C, 180 rpm for four hours before induction with 0.5 mM IPTG. Cultures were then incubated at 30°C, 150 rpm. At 24 and 48 hours, cultures were sampled for growth (OD600) and the overlay sampled for GCMS analysis.
  • the sample was analysed using GC-MS Agilent Technologies G1530A series Gas Chromatograph (GC) equipped with DB-WAXetr column coupled with 5973 Mass Selective Detector.
  • GC Gas Chromatograph
  • the oven temperature was initially held at 100°C for 50s, then ramped at 30°C/min until 250°C, where it was held for 1min.
  • Variant thioesterase sequences were created and assayed for fatty acid derived products in Escherichia coli in some cases in combination with the CAR enzyme (hence the products were in this case alcohols) as described above.
  • the amino acid encoded by the wild type CvFatBI gene (Jing et al (2011) BMC Biochemistry 12:44) produced no products.
  • Truncated variant sequences surprisingly generated measurable alcohols.
  • Variant T7 was found to be particularly good for producing octanol.
  • the variant T4 was also of interest as it showed good C10 productivity, relative to C8 ( Figure 25).
  • the entire octyl glucoside biosynthesis pathway was previously implemented in E. coli (Sattayawat et al., 2020), and demonstrated herein in figures 22-24. Here, we asked whether it could also function in Synechocystis sp. PCC 6803 with similar outcomes as observed in E. coli.
  • the octyl glucoside pathway was implemented in Synechocystis and cultured in the presence of solvent overlay in order to monitor both 1 -octanol (quantified in the solvent overlay) and octyl glucoside (quantified in the aqueous phase).
  • glucosylation of 1-octanol had a major impact on the health of the cyanobacteria and also improved metabolic flux, resulting in enhanced productivity, similar to what was already observed with glucosylation in E. coli and with esterification in both E. coli and Synechocystis sp. PCC 6803.
  • strains were done according to 'Construction of cyanobacterial strains for 1-octanol and octyl acetate production' hereinabove, except by replacing the AAT-encoding gene with the Mth2-encoding gene as described in 'Construction of E. coli strains for octyl glucoside production' hereinabove.
  • the new alcohols (menthol, linalool, eugenol and cinnamyl alcohol) and corresponding esters (menthol acetate, linalyl acetate, linalyl butyrate, eugenyl acetate and cinnamyl acetate) were used, all in the range of 0-30 mM except for cinnamyl alcohol and cinnamyl acetate which were only tested in the range of 0-5 mM.
  • Atf1 Saccharomyces cerevisiae Alcohol O-acetyltransferase 1
  • CAT Escherichia coli Chloramphenicol acetyltransferase
  • SAAT Fragaria ananassa cv.
  • Elesanta Alcohol acyltransferase Elesanta Alcohol acyltransferase), VAAT (, Fragaria vesca, Alcohol acyltransferase), VpAAH ( Vasconcellea pubescens, Alcohol acyltransferase 1), AtHPFT ( Arabidopsis thaliana Omega-hydroxypalmitate O-feruloyl transferase), CcVs ( Citrus Clementina Vinorine synthase), CsVs ( Citrus sinensis Vinorine synthase-like), LaAAH ( Lavandula angustifolia Putative alcohol acyltransferase 1), LaAAt2 (, Lavandula angustifolia Putative alcohol acyltransferase 2), LaAT1 ( Lavandula angustifolia Rosmarinic acid synthase), UAAT4 (Lavandula x intermedia clone AAT-4 alcohol acetyltransferase), N
  • n-butanol ⁇ 1 g/L
  • E. coli has been reported to show only weak toxicity to n-butanol at a concentration approx. 10x that which were obtained here (Fletcher et al., 2016), i.e. 7g/L.
  • AAT-encoding genes displayed activity resulting in the biosynthesis of butyl acetate, Atf1 (SEQ ID NO: 3), SAAT (SEQ ID NO:2) , VAAT (SEQ ID NO: 103) and UAAT4 (SEQ ID NO:82).
  • strains were described according to 'Construction of E. coli strains for 1- octanol and octyl acetate production' hereinabove except the AAT-encoding genes (Atf1 , CAT, SAAT) were replaced with the alternative AATs and the C-8 specific thioesterase was replaced with that described by Pasztor et al. (2014); GenBank ID CAH09236, originating from Bacteroides fragilis (SEQ ID NO: 102) .
  • strains were described according to 'Construction of E. coli strains for 1- octanol and octyl acetate production' as above except the thioesterase was Cuphea viscosissima (CvFatB1)(Jing et al., 2011) and different N-terminal truncations were prepared by shifting the location of the primer-recognizing sequence, against the CvFatBI encoding gene, prior to BASIC-assembly of the synthetic operons. The same constructs were then also used to prepare cyanobacteria strains according to 'Construction of cyanobacterial strains for 1-octanol and octyl acetate production' hereinabove.
  • EcACP from Escherichia coli (SEQ ID NO: 83)
  • 6803ACP from Synechocystis Sp. PCC 6803
  • CIACP2 from Cuphea lanceolata (SEQ ID NO:85).
  • the negative control strain (NA) combined pET-Plac-Tes-KanR + pCDF-Sfp-CAR, whilst the ACP strains combined pET-Plac- Tes-KanR + pCDF-PrhaBAD-ACP-Sfp-CAR.
  • Tes CvFatb1-T5.
  • SEQ ID NO: 14 Amino acid sequence of AtSUSI MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTRK
  • VLDM I RLLLDLLEAPDPCTLETFLGRVPM VFN VVI LSPHGYFAQDN VLGYPDTGGQVVYI LD
  • SEQ ID NO: 27 DNA sequence of [SEQ ID NO: 1]
  • SEQ ID NO: 28 DNA sequence of [SEQ ID NO: 2]
  • AGCCAT ACCACTCCAG AG AT CAGCG AT CT GAAACT GTGT GAT CTGGT G AACCT GTT G AA
  • SEQ ID NO: 29 DNA sequence of [SEQ ID NO: 3] ATGAACGAGATCGATGAGAAGAATCAAGCACCGGTCCAGCAAGAGTGTTTGAAGGAGA TGATTCAAAATGGGCATGCACGTCGCATGGGTTCTGTCGAAGATTTGTATGTAGCACTG AACCGTCAGAACTT AT ACCGT AATTTTTGCACCT ACGGGGAGTT ATCT GACT ATT GT ACC CGT GAT CAATT G ACT CTTGCCTT ACGT GAAATCTGCCT G AAG AATCCCACATT GTT ACAC ATCGT ACTGCCAACCCGTTGGCCAAAT CACG AG AATT ACT ATCGCTCCAGT G AGT ACTA CT CACGCCCTCATCCCGTGCACG ATT AT AT CT CT GTGCTT CAAG AACT G AAATT AT CAG GCGT AGTCCTT AAT GAACAACCGGAGT ATTCTGCGGTT AT GAAGCAGATTTTGGAGGAG TTT AAG AAT AGT AAGGGCT CTT AT ACAGC
  • SEQ ID NO: 30 DNA sequence of [SEQ ID NO: 4]
  • SEQ ID NO: 31 DNA sequence of [SEQ ID NO: 5]
  • SEQ ID NO: 32 DNA sequence of [SEQ ID NO: 6]
  • SEQ ID NO: 33 DNA sequence of [SEQ ID NO: 7]
  • SEQ ID NO: 34 DNA sequence of [SEQ ID NO: 8]
  • SEQ ID NO: 35 DNA sequence of [SEQ ID NO: 9]
  • SEQ ID NO: 36 DNA sequence of [SEQ ID NO: 10]
  • SEQ ID NO: 37 DNA sequence of [SEQ ID NO: 11]
  • SEQ ID NO: 38 DNA sequence of [SEQ ID NO: 12]
  • SEQ ID NO: 39 DNA sequence of [SEQ ID NO: 13]
  • SEQ ID NO: 40 DNA sequence of [SEQ ID NO: 14]
  • AAAG ACAAAAAAAAACCG ATCCT GTT CACCATGGCT CGTCTGG ACCGT GTT AAAAAC
  • SEQ ID NO:82 UAAT4 (Lavandula x intermedia clone, AAT-4 alcohol acetyltransferase)
  • SEQ ID NO: 846803ACP Synechocystis Sp. PCC 6803 Acyl Carrier Protein
  • CIACP2 Cuphea lanceolata Acyl Carrier Protein
  • VpAATI Vasconcellea pubescens Alcohol acyltransferase 1
  • AtHPFT Arabidopsis thaliana Omega-hydroxypalmitate O-feruloyl transferase
  • PQFLAFASPNPSILLFDVCRF SEQ ID NO:107 CsVS ( Citrus sinensis Vinorine synthase-like)
  • LaAT1 Lavandula angustifolia Rosmarinic acid synthase (LaAT1)
  • SEQ ID NO:114 SsMMT ( Salvia splendens Malonyl-coenzyme:anthocyanin 5-0- glucoside-6'"-0-malonyltransferase)

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Abstract

The present invention relates to a method of producing derivatives of medium or short chain length alcohols. In particular, the present invention relates to a method of producing derivatives of 1- octanol, in particular octyl acetate and octyl glucoside using alcohol acetyltransferase and glucosyltransferases, for reducing toxicity of octanol, as well as microorganisms and expression vectors for use in said method. The present invention also relates to novel thioesterase enzymes for improved 1-octanol production as well as using sucrose synthase to improve UDP-glucose production for enhanced glucoside production.

Description

Bio-based Production of Toxic Chemicals
FIELD OF THE INVENTION
The present invention relates to a method of producing derivatives of medium or short chain length compounds, such as medium or short chain length alcohols. In particular, the present invention relates to a method of producing a derivative of 1-octanol, as well as microorganisms and expression vectors for use in said method. The present invention also relates to novel thioesterase enzymes. The thioesterase enzymes may be particularly useful in the method of the invention. Accordingly, the present invention also relates to the use of the thioesterase enzymes in the method of the invention.
The project leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 640720.
BACKGROUND TO THE INVENTION
Microbial biotechnology offers an attractive method for renewable production of chemicals that replace those currently sourced from fossil fuel feedstocks (1) (e.g. monomers for plastic or textile polymer synthesis) or nature (2, 3) (e.g. vanillin). A critical factor determining success with such a process is the compatibility between the engineered metabolism and its microbial host (4). If the target molecule, or its metabolic intermediates, are toxic to the host organism, the maximum potential for cost-effective production is likely not achieved. And, for lower value chemicals, economics really matter in the face of competition from fossil fuels (5). Nonetheless, when strategies to enhance product tolerance have been implemented, this has been found to improve productivity (6).
In nature, many organisms naturally synthesize very toxic molecules (7), yet, have survived throughout evolution, perhaps even prospered because of this. If we look closer, however, in many cases these chemicals accumulate as chemical derivatives, for example glucosides that are synthesized by plants (8, 9). These detoxification mechanisms are in some cases so effective, that chemicals (e.g. cyanogenic glucosides) which without derivatization would certainly kill the plant itself can accumulate up to 30% dry weight (10). Another example are esters synthesized by yeasts using native alcohol acyltransferases (AATs). It has been argued that also ester synthesis is a detoxification mechanism to convert more toxic metabolites into those that are less harmful (11).
SUMMARY OF THE INVENTION Accordingly, the present invention provides methods for the production of chemicals, as well as microorganisms for use in the methods.
Thus, the invention provides a method of producing a chemical comprising expressing an enzyme in a microorganism, wherein the microorganism produces a first chemical and the enzyme converts the first chemical into a second chemical. The conversion may be through the formation of a hydrophobic (e.g. esterification) or hydrophilic (e.g. glycosylation) derivative. The second chemical may then be removed from the microorganism cell, and optionally converted back into the first chemical or, alternatively, optionally converted into a third chemical. The removal may, for example, be through secretion from the cell, followed by harvesting of the secreted chemical, or by extraction of the second chemical from stores in the cell. The removal may involve the use of a solvent overlay, for example to capture volatile products.
The enzyme is artificially expressed in the cell, i.e. the enzyme would not be expressed at appropriate levels in the cell in the absence of steps taken in order to carry out the invention. In particularly preferred methods, expression of the enzyme in the cell will be due to the presence of a nucleic acid (for example heterologous nucleic acid) sequence encoding the enzyme, for example on a plasmid construct. The enzyme may be a glycosyltransferase or an alcohol acetyltransferase.
The first and second chemicals are organic compounds, and preferably of medium or short chain length (C4 to C10). The first chemical has a functional group that is modified by the enzyme, for example a hydroxyl group, OH-. The bioderivatization of the first chemical, i.e. the modification of the functional group by the enzyme, may reduce the toxicity of the first chemical, such that the second chemical is less toxic to the cell than the first chemical, and/or it may change the chemical properties of the first chemical, e.g. increase or decrease the water solubility such that the second chemical is more or less water soluble than the first chemical, and/or it may protect the molecule from further conversion, e.g. by oxidation of the functional group so that the second chemical is less reactive than the first chemical.
In one aspect, the present invention provides a method of producing a derivative of a medium or short chain length compound, comprising: providing a first enzyme to a microorganism that produces a medium or short chain length compound, wherein the first enzyme modifies the hydroxyl group of the medium or short chain length compound to form a derivative of the medium or short chain length compound; and harvesting the derivative of the medium or short chain length compound from the microorganism. In a suitable embodiment, the medium or short chain length compound is a medium or short chain length alcohol. Suitably the alcohol may comprise 4, 5, 6, 7, 8, 9, or 10 carbons.
In some embodiments, the short chain length alcohol is n-butanol. This embodiment may give rise to a particularly preferred aspect of the invention which provides a method of producing a derivative of n-butanol, comprising: providing a first enzyme to a microorganism that produces n-butanol, wherein the first enzyme modifies the hydroxyl group of the n-butanol to form a derivative of n-butanol; and harvesting the derivative of n-butanol from the microorganism.
In some embodiments the medium chain length alcohol is 1-octanol. This embodiment may give rise to a particularly preferred aspect of the invention which provides a method of producing a derivative of 1-octanol, comprising: providing a first enzyme to a microorganism that produces 1-octanol, wherein the first enzyme modifies the hydroxyl group of the 1-octanol to form a derivative of 1-octanol; and harvesting the derivative of 1-octanol from the microorganism.
In some embodiments the medium chain length alcohol is 1-decanol. This embodiment may give rise to a particularly preferred aspect of the invention which provides a method of producing a derivative of 1-decanol, comprising: providing a first enzyme to a microorganism that produces 1-decanol, wherein the first enzyme modifies the hydroxyl group of the 1-decanol to form a derivative of 1-decanol; and harvesting the derivative of 1-decanol from the microorganism.
In a suitable embodiment, the microorganism is a bacterium.
In a suitable embodiment, the bacterium may be a heterotrophic bacterium.
In a suitable embodiment, the bacterium may be selected from the group consisting of Escherichia, Halomonas and Cyanobacterium.
Suitably the Escherichia may be Escherichia coli (E. coli), for example B strain C43 (DE3) or K-12 strain BW25113.
In a suitable embodiment, the cyanobacterium may be Synechocystis sp (for example PCC 6803).
In a suitable embodiment, two or more first enzymes are provided.
In a suitable embodiment, the first enzyme may be heterologous or homologous. In a suitable embodiment, the first heterologous enzyme may be selected from the group consisting of an alcohol acetyltransferase and a glycosyltransferase.
In a suitable embodiment, the alcohol acetyltransferase may be selected from the group consisting of CAT (SEQ ID NO: 1), SAAT (SEQ ID NO: 2) and ATF1 (SEQ ID NO: 3), or a variant thereof.
In an alternative embodiment, the alcohol acetyltransferase may be selected from the group consisting of ATF1 (SEQ ID NO: 3), VAAT (SEQ ID NO:103) or UAAT4 (SEQ ID NO:82). Suitably in such embodiments, the alcohol may be n-butanol. In one such embodiment, the alcohol acetyltransferase is UAAT4 (SEQ ID NO:82).
In a suitable embodiment, the glycosyltransferase may be selected from the group consisting of AdGT4 (SEQ ID NO: 4), VvGT1 (SEQ ID NO: 5), MtGT1 (SEQ ID NO: 6), AtGT1 (SEQ ID NO: 7) and MtH2 (SEQ ID NO: 8), or a variant thereof.
In one embodiment, the glycosyltransferase is MtH2 (SEQ ID NO: 8), or a variant thereof.
In a suitable embodiment, the derivative of 1-octanol may be octyl acetate and/or octyl glucoside.
In a suitable embodiment, the derivative of n-butanol may be butyl acetate and/or butyl glucoside.
In a suitable embodiment, the derivative of 1-decanol may be decyl acetate and/or decyl glucoside.
In a suitable embodiment, the method may further comprise providing a second enzyme to the microorganism.
In a suitable embodiment the second enzyme may be heterologous.
In a suitable embodiment, the second heterologous enzyme may be selected from the group consisting of thioesterase, carboxylic acid reductase, and phosphopantetheinyl.
In a suitable embodiment, the thioesterase may be a C8-preferring thioesterase.
In a suitable embodiment, the C8-preferring thioesterase may be selected from the group consisting of CpFatBI (SEQ ID NO: 9), CaFatB3 (SEQ ID NO:10), Tes3 (SEQ ID NO: 11), or a variant thereof.
In a suitable embodiment, the variant may be CpFatB1-4 (SEQ ID NO: 12) or CaFatB3-5 (SEQ ID NO: 13), or a variant thereof.
In one embodiment, the thioesterase is CaFatB3-5 (SEQ ID NO: 13) or variant thereof. In an alternative embodiment, the thioesterase may be a C10-preferring thioesterase. In a suitable embodiment, the thioesterase enzyme comprises or consists of a truncated SEQ ID NO: 15 or SEQ ID NO: 16, or a variant thereof. Suitably, such a thioesterase may be a C8- preferring thioesterase or a C 10- preferring thioesterase.
In a suitable embodiment, the thioesterase enzyme comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO: 90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, and SEQ ID NO:102, or a variant thereof. In a suitable embodiment, the thioesterase enzyme comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22, and SEQ ID NO:23. Suitably, such a thioesterase may be a C8-preferring thioesterase or a C 10- preferring thioesterase.
Suitably a C8-preferring thioesterase is SEQ ID NO:23 or 25, or variants thereof.
Suitably a C10-preferring thioesterase is selected from SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22, SEQ ID NO:25 or a variant thereof.
In one embodiment, the microorganisms is a cyanobacterium and the thioesterase is a C10- preferring thioesterase. Suitably in such an embodiment, the C10-preferring thioesterase is selected from SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22 or a variant thereof.
In an alternative embodiment, the thioesterase may be a C4-preferring thioesterase.
In a suitable embodiment, the C4-preferring thioesterase may be CAH09236 from B.fragilis (SEQ ID NO: 102).
In a suitable embodiment, the second enzyme may be homologous.
In a suitable embodiment, the second homologous enzyme may be aldehyde reductase.
In a suitable embodiment, the method may further comprise providing a third enzyme to the microorganism.
In a suitable embodiment, the third enzyme may be a heterologous enzyme.
In a suitable embodiment, the third heterologous enzyme is sucrose synthase.
In a suitable embodiment, the sucrose synthase may be Arabidopsis thaliana sucrose synthase. In a suitable embodiment, the sucrose synthase may be Arabidopsis thaliana sucrose synthase is according to SEQ ID NO: 14, or a fragment or variant thereof.
In a suitable embodiment, the third enzyme may be homologous.
In a suitable embodiment, the first, second, and/or third enzyme may be provided to the microorganism directly or indirectly.
In a suitable embodiment, the first, second, and/or third enzyme may be provided to the microorganism indirectly by an expression vector comprising a nucleic acid encoding the enzyme.
In a further embodiment, the method may further comprise providing a fourth protein to the microorganism. In a suitable embodiment, the fourth protein is an acyl-carrier protein (ACP). Suitably the fourth protein may be homologous or heterologous, suitably it is heterologous. Suitably the acyl-carrier protein is selected from EcACP (SEQ ID NO:83) , 6803ACP (SEQ ID NO:84), and CIACP2 (SEQ ID NO:85). Suitably the acyl-carrier protein comprises or consists of a sequence selected from SEQ ID NO:83, SEQ ID NO:84, and SEQ ID NO:85. Suitably the acyl-carrier protein is overexpressed in the microorganism. Advantageously, the provision of overexpressed ACP increases metabolic flux through the enzymatic pathway.
In a suitable embodiment, the fourth protein may be provided to the microorganism indirectly by an expression vector comprising a nucleic acid encoding the protein.
In a suitable embodiment, the method may further comprise supplying the microorganism with a precursor of the medium or short chain length alcohol.
In a suitable embodiment, the method may further comprise supplying the microorganism with a precursor of 1-octanol, n-butanol or 1-decanol. Suitably, the precursor may be an internal or external substrate.
In a suitable embodiment, the precursor may be selected from the group consisting of octanoic acid, butanoic acid, decanoic acid, glucose, octanoyl-ACP, butanoyl-ACP, decanoyl-ACP, octanal, butanal and decanal.
In one aspect, the invention provides a vector for use in a method of the invention.
In a suitable embodiment, the expression vector may comprise a nucleic acid encoding a first enzyme, and optionally a nucleic acid encoding a second and/or third enzyme, and optionally a fourth protein, wherein the first enzyme modifies the hydroxyl group of a medium or short chain length compound to form a derivative of the medium or short chain length compound.
In a suitable embodiment, the expression vector may comprise a nucleic acid encoding a first enzyme, and optionally a nucleic acid encoding a second and/or third enzyme, and optionally a fourth protein, wherein the first enzyme modifies the hydroxyl group of the 1-octanol to form a derivative of 1-octanol.
In one aspect, the invention provides a microorganism for use in a method of the invention.
In a suitable embodiment the microorganism may comprise:
(i) a first enzyme, and optionally a second and/or third enzyme, and optionally a fourth protein, wherein the first enzyme modifies the hydroxyl group of a medium or short chain length compound to form a derivative of the medium or short chain length compound; or
(ii) an expression vector comprising a nucleic acid encoding a first enzyme, and optionally a nucleic acid encoding a second and/or third enzyme, and optionally a fourth protein, wherein the first enzyme modifies the hydroxyl group of the medium or short chain length compound to form a derivative of the medium or short chain length compound; or
(iii) a combination of (i) and (ii).
In a suitable embodiment the microorganism may comprise:
(i) a first enzyme, and optionally a second and/or third enzyme, and optionally a fourth protein, wherein the first enzyme modifies the hydroxyl group of the 1-octanol to form a derivative of 1-octanol; or
(ii) an expression vector comprising a nucleic acid encoding a first enzyme, and optionally a nucleic acid encoding a second and/or third enzyme, and optionally a fourth protein, wherein the first enzyme modifies the hydroxyl group of the 1-octanol to form a derivative of 1-octanol; or
(iii) a combination of (i) and (ii).
In one aspect, the invention provides a thioesterase enzyme, wherein the thioesterase comprises or consists of a truncated SEQ ID NO: 15 or SEQ ID NO: 16, or a variant thereof.
In a suitable embodiment, the thioesterase enzyme may comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21 , SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO: 90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, or a variant thereof. In one aspect, the present invention provides use of the thioesterase enzyme of the invention in the method of the invention. Except for where the context requires otherwise, the considerations set out in this disclosure should be considered to be applicable to methods, vectors and microorganisms of the invention.
DETAILED DESCRIPTION
The present invention is based upon the purposeful in vivo transformation of chemicals into chemical derivatives by modification of functional groups, defined as bioderivatization. Often, these functional groups (for example hydroxyl groups, OH-) are central to rendering a chemical toxic towards cells, including cells that produce the chemical.
At the same time, bioderivatization may also change the chemical properties (e.g. water solubility) of the target chemical, and/or protect the molecule from further conversion (e.g. oxidation). Bioderivatization could also open new opportunities for strategic product: process separation that is more cost-efficient. Once the derivative has been isolated outside of the biological host, it could then be converted back to its original form, unless the particular derivative in question is also an attractive product. Thus, the method of the invention may comprise the step of converting a harvested derivative of a medium or short chain length compound (such as a derivative of 1-octanol) to the corresponding medium or short chain length compound (for example 1-octanol). Suitably, this step may be performed ex vivo.
A general bioderivatization concept according to the invention is illustrated in Figure 1.
The present invention is based on the inventors’ development of an in vivo method for the transformation of chemicals into chemical derivatives that have more desirable properties, for example that are less toxic or more soluble. Specifically, the methods of the invention allow the transformation of 1-octanol into less toxic derivatives of 1-octanol, such as octyl acetate. The inventors have surprisingly found that whilst E. coli cells are unable to grow when the concentration of 1-octanol is above 0.75mM, the cells are able to grow in the presence of 1- octanol derivatives even when those are present at a much higher concentration which speaks to the reduced toxicity of the derivatives. For example, the cells are able to grow in the presence of octyl acetate at a concentration as high as 50mM, or the in the presence of octyl glucoside at a concentration as high as 2.5mM. It will be appreciated that as a result of the cells’ ability to grow under these increased concentrations of derivatives, the cells are able to produce more of the derivatives. The derivatives may be then converted ex vivo back into 1- octanol, allowing for increased amounts of 1-octanol to be ultimately produced.
Accordingly, in one aspect, the present invention provides a method of producing a derivative of a medium or short chain length compound, comprising: providing a first enzyme to a microorganism that produces a medium or short chain length compound, wherein the first enzyme modifies the hydroxyl group of the medium or short chain length compound to form a derivative of the medium or short chain length compound; and harvesting the derivative of the medium or short chain length compound from the microorganism.
The term “medium chain length compound” as used herein refers to a compound having a medium length carbon chain. A medium length carbon chain may have 6, 7, 8, 9, 10, 11, or 12 carbons. The term “short chain length compound” as used herein refers to a compound having a short length carbon chain. A short length carbon chain may have 1, 2, 3, 4 or 5 carbons. Suitably, the compound may be an organic compound. Suitably, the compound may be an alcohol. Suitably, the alcohol may be butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol or dodecanol. Suitably, the alcohol may be octanol. Suitably, the alcohol may be butanol. Suitably, the alcohol may be decanol.
Suitably the alcohol may be a terpene alcohol, for example linalool, menthol, eugenol or cinnamoyl alcohol.
In one aspect, the present invention provides a method of producing a derivative of 1-octanol, n-butanol or 1-decanol. Suitably the method comprises providing a first enzyme to a microorganism that produces 1-octanol, n-butanol or 1-decanol, wherein the first enzyme modifies the hydroxyl group of the 1-octanol, n-butanol or 1-decanol to form a derivative of 1- octanol, n-butanol or 1-decanol. Suitably, the method may further comprise the step of harvesting the derivative of 1-octanol from the microorganism.
In the context of the present invention the term “derivative of the medium or short chain length compound” or “derivative”, refers to a compound obtained from the medium or short chain length compound, wherein the compound has a modified hydroxyl group as compared to the corresponding compound that it is derived from.
By the same token, in the context of the present invention the term “derivative of 1-octanol” refers to a compound obtained from 1-octanol, wherein the compound has a modified hydroxyl group as compared to 1-octanol. In the present application, the derivative of the medium or short chain length compound, such as for example the derivative of 1-octanol, n-butanol or 1- decanol, may be referred to as “a target molecule”.
Suitably, the derivative may be hydrophobic or hydrophilic. A hydrophobic derivative may be obtained, for example, by esterification of the hydroxyl group. A hydrophobic derivative may be obtained, for example, by esterification of the hydroxyl group of 1-octanol, resulting in the production of octyl acetate.
A hydrophilic derivative may be obtained, for example, by glycosylation of the hydroxyl group. A hydrophilic derivative may be obtained, for example, by glycosylation of the hydroxyl group of 1-octanol, resulting in the production of octyl glucoside.
Thus, in a suitable embodiment, the derivative of 1-octanol may be selected from the group consisting of octyl acetate, octyl glucoside, glycosides with other glycosyl groups and esters with other acyl groups.
Thus, in a suitable embodiment, the derivative of n-butanol may be selected from the group consisting of butyl acetate, butyl glucoside, glycosides with other glycosyl groups and esters with other acyl groups.
Thus, in a suitable embodiment, the derivative of 1-decanol may be selected from the group consisting of decyl acetate, decyl glucoside, glycosides with other glycosyl groups and esters with other acyl groups.
Thus, in a suitable embodiment, the derivative of the terpene alcohols may be selected from the group consisting of menthyl acetate, linalyl acetate, linalyl butyrate, eugenyl acetate, and cinnamyl acetate.
As mentioned, the method of the invention may be particularly useful in the context of producing compounds that are toxic to the microorganisms which produce said compounds. It will be appreciated that in the context of the present invention, the derivative (such as derivative of 1-octanol) may be less toxic than the compound from which the derivative is produced from (for example 1-octanol). Toxicity may be determined, for example, by determining the compounds effect on cell growth, proliferation and/or genetic stability. Method for determining a compounds effect on cell growth, proliferation and/or genetic stability will be known to those skilled in the art.
The method of the invention may involve the use or production of unstable compounds. Unstable compounds may be reactive in the environment and/or during normal use. Compounds may be unstable as a result of being reactive.
In embodiments where unstable, toxic or poorly soluble compounds are used or produced, the method may comprise the use of a solvent overlay. The solvent overlay may be provided by adding the solvent to the culture medium for culturing the microorganism. The use of a solvent overlay may increase the amount of derivative produced and/or the amount of derivative harvested. Suitably, the solvent may be hexadecane and/or pentadecane, but other suitable solvents will be known to those skilled in the art. It will also be appreciated by those skilled in the art that the choice of a solvent for use in a solvent overlay may depend upon the type of derivative produced.
The term "first enzyme” as used herein refers to an enzyme which converts a medium or short chain length compound to a derivative of the medium or short chain length compound by modifying the hydroxyl group of the medium or short chain length compound. Suitably, the first enzyme may convert 1-octanol, n-butanol or 1-decanol respectively to a derivative of 1- octanol, n-butanol or 1-decanol respectively by modifying the hydroxyl group of 1-octanol, n- butanol or 1-decanol respectively.
Suitably, the first enzyme may be heterologous. Herein, such an enzyme may be referred to as “the first heterologous enzyme”. In the context of the present specification, in particular in the context of the first, second and third enzymes described herein, “a heterologous enzyme” is one which does not naturally exist in the microorganism to which the enzyme is provided.
Alternatively, the first enzyme may be homologous. Herein, such an enzyme may be referred to as “the first homologous enzyme”. In the context of the present specification, in particular in the context of the first, second and third enzymes described herein, “a homologous enzyme” is one which naturally exists in the microorganism to which the enzyme is provided. A heterologous enzyme may be provided to produce a derivative of 1-octanol that might naturally be not produced by the microorganism.
A homologous enzyme may be provided to increase the amount of an enzyme that naturally exists in the microorganism. Increasing the amount of an enzyme may be useful to increase the amount of the derivative (for example derivative of 1-octanol) being produced, or when the amount of the naturally existing enzyme is insufficient to produce the derivative (for example derivative of 1-octanol).
In a suitable embodiment, the microorganism may be provided with one or more first enzyme. Suitably, the microorganism may be provided with one, two, three, four, five, six, or more first enzymes. Suitably, some or all of the first enzymes may be heterologous or homologous.
In an embodiment where the microorganism is provided with two or more first enzymes (for example two or more heterologous enzymes), the enzymes may produce different derivatives, for example derivatives of 1-octanol, such as acetyltransferase and glycosyltransferase. Alternatively, they may produce the same derivative, such as acetyltransferase or glycosyltransferase. Suitably, the first heterologous enzyme may be an alcohol acetyltransferase or glycosyltransferase. In an embodiment, where the microorganism is provided with more than one first heterologous enzyme it may be provided, for example, with one or more alcohol acetyltransferase and one or more glycosyltransferase, or two or more different alcohol acetyltransferase, or two or more different glycosyltransferases.
By way of example, a suitable alcohol acetyltransferase may be selected from the group consisting of CAT (SEQ ID NO: 1), SAAT (SEQ ID NO: 2) and ATF1 (SEQ ID NO: 3).
By way of example, a suitable glycosyltransferase may be selected from the group consisting of AdGT4 (SEQ ID NO: 4), VvGT1 (SEQ ID NO: 5), MtGT1 (SEQ ID NO: 6), AtGT1 (SEQ ID NO: 7) and MtH2 (SEQ ID NO: 8).
In an alternative embodiment, the alcohol acetyltransferase may be selected from the group consisting of ATF1 (SEQ ID NO: 3), VAAT (SEQ ID NO:103) or UAAT4 (SEQ ID NO:82).
It will be appreciated that the first enzyme provided to the microorganism may dictate what derivative, for example what derivative of 1-octanol, is produced by the microorganism. Thus, by way of example, in an embodiment where the first enzyme is an alcohol acetyltransferase, the derivative produced may be octyl acetate, whereas when the first enzyme is a glycosyltransferase the derivative produced may be octyl glucoside. This is the same for any other alcohol described herein.
The term “providing” or “provided” as used herein refers to any techniques by which the microorganism will receive a first, second and/or third enzyme. The enzyme may be provided to the microorganism either indirectly or directly.
In an embodiment where the enzyme is provided to the microorganism indirectly, it may be provided in the form of a nucleic acid encoding such an enzyme. A nucleic acid encoding the enzyme may be provided to the microorganism, for example, through the use of an expression vector comprising a nucleic acid sequence encoding such an enzyme.
In an embodiment where the enzyme is provided to the microorganism directly, it may be provided in the form of the protein itself. In such an embodiment, the microorganism may be cultured in the presence of the enzyme. In addition, the microorganism may be cultured in the presence of an agent which may facilitate the transport of the enzyme into the microorganism.
The term “expression vector” as used herein refers to an isolated DNA molecule which upon transfection into the microorganism provides for expression of the enzyme within the microorganism. The enzyme may be heterologous or homologous. In addition to the DNA sequence coding for the enzyme the expression vector may comprise regulatory DNA sequences that are required for an efficient transcription of the DNA coding sequence into mRNA and for an efficient translation of the mRNAs into enzymes in the microorganism. The expression vector may comprise a DNA sequence that encodes the first, second and/or third enzyme.
The expression vector may be viral or non-viral. More suitably, the expression vector may be viral. By way of example, a suitable viral expression vector may be derived from a virus selected from the group consisting of paramyxovirus, retrovirus, adenovirus, lentivirus, pox virus, alphavirus, and herpes virus. Methods of delivering expression vectors to a cell are well known in the art. Merely by way of example, such methods include viral transfection, electroporation and sonoporation.
Suitable non-viral expression vectors may be selected from the group consisting of inorganic particle expression vectors (such as calcium phosphate, silica, and gold), lipid based particle expression vectors (for example cationic lipids, lipid nano emulsions, and solid lipid nanoparticles) and polymer based particle expression vectors (for example peptides, polyethylenimine, chitosan, and dendimers). Other suitable non-viral expression vectors will be known to those skilled in the art.
Exemplary suitable expression vectors are provided in the Examples section of the present specification.
The term "microorganism" as used herein refers to any prokaryotic or eukaryotic organism, such as a bacterium, protozoa, a virus or any kind of higher organism, such as a fungus (for example yeast), a plant, or an animal, which can be maintained in the form of a cell suspension or cell culture, and which produces a medium or short chain length compound, for example 1- octanol, n-butanol or 1-decanol.
Suitably, the microorganism is a bacterium. Suitably the bacterium may be heterotrophic. Suitably, the bacterium may be selected from the group consisting of Escherichia, Halomonas, Cyanobacterium. Suitably the Escherichia may be Escherichia coli (E. coli).
Suitably, the E. coli may be B strain C43 (DE3) or K-12 strain BW25113. Suitably the cyanobacterium may be Synechocystis sp (for example PCC 6803). It will be appreciated that these are merely exemplary microorganisms, and other suitable microorganisms, such as other suitable bacteria will be known to those skilled in the art.
The microorganism may be capable of producing the medium or short chain length compound, such as 1-octanol, n-butanol or 1-decanol on its own (i.e. without the need of being supplied with a precursor of 1-octanol, n-butanol or 1-decanol), or alternatively or additionally, the microorganism may be capable of producing the medium or short chain length compound (such as 1-octanol, n-butanol or 1-decanol) from a precursor of the medium or short chain length compound (such as a precursor of 1-octanol n-butanol or 1-decanol) that has been supplied to the microorganism.
Suitably, the microorganism may comprise a first enzyme, and optionally a second and/or third enzyme, or an expression vector comprising a nucleic acid encoding a first enzyme, and optionally a second and/or third enzyme. Suitably, the first, second and/or third enzyme is heterologous.
The term “precursor of medium or short chain length compound” as used herein refers to a compound that can be converted into a medium or short chain length compound. The precursor may be converted into a medium or short chain compound via a single step reaction or via a multistep reaction. Suitably the precursor may be selected from the group consisting of a lignocellulosic substrate, sucrose, starch, and amylose.
By the same token, the term “precursor of 1-octanol” as used herein refers to a compound that can be converted into 1-octanol. The precursor may be converted into 1-octanol via a single step reaction or via a multistep reaction. Suitably the precursor may be selected from the group consisting of a lignocellulosic substrate, sucrose, starch, and amylose. Suitably, the precursor may be an internal or external substrate. Suitably the precursor may be selected from the group consisting of octanoic acid, CO2, formate, glucose, octanoyl-ACP, octanoyl- CoA, octanal, and hexose.
It will be appreciated that the precursor may be one that naturally exists in the microorganism or one that doesn’t naturally exist in the microorganism. Providing the microorganism with the precursor may allow the production of medium or short chain length compound, for example 1-octanol, n-butanol or 1-decanol, (if the precursor does not naturally exist in the microorganism) or may increase the production of medium or short chain length compound, for example 1-octanol, n-butanol or 1-decanol (if the precursor naturally exists in the microorganism).
Accordingly, a method of the invention may comprise the step of supplying the microorganism with a precursor medium or short chain length compound. The precursor may be supplied to the microorganism by adding the precursor to the medium in which the microorganism is cultured.
Accordingly, a method of the invention may comprise the step of supplying the microorganism with a precursor of 1-octanol, n-butanol or 1-decanol (for example octanoic acid). The precursor may be supplied to the microorganism by adding the precursor to the medium in which the microorganism is cultured. In some embodiments, the method of the invention may further comprise providing the microorganism with a second enzyme, wherein the enzyme will convert the precursor into medium or short chain length compound or into a different precursor of medium or short chain length compound and/or the medium chain length compound.
Suitably, the precursor might not comprise a hydroxyl group and/or the precursor may for example comprise a carboxylic acid group. In some embodiments, the method of the invention may further comprise providing the microorganism with a second enzyme.
Suitably, the second enzyme may convert the precursor into the medium or short chain length compound. For example, the enzyme may convert the precursor into 1-octanol, n-butanol or 1-decanol, or into a different precursor of 1-octanol, n-butanol or 1-decanol.
Suitably, the second enzyme maybe heterologous or homologous. Providing the second enzyme may be particularly desirable when the method of the invention comprises the step of supplying the microorganism with a precursor of 1-octanol, n-butanol or 1-decanol (for example octanoic acid, butanoic acid, or decanoic acid respectively). Suitably, the second enzyme may convert the precursor into 1-octanol, n-butanol or 1-decanol, or into a different precursor of 1-octanol, n-butanol or 1-decanol. Suitably, the method of the invention may comprise providing the microorganism with one or more second enzyme. For example, the method may comprise providing the microorganism with one, two, three, or more second enzymes. Suitably, some or all of the second enzymes may be heterologous or homologous.
By way of example the second homologous enzyme may be aldehyde reductase.
By way of example, the second heterologous enzyme may be selected from the group consisting of thioesterase, carboxylic acid reductase, and phosphopantetheinyl. Suitably, the thioesterase may be a C8-preferring thioesterase. Suitably, the thioesterase may be a C4- preferring thioesterase. Suitably, the thioesterase may be a C 10- preferring thioesterase.
Suitably, the C8-preferring thioesterase may be selected from the group consisting of ‘CpFatBI (SEQ ID NO: 9), ‘CaFatB3 (SEQ ID NO: 10), Tes3 (SEQ ID NO: 11), or variants thereof. Merely by way of example, a suitable variant of ‘CpFatBI is ‘CpFatB1-4 (SEQ ID NO:12). Merely by way of example, a suitable variant of ‘CaFatB3 is ‘CaFatB3-5 (SEQ ID NO: 13). Other suitable exemplary variants are disclosed in US2019284588, which is herein incorporated by reference.
In a suitable embodiment, the thioesterase enzyme comprises or consists of a truncated SEQ ID NO: 15 or SEQ ID NO: 16, or a variant or derivative thereof. Suitably, such a thioesterase may be a C8-preferring thioesterase. In the context of present disclosure, when the thioesterase enzyme comprises a truncated SEQ ID NO: 15 or SEQ ID NO: 16, it will be appreciated that the enzyme does not comprise or consist of the full-length sequence according to SEQ ID NO: 15 or SEQ ID NO: 16.
Merely by way of example, the thioesterase enzyme that comprises or consists of a truncated SEQ ID NO: 15 or SEQ ID NO: 16, may comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21 , SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO: 90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, or a variant or derivative thereof. Suitably, the thioesterase enzyme that comprises or consists of a truncated SEQ ID NO: 15 or SEQ ID NO: 16, may comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21 , SEQ ID NO: 22, and SEQ ID NO:23. Such thioesterases may be C8-preferring thioesterases, or C 10- preferring thioesterases.
The present inventors have found that thioesterase enzymes that comprises or consists of a truncated SEQ ID NO: 15 or SEQ ID NO: 16, such as thioesterase enzymes that comprise or consist of SEQ ID NO: 17, SEQ ID: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21 , SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO: 90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 or a variant or derivative thereof may increase the production of fatty acids in the microorganisms, and/or fatty acid derivatives as compared to the wild-type enzymes according to SEQ ID NO: 15 or 16. Suitably, the fatty acid may be a C6, C8, C10, C12, C14, C16 or C18 fatty acid. More suitably a C8 or C10 fatty acid. Suitably, the fatty acid derivative may be an alcohol. Suitably, the alcohol has a 6, 8, 10, 12, 14, 16 or 18 carbon backbone. More suitably the alcohol is an alcohol that has an 8 or 10 carbon backbone.
In one embodiment, the microorganisms is a cyanobacterium and the thioesterase is a C10- preferring thioesterase. Suitably in such an embodiment, the C10-preferring thioesterase is selected from SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22 or a variant thereof.
In an alternative embodiment, the thioesterase may be a C4-preferring thioesterase.
In a suitable embodiment, the C4-preferring thioesterase may be CAH09236 from B.fragilis (SEQ ID NO: 102).
Thioesterase enzymes described herein that comprise or consist of a truncated SEQ ID NO: 15 or SEQ ID NO: 16 have been developed by the present inventors, and surprisingly found to increase the production of fatty acids or fatty acid derivatives, such as alcohols. This finding gives rises to a further aspect of the invention, directed to the novel thioesterase enzymes themselves.
Accordingly, in one aspect, the invention provides a thioesterase enzyme, wherein the thioesterase comprises or consists of a truncated SEQ ID NO: 15 or SEQ ID NO: 16, or a variant thereof. In a suitable embodiment, the thioesterase enzyme comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO: 90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, or a variant or derivative thereof. In a suitable embodiment, the thioesterase enzyme comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22, and SEQ ID NO:23.
A thioesterase enzyme that is particularly useful in the context of increased fatty acid production or fatty acid derivative production may comprise or consist of SEQ ID NO: 23 or SEQ ID NO: 25.
The term “variant” as used in the present description refers to an amino acid sequence in which one or more amino acids have been replaced by different amino acids as compared to the corresponding amino acid sequence. Suitably, the variant may be at least 70%, at least 75%, at least 80%, be at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to the corresponding sequence.
It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the peptide (conservative substitutions). Generally, the substitutions which are likely to produce the greatest changes in a peptide's properties are those in which (a) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g. Leu, lie, Phe or Val); (b) a cysteine or proline is substituted for, or by, any other residue; (c) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp) or (d) a residue having a bulky side chain (e.g., Phe or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala, Ser) or no side chain (e.g., Gly).
In a further aspect, the present invention provides use of the thioesterase enzyme of the invention in the method of the invention.
The method of the invention may further comprise the step of providing a third enzyme to the microorganism. Suitably, the third enzyme may generate a substrate involved in producing a derivative of the medium or short chain length compound, such as a derivative of 1-octanol, n-butanol or 1-decanol. Suitably the third enzyme may be heterologous or homologous (herein also referred to as the third heterologous enzyme or third homologous enzyme, respectively). Suitably, the method of the invention may comprise providing the microorganism with one or more third enzyme. For example, the method may comprise providing the microorganism with one, two, three, or more second enzymes. Suitably, some or all of the third enzymes may be heterologous or homologous.
Suitably, the third heterologous enzyme may be sucrose synthase, for example Arabidopsis thaliana sucrose synthase (AtSUSI). Suitably the Arabidopsis thaliana sucrose synthase may be according to SEQ ID NO: 14 or a fragment or variant thereof.
The term “fragment” as used herein refers to an amino acid that consists of a truncation in the corresponding amino acid sequence (for example SEQ ID NO: 14).
Providing a third enzyme, such as sucrose synthase (for example AtSUSI), may be particularly useful in an embodiment of the method of the invention in which octyl glucoside is the produced derivative of 1-octanol. Those skilled in the art will appreciate that insufficient glycosylation may be due to a limitation in the supply of a substrate required for the production of 1-octanol. The substrate may be UDP-glucose. As shown in Figure 3, UDP-glucose in produced by sucrose synthase from UDP and sucrose, rendering sucrose synthase particularly useful in the context of producing octyl glucoside.
The term “harvesting” as used herein refers to removing and/or isolating the derivative of medium or short chain length alcohol, for example derivative of 1-octanol, from the microorganism or culture medium in which the microorganism is grown. Methods of harvesting the derivative, such as derivative of 1-octanol will be known to those skilled in the art. Merely by way of example harvesting may be by distillation. The harvesting may involve the use of a solvent overlay, for example to capture volatile products.
In a further embodiment, the method may further comprise providing a fourth protein to the microorganism. In a suitable embodiment, the fourth protein is an acyl-carrier protein (ACP). Suitably the fourth protein may be homologous or heterologous, suitably it is heterologous. Suitably the acyl-carrier protein is selected from EcACP (SEQ ID NO:83), 6803ACP (SEQ ID NO:84) , and CIACP2 (SEQ ID NO:85) . Suitably the acyl-carrier protein is overexpressed in the microorganism. Advantageously, the provision of overexpressed ACP increases metabolic flux through the enzymatic pathway.
In a suitable embodiment, the fourth protein may be provided to the microorganism indirectly by an expression vector comprising a nucleic acid encoding the protein. In a suitable embodiment, the method may further comprise supplying the microorganism with a precursor of the medium or short chain length alcohol.
Suitably, once harvested, the derivative, for example derivative of 1-octanol may be reconverted to 1-octanol. Suitably by hydrolysis. A derivative of 1-octanol may be reconverted into 1-octanol by hydrolysis. Alternatively, the derivative may be converted into a different compound, for example by oxidation. Alternatively, the derivative of 1-octanol may be converted to a different compound, such as its corresponding aldehyde, octanal. 1-octanol may be converted to octanol by oxidation.
In one embodiment, the method of the invention is carried out in a cyanobacteria, and the cyanobacteria comprises a thioesterase and a glycosyltransferase, wherein the thioesterase is CaFatB3-5, and the glycosyltransferase is MtH2. Suitably the cyanobacteria is Synechocystis sp (for example PCC 6803). Optionally, the cyanobacteria may further comprise an acyl-carrier protein, suitably an overexpressed acyl-carrier protein.
In one embodiment, the method of the invention is carried out in E.coli, and the E.coli comprises a thioesterase and an alcohol acetyltransferase, wherein the thioesterase is CaFatB3-5, and the alcohol acetyltransferase is ATF1.
In one embodiment, the alcohol is n-butanol and the microorganism comprises a thioesterase and an alcohol acetyltransferase, wherein the thioesterase is a C4-preferring thioesterase and the alcohol acetyltransferase is selected from the group consisting of ATF1 (SEQ ID NO: 3), VAAT (SEQ ID NO: 103) or UAAT4 (SEQ ID NO:82). Suitably the C4-preferring thioesterase is CAH09236 from B.fragilis (SEQ ID NO: 102).
In one embodiment, the method of the invention is carried out in E.coli, and the E.coli comprises a thioesterase, wherein the thioesterase is a C8-preferring thioesterase selected from SEQ ID NO:23 or 25, or variants thereof.
In one embodiment, the method of the invention is carried out in a cyanobacteria, and the cyanobacteria comprises a thioesterase, wherein the thioesterase is a C 10- preferring thioesterase selected from SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22 or a variant thereof. Suitably the cyanobacteria is Synechocystis sp (for example PCC 6803).
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.
All documents mentioned in this specification are incorporated herein by reference in their entirety.
BRIEF DESCRIPTION OF THE FIGURES
The invention will now be described in detail with reference to a specific embodiment and with reference to the accompanying drawings, in which:
Figure 1 shows an overview of the bioderivatization process. (A) Concept scheme. Inside the cell, the target molecule is enzymatically converted into a derivative (e.g. ester) (1), with potential consequences for both toxicity and purification. After the product has been excreted from the cell (2), the original target molecule can be recovered by enzymatic or chemical processes that remove the conjugate group (3). The yellow box illustrates a cell or a production system. The filled circle represents the conjugate, e.g. acid. 'Met' represents the target chemical in question. (B) Non-stoichiometric overview of the two derivatization processes considered in this study, (1) esterification and (2) O-glucosylation. Abbreviations: FAS = fatty acid synthesis, GT = glycosyltransferase, AAT = alcohol acetyltransferase. (C) The varying effect of bioderivatization on the water solubility of the target molecule.
Figure 2 shows toxic effect comparison of 1-octanol, octyl acetate and octyl glucoside based on specific growth rates. E. coli C43 (DE3) was cultivated in M9 minimal media and different concentrations of compounds of interest (0-50 mM) were added at the beginning of the cultivation. The specific growth rate was determined by calculating the slopes of three biological replicates from average growth curves and only considered data in the range of 1- 4 hours. The empty symbols represent the mean average from 3 biological replicates and each filled circle represents data from independent biological replicates.
Figure 3 shows comparison of toxicity of externally added octyl acetate or 1-octanol to Synechocystis sp. PCC 6803. Twenty-five millilitre of Synechocystis sp. PCC 6803 lacking acyl-ACP synthetase was cultivated in a 100-ml Erlenmeyer flask with a starting OD730 0.2 at 30°C, 180 rpm (60 mhioI photons/m2. s, 1% CO2) in AlgaeTron AG 230 (Photon Systems
Instruments). On day 2 onwards, 25 mg/L 1-octanol or octyl acetate was added exogenously every 24 h to the liquid culture in the absence of 10% (v/v) hexadecane solvent overlay (A) and in the presence of 10% (v/v) hexadecane solvent overlay (B). OD730 was monitored every 24 h.
Figure 4 shows pathway engineering for the synthesis of octyl glucoside and octyl acetate. (A) Metabolic scheme depicting a novel synthetic pathway for in vivo production of octyl glucoside and octyl acetate extended from ACP-dependent 1-octanol pathway (TPC3) (17). FAS, Fatty Acid Synthesis; Tes3, ‘CpFatBI , ‘CaFatB3, ‘CpFatB1-4, ‘CaFatB3-5, acyl-ACP thioesterase; CAR, carboxylic acid reductase; Sfp, phosphopantetheinyl transferase; AHR, aldehyde reductase; CAT, SAAT, ATF1 , alcohol acyltransferase; AdGT4, AtGT1, MtG1, MtH2, VvGT1 , glycosyltranferase; AtSUSI , sucrose synthase. (B) Schematic diagram of plasmids used to generate Strain No. 1 to 5 as shown in Table 1. (C) Production of octyl actate by introducing various AAT enzymes in E. coli C43 (DE3). (D) Chromatogram of overlay obtained from Strain No. 2 (Tes3-Sfp-CAR) and Strain No. 5 (Tes3-Sfp-CAR + ATF1) at 48 h. Peak identification: (1) 1-octanol and (2) octyl acetate. (E) Growth curves of the strains in Figure 3C during 48 h of cultivation. E. coli C43 (DE3) Strains No. 1 to 5 (Table 1) were cultivated in M9 media with 2% (w/v) glucose, 0.5 mM IPTG induction and 10% (v/v) hexadecane overlay for 48 h. The asterisk indicates significance by Student’s t-test (*, P£0.05; ***, P£0.005). All data are shown as the average from 3 biological replicates and circles represent data from independent biological replicates.
Figure 5 shows identification of limiting substrates in 1-octanol and octyl acetate production by substrate feeding cultivation and effect of bioderivatization on E. coli growth and glucose consumption without overlay use. E. coli C43 (DE3) (A) Strain No. 2 (Tes3-Sfp-CAR) (Table 1) (B) Strain No. 4 (Tes3-Sfp-CAR + SAAT) (Table 1) were cultivated in M9 media with 2% (w/v) glucose, 0.5 mM IPTG induction, 10% (v/v) hexadecane overlay and different concentrations of octanoic acid were added to the cultures. Growth and metabolism of E. coli C43 (DE3) when fed with (C,E) 0 mM octanoic acid and (D,F) 1.5 mM octanoic acid in the absence of solvent overlay. Three strains were tested: Strain No. 1 (negative control; indicated as open grey triangles), Strain No. 2 (Tes3-Sfp-CAR; indicated as open black circles), and Strain No. 4 (Tes3-Sfp-CAR + SAAT; indicated as open red squares) (Table 1). All the strains were cultivated in M9 minimal media and octanoic acid was spiked to the liquid media in the absence of solvent overlay. The empty symbols represent the average from 3 biological replicates and each filled circle represents data from independent biological replicates in panels C to F. The photographs of liquid cultures taken at 48 h when supplemented with (G) 0 mM octanoic acid and (H) 1.5 mM octanoic acid in the absence of solvent overlay.
Figure 6 shows average growth curves of E. coli C43 (DE3) cultivated in M9 media with varying concentrations of (A) 1-octanol (0 - 50 mM), (B) octyl acetate (0 - 50 mM), and (C) octyl glucoside (0 - 100 mM). All cells were cultivated in 96-well microtiter plates at 37°C, and 432 rpm. Data are the average ± standard deviation from 3 replicates.
Figure 7 shows amino acid sequence alignments of CpFatB1_wild type (SEQ ID NO: 100), ‘CpFatBI (SEQ ID NO: 9), OpFatB1-4 (SEQ ID NO: 12), CaFatB3_ wild type (SEQ ID NO: 101), ‘CaFatB3 (SEQ ID NO: 10) and QaFatB3-5 (SEQ ID NO: 13).
Figure 8 shows selection of thioesterases and IPTG optimization for 1-octanol production and effect of bioderivatization on E. coli growth and metabolism in the absence of solvent overlay. Yield (mmol/mol glucose) from in vivo production of 1-octanol from E. coli BW25113 Strain No. 7 to 11 (Table 1) at 24 h and 48 h when cultivated in M9 media overlaid with 10% (v/v) hexadecane overlay with different IPTG used to induce at (A) 0.02 mM IPTG, (B) 0.05 mM IPTG, (C) 0.2 mM IPTG, and (D) 0.5 mM IPTG. Data are the average from 3 biological replicates and circles represent data from independent biological replicates. (E) Average growth curves of E. coli BW25113 Strain No. 12 (‘CpFatB1-4-Sfp-CAR; indicated as open black circles) and 14 (‘CpFatBI -4-Sfp-CAR + ATF1; indicated as open red squares) (Table 1) and (F) glucose consumptions when cultivated in M9 minimal media and 0.05 mM IPTG in the absence of solvent overlay. The empty symbols represent the mean average from 3 biological replicates and each filled circle represents data from independent biological replicates.
Figure 9 shows toxic effect of octyl acetate on growth. Specific growth rates when E. coli C43 (DE3) was cultivated in M9 minimal media with 2% (w/v) glucose and different concentrations (50-200 mM) of octyl acetate were added at the beginning of the cultivation. The specific growth rate was calculated using slopes from average growth curves and only considered data in the range of 1-4 h. Data are the average ± standard deviation from 3 replicates.
Figure 10 shows growth from octyl acetate producing strains and 1-octanol producing strains. E. coli Strain No. 12 (‘CpFatBI -4-Sfp-CAR), 14 (‘CpFatB1-4-Sfp-CAR + ATF1), 13 (‘CaFatB3- 5-Sfp-CAR) and, 15 (‘CaFatB3-5-Sfp-CAR + ATF1) (Table 1) were cultivated in M9 minimal media with 2% (w/v) glucose and 10% (v/v) hexadecane overlay. Different concentrations of IPTG were used to induce the cultures (A) 0.05, (B) 0.2 and, (C) 0.5 mM.
Figure 11 shows glucose consumption from octyl acetate producing strains and 1-octanol producing strains. E. coli Strain No. 12 (‘CpFatBI -4-Sfp-CAR), 14 (‘CpFatBI -4-Sfp-CAR + ATF1), 13 (CaFatB3-5-Sfp-CAR) and, 15 (‘CaFatB3-5-Sfp-CAR + ATF1) (Table 1) were cultivated in M9 minimal media with 2% (w/v) glucose and 10% (v/v) hexadecane overlay. Different concentrations of IPTG were used to induce the cultures (A) 0.05, (B) 0.2 and, (C) 0.5 mM.
Figure 12 shows comparison of in vivo 1-octanol and octyl acetate production from E. coli BW25113 in the presence of solvent overlay. The 1-octanol and octyl acetate production from E. coli Strain No. 12 (‘CpFatB1-4-Sfp-CAR) and No. 14 (‘CpFatB1-4-Sfp-CAR + ATF1) at (A) 24 h, (B) 48 h, and (C) 72 h. The 1-octanol and octyl acetate production from E. coli Strain No. 13 (‘CaFatB3-5-Sfp-CAR) and No. 15 (‘CaFatB3-5-Sfp-CAR + ATF1) at (D) 24 h, (E) 48 h, and (F) 72 h (Table 1) were cultivated in M9 minimal media with 2% (w/v) glucose and 10% (v/v) hexadecane overlay. Different concentrations of IPTG were used to induce the cultures. The asterisk indicates significance by Student’s t-test (*, P£0.05; **, P£0.01 ***, P£0.005). The bar graphs are shown as the average from 3 biological replicates and circles represent data from independent biological replicates.
Figure 13 shows GC-MS chromatogram of (A) 1-octanol and (B) octyl acetate peak. One millimolar of 1-octanol or octyl acetate was spiked into 25 ml of M9 liquid media containing 2% (w/v) glucose in the presence of 10% (v/v) hexadecane overlay. Flasks were incubated at 30°C, 150 rpm for 24 h. The hexadecane overlay was sampled and analyzed by GC-MS, whilst the aqueous phase was extracted by 10% (v/v) hexadecane before the analysis.
Figure 14 shows evaluation of 1-octanol production from 2 E. coli strain backgrounds BW25113 and C43 (DE3). The strains harboring pET-PA1lacO-1-TPC3 were cultivated in M9 minimal media with 2% (w/v) glucose, induced with 0.5 mM IPTG and overlaid with 10% (v/v) hexadecane overlay for 48 h. Data are the average from 3 biological replicates.
Figure 15 shows cellular health of E. coli strains harboring different thioesterases. Average growth of (A) Strain No. 10 (CpFatB1-4) and (B) 11 (CaFatB3-5) (Table 1) when cultivated in M9 media with 2% (w/v) glucose overlaid with 10% (v/v) hexadecane overlay with different IPTG used to induce the cultures (0.02, 0.05, 0.2 and 0.5 mM). Data are the average ± standard deviation from 3 replicates.
Figure 16 shows growth from 1-octanol producing and octyl acetate producing strains in 24- well plate with solvent overlay. E. coli Strain No. 12 (CpFatB1-4-Sfp-CAR), 14 (‘CpFatB1-4- Sfp-CAR + ATF1), 13 (CaFatB3-5-Sfp-CAR) and 15 (‘CaFatB3-5-Sfp-CAR + ATF1) (Table 1) were cultivated in M9 minimal media with 2% (w/v) glucose in 24-well microtiter plate at 30°C and 432 rpm. IPTG was used to induce the strains at 0.2 mM and 10% (v/v) hexadecane overlay was applied at the beginning of the incubation. Data are the average ± standard deviation from 4 replicates.
Figure 17 shows conversion of 1-octanol into octyl acetate resulted in enhanced titer, growth, and yield. (A) Growth profile of 1-octanol producing strain (Table 2, Strain No. 3, Aaas-PnrsB- Sfp-CAR-Pcoa-‘CpFatB1-4) vs octyl acetate producing strain (Table 2, Strain No. 4, Aaas- PnrsB-Sfp-CAR-Pcoa-‘CpFatB1-4-SAAT) in the absence of overlay for 20 days. All strains were induced to express recombinant proteins on day 2 with 625 nM cobalt and 15 uM nickel. After 20 days of cultivation in the absence of solvent overlay, each culture was used to inoculate fresh cultures with solvent overlay that were induced to express recombinant proteins as above. (B) The photographs of 1-octanol strain (-AAT) and octyl acetate strain (+AAT) taken on day 10 when cultivated in the absence of hexadecane solvent overlay. (C) The photographs of 1-octanol strain (-AAT) and octyl acetate strain (+AAT) taken on day 10 when cultivated in the presence of hexadecane solvent overlay (30% (v/v)). (D) Product titer, (E) biomass accumulation and (F) product yield in solvent overlay cultures sampled on day 16. Student’s t-test analysis on all data shown in panels D to F were statistically significant (P£0.01). The bar graphs are shown as the average from 3 biological replicates and white circles in panels D to F represent data from independent biological replicates.
Figure 18 shows engineering a synthetic pathway for octyl glucoside biosynthesis with pathway enhancement by introducing AtSUSI for in-situ UDP-glucose regeneration and pathway flux comparison. (A) Schematic diagram of plasmids used to generate Strain No. 13,
16 to 22 as shown in Table 1. (B) Selection of GT for octyl glucoside production from E. coli BW25113 Strain No. 13 (‘CaFatB3-5-Sfp-CAR), No. 16 (‘CaFatB3-5-Sfp-CAR + AdGT4), No.
17 (‘CaFatB3-5-Sfp-CAR + AtGT1), No. 18 (‘CaFatB3-5-Sfp-CAR + MtG1), No. 19 (‘CaFatB3- 5-Sfp-CAR + MtH2), and No. 20 (‘CaFatB3-5-Sfp-CAR + VvGT1). The strains were cultivated in M9 media with 2% (w/v) glucose and 0.5 mM IPTG for 48 h. (C, D) The use of AtSUSI to enhance octyl glucoside production from E. coli Strain No. 13 (‘CaFatB3-5-Sfp-CAR), No. 19 (‘CaFatB3-5-Sfp-CAR + MtH2), No. 21 (‘CaFatB3-5-Sfp-CAR + AtSUSI), and No. 22 (‘CaFatB3-5-Sfp-CAR + MtH2 + AtSUSI). The strains were cultivated in M9 media with 2% (w/v) glucose and 0.5 mM IPTG supplemented with (C) 15 mM (D) 100 mM sucrose. (E) Chromatogram obtained from Strain No. 13 (‘CaFatB3-5-Sfp-CAR) (i), No. 21 (‘CaFatB3-5- Sfp-CAR + AtSUSI) (ii), No. 19 (‘CaFatB3-5-Sfp-CAR + MtH2), and No. 22 (‘CaFatB3-5-Sfp- CAR + MtH2 + AtSUSI) at 48 h. (F,G) Comparison of in vivo 1-octanol and octyl glucoside production from E. coli BW25113 in the presence of solvent overlay. E. coli Strain No. 13 (‘CaFatB3-5-Sfp-CAR), No. 21 (‘CaFatB3-5-Sfp-CAR + AtSUSI), No. 19 (‘CaFatB3-5-Sfp- CAR + MtH2), and No. 22 (‘CaFatB3-5-Sfp-CAR + MtH2 + AtSUSI) were cultivated in M9 media with 2% (w/v) glucose, 0.5 mM IPTG, overlaid with 10% (v/v) hexadecane, and supplemented with 15 mM sucrose at (F) 24 h (G) 48 h. The asterisk indicates significance by Student’s t-test (*, P£0.05; **, P£0.01 ***, P£0.005). Data are the average from 3 biological replicates and circles represent data from independent biological replicates.
Figure 19 shows comparison of in vivo 1-octanol and octyl acetate production from E. coli BW25113 (titers). E. coli Strain No. 12 (‘CpFatB1-4-Sfp-CAR), 14 (‘CpFatB1-4-Sfp-CAR + ATF1) at (A) 24 h, (B) 48 h, (C) 72 h and Strain No. 13 (‘CaFatB3-5-Sfp-CAR), 15 (‘CaFatB3- 5-Sfp-CAR + ATF1) at (D) 24 h, (E) 48 h and (F) 72 h (Table 1) were cultivated in M9 minimal media with 2% (w/v) glucose and 10% (v/v) hexadecane overlay. Different concentrations of IPTG were used to induce the cultures (0.02, 0.2 and, 0.5mM). ** indicates significant difference between 2 treatments (p£0.01). Data are the mean from 3 biological replicates.
Figure 20 shows the ratios between product yields from octyl acetate producing strains and 1-octanol producing strains. E. coli Strain No. (A) 12 (‘CpFatB1-4-Sfp-CAR), 14 (‘CpFatB1-4- Sfp-CAR + ATF1) and (B) 13 (‘CaFatB3-5-Sfp-CAR), 15 (‘CaFatB3-5-Sfp-CAR + ATF1) (Table 1) were cultivated in M9 minimal media with 2% (w/v) glucose and 10% (v/v) hexadecane overlay. Different concentrations of IPTG were used to induce the cultures (0.05, 0.2 and 0.5 mM).
Figure 21 shows the ratios between product titers from octyl acetate producing strains and 1- octanol producing strains. E. coli Strain No. (A) 12 (‘CpFatB1-4-Sfp-CAR), 14 (‘CpFatB1-4- Sfp-CAR + ATF1) and (B) 13 (‘CaFatB3-5-Sfp-CAR), 15 (‘CaFatB3-5-Sfp-CAR + ATF1) (Table 1) were cultivated in M9 minimal media with 2% (w/v) glucose and 10% (v/v) hexadecane overlay. Different concentrations of IPTG were used to induce the cultures (0.05, 0.2 and 0.5 mM).
Figure 22 shows localization of octyl glucoside in the absence of solvent overlay. E. coli BW25113 Strain No. 13 (‘CaFatB3-5-Sfp-CAR) and 19 (‘CaFatB3-5-Sfp-CAR + MtH2) were cultivated in M9 media with 2% (w/v) glucose with 0.5 mM IPTG. The cultures were harvested at centrifuged (17,000 x g, 10 min) to separate culture media and cells. The supernatant was analysed with HPLC. The cell fraction was suspended and lyzed with glass beads (Sigma Aldrich) at 30 hertz for 6 min on Tissuelyser II (QIAGEN). After that, the liquid fraction was analysed with HPLC.
Figure 23 shows comparison of in vivo 1-octanol and octyl glucoside production from E. coli BW25113 (titers). E. coli Strain No. 13 (‘CaFatB3-5-Sfp-CAR), 21 (‘CaFatB3-5-Sfp-CAR + AtSUSI), 19 (‘CaFatB3-5-Sfp-CAR + MtH2) and 22 (‘CaFatB3-5-Sfp-CAR + MtH2 + AtSUSI) were cultivated in M9 media with 2% (w/v) glucose supplemented with 15 mM sucrose at (A) 24 h (B) 48 h. The asterisk indicates significance by Student’s t-test (**, P£0.01 ;***, P£0.005). All data are shown as the average from 3 biological replicates and circles represent data from independent biological replicates.
Figure 24 shows growth from octyl glucoside producing strains and 1-octanol producing strains in the presence of solvent overlay. E. coli Strain No. 13 (‘CaFatB3-5-Sfp-CAR), No. 19 (‘CaFatB3-5-Sfp-CAR + MtH2), No. 21 (‘CaFatB3-5-Sfp-CAR + AtSUSI), and No. 22 (‘CaFatB3-5-Sfp-CAR + MtH2 + AtSUSI) (Table 1) were cultivated in M9 minimal media with 2% (w/v) glucose, 0.5 mM and 10% (v/v) hexadecane overlay. Figure 25 shows alcohol yield generated by wild type and novel truncated thioesterase enzymes. (A) alcohol yield by truncated CvFatBI thioesterases. (B) Fatty acid and fatty alcohol yield by truncated ChoFatB2 thioesterases.
Figure 26 shows (A) Photosynthetic growth in shake flasks of Synechocystis sp. PCC 6803 strains engineered to contain either (i) the 1-octanol pathway ('CaFatB3-5') or (ii) the 1-octanol pathway + glucosyltransferase ('CaFatB3-5-MtH2'); (B) molar product titer of engineered Synechocystis sp. PCC 6803 strains producing either 1-octanol (CaFatB3-5) or octyl glucoside (CaFatB3-5-Mth2) in the presence of solvent overlay; (C) Cultivation of 1-octanol and octyl glucoside accumulating Synechocystis sp. PCC 6803 strains in the absence of solvent overlay, left panel shows the weight of the biomass whilst the right panel indicates the visual density of the cultures.
Figure 27 shows different concentrations of alcohol or ester that were used to spike Escherichia coli cultures grown in 24-well plates with LB media. After 15h the OD600 of each culture was measured. In each panel, the white bar represent the alcohol whilst the grey bar(s) represent the corresponding acetyl ester, or in the case of linalool, also the butyryl ester (light grey).
Figure 28 shows (A) Sixteen different AAT-encoding genes were tested for acetylation of n- butanol in vivo in E. coli DfadE expressing a n-butanol pathway with a solvent overlay to trap butyl acetate. Only four AAT-encoding genes displayed activity resulting in the biosynthesis of butyl acetate, Atf 1 , SAAT, VAAT and UAAT4. The Y-axis displays the titer of butyl acetate after 48h of cultivation. (n=3, standard deviation); (B) The top six AAT-encoding genes were also evaluated in an alternative E. coli strain (G4165 [DglyA::aad D(tdh-kbl) DltaE AgcvR AkdgK :: ftfL(C.kluyveri) Agcv::erm]) and this time also the n-butanol (dark grey) content was measured. Hence, the conditions were similar to those in A, but also the aqueous fraction was used to quantify n-butanol (dark grey) using HPLC, whilst the solvent overlay (hexadecane) fraction was used to quantify the butyl acetate (light grey) content using GC- MS. All measurements after 48h of induction in minimal M9 media supplemented with 10mM glycine, 10mM formate and 2% glucose; (C) Final cell density of the seven strains tested in B after 48h.
Figure 29 shows (A) Chain-length specificity of CvFatBI truncations in E. coli after cultivation in minimal M9 media for 48 hours. All E. coli strains also express the CAR enzyme and helper Spf protein, resulting in conversion of released "free"' fatty acids into corresponding alcohols; (B) Chain-length specificity of CvFatBI truncations in Synechocystis sp. PCC 6803 after cultivation in BG11 media for 10 days. All Synechocystis strains also express the CAR enzyme and helper Spf protein, resulting in conversion of released "free"' fatty acids into corresponding alcohols.
Figure 30 shows co-expression of acyl-carrier proteins (ACPs) from different species together with the CvFatB1-T5 thioesterase in E. coli after cultivation in minimal M9 media for 48 hours. All E. coli strains also express the CAR enzyme and helper Spf protein, resulting in conversion of released "free"' fatty acids into corresponding alcohols. NA = strains that do not over express any ACP.
EXAMPLES
Experimental Results
There are several instances where bioderivatization has been implemented without a rationale (e.g. vanillin glucoside biosynthesis as opposed to synthesizing plain vanillin (2)) or happened by chance through interactions with native metabolism in the biotechnological host (e.g. geraniol synthesis results in geranyl acetate formation through the action of native AATs (13)). To our knowledge, however, the impact of this process on biotechnological objectives has not yet been studied.
We assumed that only toxic or labile products, or those which are expensive to separate (e.g. organic acids (14)), were likely to benefit from the strategy. For example, shorter chain-length alcohols such as n-butanol and ethanol have relatively low toxicity and effective production systems are already in place. In the present work, the hypothesis that bioderivatization offers benefits for a microbial biotechnological process was therefore tested using 1-octanol (15, 16) as the model product and toxicity as the primary focus using two different model biotechnological hosts, Escherichia coli and Synechocystis sp. PCC 6803. As only efficient pathways are likely to result in the accumulation of sufficient concentration(s) of compounds to elicit a toxic response, the metabolic systems were first optimized. An additional point of interest in this study was the role of solvent overlay. For hydrophobic products, solvent overlay offers an opportunity to reduce toxicity, most likely simply by facilitating product removal. However, situations may arise when solvent overlay is undesirable, for example when attempting to separate a volatile product in the off-gas or when the energetic cost of solvent- product separation is excessive. The presence or absence of solvent overlay was therefore also evaluated.
Material and Methods
Strains and plasmid E. coli DH5a (Thermo Fisher Scientific) was used to propagate all the plasmids used in this work. Two strains of E. coli (E. coli C43 (DE3) (Lucigen) and BW25113 (Keio collection)) and one species of cyanobacteria ( Synechocystis sp. PCC 6803) were used as hosts for 1-octanol and octyl acetate production. All E. coli and cyanobacterial strains used in this study are listed in Table 1 and Table 2, respectively. The genes encoding Tes3, Sfp, and CAR were obtained from plasmid pET-TPC3 (17), whereas the chloramphenicol acyltransferase (cat) was amplified from plasmid pACYC-petF-fpr (18). A strawberry alcohol acetyltransferase gene (. saat ) from Fragaria x ananassa (19) (UniProtKB: Q9FVF1), an alcohol O-acetyltransferase gene ( atf1 ) from Saccharomyces cerevisiae (UniProtKB: P40353), and C8-preferring thioesterases (CpFatBI from Cuphea palustris and CaFatB3 from Cuphea avigera pulcherrima ) including their variants (‘CpFatBI -4 and ‘CpFatB3-5) were chemically synthesized from Integrated DNA Technologies (IDT) and codon optimized for E. coli. Five genes encoding glycosyl transferases and one gene encoding sucrose synthase (Table 3) were also synthesized from IDT for octyl glucoside production. Plasmids used for gene expression were constructed using Biopart Assembly Standard for Idempotent Cloning (BASIC) (20) or traditional restriction enzyme ligase based cloning. The details of strain and plasmid construction method are described below. The UniProtKB ID for all the genes used in this study is listed in Table 3; note that if the genes were from foreign organisms, codon optimization was carried out prior to synthesis in order to overexpress in bacteria. Primers used for polymerase chain reaction (PCR) are provided in Table 4. All linkers used for BASIC are listed in Table 5. The amino acid sequence alignments of the C8-preferring thioesterases are shown in Figure 7.
Table 1. List of E. coli strains used in this study
Figure imgf000029_0001
Figure imgf000030_0001
Table 2. List of Synechocystis sp. PCC 6803 strain used in this study
Figure imgf000030_0002
Table 3. Source organisms of overexpressed genes in this study
Gene Source organism UniProtKB Reference tes3 Anaerococcus tetradius C2CIR4 (Akhtar et al., 2015) sfp Bacillus subtilis P39135 (Akhtar et al., 2015) car Mycobacterium marinum B2HN69 (Akhtar et al., 2015) cat Escherichia coli P62577 (Rottig and SteinbOchela, 2013) saat Fragaria ananassa cv. Q.9FVF1 (Aharoni et al., 2000) Elsanta atfl Saccharomyces cerevisiae P40353 (Verstrepen et al., 2003)
CpFatBl Cuphea palustris 0.39554 (Dehesh et al., 1996) (wild type)
CaFatB3 Cuphea avigera pulcherrima V9MFIU7 (Tjellstrom et al., 2013) (wild type)
'CpFatBl-4 Cuphea palustris (Flernandez Lozada et al., 2018) 'CaFatB3-5 Cuphea avigera pulcherrima Manuscript submitted
AdGT4 Actinidia deliciosa A0A077EMP8 (Yauk et al., 2014)
VvGTl Vitis vinifera P51094 (Christopher M. Ford et al., 1998)
AtGTl Arabadopsis thaliana 0.9 M 156 (Lim et al., 2002)
MtGl Medicago truncatula Q5IFH7 (Shao et al., 2005)
MtH2 Medicago truncatula A6XNC5 (Li et al., 2007)
AtSUSl Arabidopsis thaliana P49040 (Zheng et al., 2011)
Construction of E. coli strains for 1-octanol and octyl acetate production
To obtain Strain No. 3 to 5 in Table 1, E. coli strain C43 (DE3) was transformed with plasmid pET-TPC3 (Akhtar et al., 2015) harboring tes3, sfp, and car genes. Plasmid pACYC-petF-fpr plasmid (Kallio et al., 2014) was used as a template to amplify the cat gene. The strawberry alcohol acetyltransferase gene ( saat ) from Fragaria x ananassa (Aharoni et al., 2000) (UniProtKB: Q9FVF1) and the alcohol O-acetyltransferase gene ( atf1 ) from Saccharomyces cerevisiae (UniProtKB: P40353) were chemically synthesized from Integrated DNA Technologies (IDT) and codon optimized for E. coli. The saat and atf1 genes were individually stored in a blunt-ended pJET1.2 plasmid (Thermo Fisher Scientific), resulting in pJET-SAAT and pJET-ATF1 plasmids, respectively. Next, pCDF-GFP plasmid harboring T7 promoter was constructed as described in Table 8. The cat, saat, and atf1 genes were then sub-cloned into pCDF-GFP plasmid using Ssal restriction site which was introduced by PCR using the oligonucleotide primers listed in Table 4. The resulting plasmid was transformed into the E. coli C43 (DE3) strain carrying the pET-TPC3 plasmid, generating Strain No. 3 to 5 in Table 1.
Table 4 List of templates and primers used for gene(s) amplification
Figure imgf000031_0001
Figure imgf000032_0001
aKallio et al., 2014, Nature communications
Next, BASIC method was used to construct plasmids in Strain No. 7 to 15 in Table 1. First, plasmid pET-PA1lacO-1-GFP and pCDF-PA1lacO-1-GFP harboring PA1lacO-1 promoter were constructed as shown in Table 7 No. 1 and 2, respectively. Next, the tes3 and sfp-car genes were amplified from plasmid pET-TPC3 (Akhtar et al., 2015) using primers listed in Table 4 and cloned into a blunt-ended plasmid pJET1.2 (Thermo Fisher Scientific). Other genes encoding thieosterases: ‘CpFatBI, ‘CaFatB3,’CpFatB1-4, and ‘CaFatB3-5 genes were chemically synthesized as gBIocks from Integrated DNA Technologies (IDT) and cloned into pJET1.2 blunt. The resulting plasmids were used to create plasmids used in Strain No. 7 to 15 in Table 1. The linkers and plasmids used for BASIC method are listed in Table 7 No. 3 to 9. The amino acid sequence of ‘CpFatBI (SEQ ID NO: 9), ‘CaFatB3 (SEQ ID NO: 10), ‘CpFatBI -4 (SEQ ID NO: 12), and ‘CaFatB3-5 (SEQ ID NO: 13) and their wild types are shown in Figure 7. Construction of E. coli strains for octyl glucoside production
To obtain Strain No. 16-22, E. coli strain BW25113 was transformed with pET-‘CaFatB3-5- Sfp-CAR. The glycosyltransferase (GT) from Actinidia deliciosa ( AdGT4 ), Vitis vinifera ( VvGT1 ), Medicago truncatula ( MtGT1 ), Arabidopsis thaliana ( AtGT1 ) and Medicago truncatula (MtH2) and the gene encoding sucrose synthase from Arabidopsis thaliana ( AtSUSI ) were chemically synthesized as gBIocks from Integrated DNA Technologies (IDT) and codon-optimized for E. coli. The UniProtKB ID for all the genes used in this study is listed in Table 3. All gBIocks were cloned into blunt-ended pJET1.2 plasmid (Thermo Fisher Scientific). The resulting plasmids are listed in Table 6 and used for Biopart Assembly Standard for Idempotent Cloning (BASIC) method (Storch et al. , 2015) in Table 7. All the linkers (Table 5) and plasmids used to create CloDF13-based plasmids harboring the glycosyltransferase and sucrose synthase under the PA1lac01 promoter are provided in Table 7. The resulting plasmid was then transformed into E. coli BW25113 strain carrying pET-‘CaFatB3-5-Sfp-CAR plasmid.
Table 5 DNA linkers used to generate plasmid constructs by BASIC assembly
Name Prefix linker
Linker sequence (5’ to 3’) Adapter sequence (5’ to 3’)
1 MP GGACAGAGACCCACCAGAT AAT AGT GTTTCCACGAAGT G (SEQ ID NO: TCTGGTGGGT/iMe-dC/TCT (SEQ ID NO: 60) 53)
2MP GG ACG ATT CCG AAGTT ACACCAG ATT G G ACTGTT ATT AC (SEQ ID NO: 54) AACTTCGGAATC (SEQ ID NO: 61)
1 P GGACT AGTTCAAT AAAT ACCCTCT GACT GTCTCGGAG (SEQ ID NO: 55) TTT ATT GAACT A (SEQ ID NO: 62)
2P GGACAGGTAATAAGAACTACACGACTGGATACTGACT (SEQ ID NO: 56) TTCTTATTACCT (SEQ ID NO: 63)
3P GGACTCT GT AAT AACAAT ACCGAT AAAGCAACGAGT G (SEQ ID NO: 57) TGTT ATT ACAGA (SEQ ID NO: 64)
LRBS1-3P GGACT ATTT CTCCT CTTTTT ACAACT GAT ACTTACCT GA (SEQ ID NO: 58) AAAGAGGAGAAAT A (SEQ ID NO: 65)
LRBS2-3P GGACTATTTCTCCTCTTTTTTCTGCTACCCTTATCTCAG (SEQ ID NO: 59) AAAGAGGAGAAAT A (SEQ ID NO: 65)
Name Suffix linker
Linker sequence (5’ to 3’) Adapter sequence (5’ to 3’)
1 MS CTCGGGTAAGAACTCGCACTTCGTGGAAACACTATTA (SEQ ID NO: 66) CGAGTTCTTACC (SEQ ID NO: 72)
2MS nTATCGGTAATAACAGTCCAATCTGGTGT (SEQ ID NO: 67) CGATAGGT/iMe-dC/TCC (SEQ ID NO: 73)
1 S CTCGTTACTTACGACACTCCGAGACAGTCAGAGGGTA (SEQ ID NO: 68) TGTCGTAAGTAA (SEQ ID NO: 74)
2S CTCGATCGGT GT GAAAAGT CAGT ATCCAGTCGT GTAG (SEQ ID NO: 69) TTTCACACCGAT (SEQ ID NO: 75)
3S CTCGATCACGGCACTACACTCGTTGCTTTATCGGTAT (SEQ ID NO: 70) TAGTGCCGTGAT (SEQ ID NO: 76)
LRBS1-XS CTCGTT GAACACCGT CTCAGGTAAGTATCAGTTGTAA (SEQ ID NO: 71) GACGGTGTTCAA (SEQ ID NO: 77)
LRBS2-XS CTCGT GTT ACT ATTGGCT GAGAT AAGGGT AGCAGAAA (SEQ ID NO: 80) CCAATAGTAACA (SEQ ID NO: 81)
Table 6. Plasmids used for construct preparation in this study
Figure imgf000035_0001
Figure imgf000036_0001
Table 7 Part compositions used for plasmid assembly
No. Prefix Plasmid Suffix Plasmid generated Relevant information
1 3P pJET-ColE1-rop-bom 1S pET- PA1 lacO-1-GFP A plasmid used as a backbone for BASIC assembly with ColE1 origin of
1 P pJET- PA1 lacO-1 2MS replication, pAl lacOI promoter and
2MP pJET-GFP 1 MS kanamycin resistance.
1 MP pJET-termB15 2S
2P pJET-Kan 3S
2 1 P pJET-CloDF-Spec 2S pCDF- PA1 lacO-1-GFP A plasmid used as a backbone for BASIC assembly with CloDF origin of
2P pJET- PA1 lacO-1 2MS
No. Prefix Plasmid Suffix Plasmid generated Relevant information
2MP pJET-GFP 1 MS replication, pA1 lac01 promoter and spectinomycin resistance.
1 MP pJET-termB15 1S
3 2P pET-GFP LRBS1-XS pET- PA1 lacO-1 -TPC3 A plasmid encoding Tes3, Sfp and CAR.
LRBS1-3P pJET-Tes3 LRBS2-XS
LRBS2-3P pJET-Sfp-CAR 2S
4 2P pET-GFP LRBS1-XS pET- PA1 lacO-1 -‘CpFatB! -Sfp-CAR A plasmid encoding ‘CpFatBI , Sfp and CAR.
LRBS1-3P pJET-‘CpFatB1 LRBS2-XS
LRBS2-3P pJET-Sfp-CAR 2S
5 2P pET-GFP LRBS1-XS pET- PA1 lacO-1 -‘CaFatB3-Sfp-CAR A plasmid encoding ‘CaFatB3, Sfp and CAR.
LRBS1-3P pJET-‘CaFatB3 LRBS2-XS
LRBS2-3P pJET-Sfp-CAR 2S
6 2P pET-GFP LRBS1-XS pET- PA1 lacO-1 -‘CpFatB! -4-Sfp-CAR A plasmid encoding ‘CpFatB1-4, Sfp and CAR.
LRBS1-3P pJET-‘CpFatB1-4 LRBS2-XS
LRBS2-3P pJET-Sfp-CAR 2S
7 2P pET-GFP LRBS1-XS pET- PA1 lacO-1 -‘CaFatB3-5-Sfp-CAR A plasmid encoding ‘CaFatB3-5, Sfp and CAR.
LRBS1-3P pJET-‘CaFatB3-5 LRBS2-XS
LRBS2-3P pJET-Sfp-CAR 2S
No. Prefix Plasmid Suffix Plasmid generated Relevant information
8 2P pCDF-GFP LRBS1-XS pCDF- PA1 lacO-1-ATF1 A plasmid encoding ATF1
LRBS1-3P pJET-ATFI 2S
9 2P pCDF-GFP 2S pCDF- PA1 lacO-1 -empty A negative control plasmid
10 1 P pRSF1010-Ery-Pcoa-GFP LRBS1-XS pRSF1010-Ery-Pcoa-‘CpFatB1-4 A plasmid encoding ‘CpFatBI -4
LRBS1-4P pJET-‘CpFatB1-4 1S
11 1 P pRSF1010-Ery-Pcoa-GFP LRBS1-XS pRSF1010-Ery-Pcoa-‘CpFatB1-4-SAAT A plasmid encoding ‘CpFatBI -4 and
Figure imgf000038_0001
LRBS1-4P pJET-‘CpFatB1-4 LRBS2-XS
LRBS2-4P pJET-SAAT 1S 12 2P pCDF-GFP LRBS1-XS pCDF- PA1 lacO-1-AdGT4 A plasmid encoding AdGT4
LRBS1-3P pJET-AdGT4 2S
13 2P pCDF-GFP LRBS1-XS pCDF- PA1 lacO-1-AtGT1 A plasmid encoding AtGT1
LRBS1-3P pJET-AtGT1 2S
14 2P pCDF-GFP LRBS1-XS pCDF- PA1 lacO-1-MtG1 A plasmid encoding MtG1
LRBS1-3P pJET-MtG1 2S
15 2P pCDF-GFP LRBS1-XS pCDF- PA1 lacO-1-MtH2 A plasmid encoding MtH2
LRBS1-3P pJET-MtH2 2S
16 2P pCDF-GFP LRBS1-XS pCDF- PA1 lacO-1-VvGT1 A plasmid encoding VvGT1
No. Prefix Plasmid Suffix Plasmid generated Relevant information
LRBS1-3P pJET-Vv 2S
17 2P pCDF-GFP LRBS1-XS pCDF- PA1 lacO-1-MtH2-AtSUS1 A plasmid encoding MtH2 and AtSUSI
LRBS1-3P pJET-MtH2 LRBS2-XS
LRBS2-3P pACIDT-AtSUSI 2S
18 2P pCDF-GFP LRBS1-XS pCDF- PA1 lacO-1 -AtSUSI A plasmid encoding AtSUSI
LRBS1-3P pACIDT-AtSUSI 2S
Table 8 Summary of plasmid construction via traditional restriction cloning
Plasmid used Ligated fragment Relevant information
Plasmid backbone Insert pCDF-CAT pCDF-GFP (Bsal) Cat (Bsal) A plasmid encoding CAT protein pCDF-SAAT pCDF-GFP (Bsal) Saat (Bsal) A plasmid encoding SAAT protein pCDF-ATFI pCDF-GFP (Bsal) Atfl (Bsal) A plasmid encoding ATF1 protein pCDF-empty pCDF-GFP (Bsal) A negative control plasmid pET-empty PET-TPC3 (Ncol and Avrll) A negative control plasmid
GFP gene was amplified from pJET-GFP (Yunus and Jones, 2018) using oligonucleotides with Ncol and Avrll cutting sites for the construction of pCDF-GFP plasmid backbone. The amplified GFP was then cloned into pCDF-Ahr s (Akhtar et al., 2015) by replacing Ahr s with GFP. cat was amplified from pACYC-petF-fpr with Bsal prefix and suffix, while SAAT and ATF1 were order as a gblock from IDT technology. All 3 genes were
cloned into pCDF-GFP backbone. To construct empty plasmids, pET-TPC4 was digested with Ncol and Avrll and pCDF-GFP was digested with Bsal. Both backbones were recirculated using Quick Blunting™ and Quick Ligation™ Kits from NEB after restriction digestion.
Table 9. List of DNA templates and primers used to generate genetic parts for the construction of the suicide plasmid targeting phaAB site
Primer Plasmid
Plasmid
Template Generate Relevant information
F R Contained d
Genetic Part 1 - Upstream homology region
D pJET::p/?aAB_UH .NA „ IY224 IY225 plY442 chromosome3 ^ R Plasmid containing a lad repressor gene and Pciaci43 promoter.
Genetic Part 2 - Downstream homology region
DNA pJET::p/?aAB_DH
IY226 IY227 plY443 chromosome3 R Plasmid containing PnrsB promoter.
Genetic Part 3 - Promoter DNA
PB210 PB211 plY171 pJ ETiiPnrsB chromosome3 Plasmid containing PnrsB promoter.
Genetic Part 4 - GFP dropout gene plY24 pJET ::gfp Superfold green fluorescence (GFP) gene ordered as a gBIock.
Genetic Part 5 - Spectinomycin resistance cassette
PB29 PB30 plY98 pJET::SpR Spectinomycin resistance cassette was ordered as a gBIock. Genetic Part 6 - Terminator plY67 pJET::termB15 Plasmid storage containing rrnB T1 terminator and T7Te terminator.
Genetic Part 7 & 8 - Backbone plY99 nPniFi nfn KanR Plasmid backbone for plasmid propagation in E. coli with a kanamycin P selection marker cassette and a GFP dropout gene
Plasmid backbone for plasmid propagation in E. coli with a plY23 pColE1 ::AmpR carbenicillin selection marker cassette (requested from Dr. Geoff Baldwin)
Gene of interest pET-TPC3b IY234 IY155 plY485 pJET-Sfp-CAR Plasmid storage containing Sfp and CAR.
Synechocystis sp. PCC 6803 wild-type strain bAkhtar et al. , Metabolic Engineering Communications, 2015
Table 10. List of genetic parts and linkers used to construct plasmid plY453 and plY454
Prefix Linker Plasmid Genetic part Suffix Linker Plasmid generated Relevant Information
1MP plY442 phaABJJHR 1S plY453 is the plasmid carrying the upstream genetic parts for the
1P plY171 PnrsB 2MS plY453 construction of suicide vector targeting phaAB site
2MP plY99 ColE1-KanR 1MS
1MP plY67 TermB15 1S
1P plY98 Sp ,R> 2S plY454 is the plasmid carrying the downstream genetic parts for the plY454
2P plY443 phaAB_DHR 2MS construction of suicide vector targeting phaAB site
2MP plY99 ColE1-KanR 1MS
Construction of cyanobacterial strains for 1-octanol and octyl acetate production
Strain 6803- Aaas was constructed as described in (Yunus and Jones, 2018). To create strain 6803-Aaas-PnrsB-Sfp-CAR, a suicide plasmid targeting phaA and phaB sites (encoded by slr1993 and slr1994) was first constructed. All the genetic parts used for this plasmid construction are listed in Table 9. Next, the plasmids carrying the upstream (plY453) and downstream (plY454) genetic parts were assembled using the linkers shown in Table 10. Next, the suicide plasmid (plY475) carrying a gfp dropout gene were created as shown in Table 11. Next, the sfp and car genes were then amplified using primers listed in Table 4 and sub-cloned into pJET1.2 blunt plasmid to give plasmid plY485. Finally, the sfp and car genes were cloned into plasmid plY475 using the linkers shown in Table 12. The expression of Sfp and CAR were under controlled of a nickel-inducible promoter PnrsB. Plasmid plY706 (pMB1- Amp-phaAup-PnrsB-Sfp-CAR-termB15-Sp-phaBdown) was then naturally transformed into 6803 -Aaas strain. In brief, 6803- Aaas strain was inoculated in 25 ml of BG11-Co liquid medium at 30°C with continuous illumination at 60 mhioI photons/m2. s and 1% (v/v) CO2 in the Algaetron AG 230 (Photon Systems Instruments). When the OD730 reached 0.3-0.4, the cells were harvested and resuspended in 500 mI_ fresh BG11-Co medium. One hundred microliters of concentrated liquid culture were mixed with four to seven micrograms of plasmid plY706 and incubated at at 60 pmol photons/m2. s and 1% (v/v) C02 for 12-16 h prior to plating on BG11-Co agar containing 20 pg/ml spectinomycin. The plate was then incubated at 30°C with continuous illumination at 60 pmol photons/m2. s for 1-2 weeks or until the colonies appeared. To promote segregations, individual colonies were restreaked on BG11-Co containing 50 pg/ml spectinomycin and 100 pg/ml spectinomycin. A fully segregated mutant was confirmed by PCR using primers IY293 (5’-GGCAAAGCTTTATTTGCCAATGCG-3’ (SEQ ID NO: 78)) and IY292 (5’-CCGATGACACTAATCTCAAGGCGG-3’ (SEQ ID NO: 79)) and used in the subsequent experiments.
Table 11. Linkers and genetic parts used to construct the suicide vector backbone plY475 carrying a gfp dropout gene
Prefix Selectable Suffix Plasmid
Plasmid No. Genetic part Relevant Information
Linker Marker Linker generated
1 P plY453 phaAB_UHR, PnrsB AmpR 2MS plY475 is a suicide vector targeting phaAB
2MP plY24 GFP GFP, AmpR 1 MS site containing phaAB upstream homology region, gfp dropout gene, a terminator, a termB15, SpR, plY475
1 MP plY454 SpR, KanR 2S spectinomycin selection marker cassette, a phaAB_DHR phaAB downstream homology region, and an
2P plY23 ColE1 , AmpR AmpR 1S E. coli ori with an ampicillin selection marker
Table 12. Linkers and genetic parts used to construct the suicide vector carrying sfp and car genes targeting phaAB site
Selectable
Prefix Linker Plasmid Suffix Linker Plasmid generated Relevant Information
Marker
LRBS1-4P plY485 AmpR 1S plY706 is a suicide vector carrying sfp and car genes targeting plY706
1 P plY475 GFP, KanR, AmpR LRBS1-4S phaAB site.
Next, to obtain strain 6803-Aaas-PnrsB-Sfp-CAR-Pcoa-‘CpFatB1-4 and 6803-Aaas-PnrsB- Sf p-CA R- Pcoa-‘Cp F atB 1 -4-SAAT, plasmid plY849 (Table 7 No. 10, pRSF1010-Ery-Pcoa- ‘CpFatB1-4) and plY887 (Table 7 No. 11, pRSF1010-Ery-Pcoa-‘CpFatB1-4-SAAT) were individually transformed into 6803-Aaas-PnrsB-Sfp-CAR strain by triparental conjugation. In brief, plasmid plY849 and plY887 was first transformed individually into E. coli cargo HB101 strain carrying pRL623 plasmid (Elhai et al. , 1997). Next, one hundred microliters or the cargo strain carrying both pRL623 and plY849 or plY887, conjugal strain carrying plasmid pRL443 (Elhai et al., 1997) and the 6803-Aaas-PnrsB-Sfp-CAR strain were mixed and incubated for 2 h (30°C, 60 mhioI photons/m2. s). Prior to mixing, all strains were washed with LB medium (for E. coli) or BG11-CO medium (for cyanobacteria) to remove all the antibiotics. After 2 h of incubation, the mixed culture was then plated on BG11-Co agar plates without antibiotic and incubated for two days (30°C, 60 pmol photons/m2. s). After 2 days of incubation, biomass were scraped from the agar plates and transferred onto a new agar plate containing 20 mg/ml erythromycin. Individual colonies were restreaked onto a new agar plate containing 20 mg/ml erythromycin, 10 mg/ml spectinomycin, and 10 mg/ml kanamycin and incubated in the Algaetron AG 230 for 4-7 days before it was used for subsequent experiments. Empty plasmid plY606 (Yunus and Jones, 2018) was also used as control to obtain strain 6803-Aaas-PnrsB- Sf p-CA R- Pcoa-em pty . All plasmids used in this study are listed in Table 13.
Table 13 Plasmids used for production in this study
Figure imgf000044_0001
Figure imgf000045_0001
Evaluating the effect of 1-octanol, octyl acetate and octyl glucoside on growth
E. coli strain C43 (DE3) was cultivated in 10 ml of Luria-Bertani (LB) liquid media (LB broth, Sigma Aldrich) overnight at 37°C, 180 rpm. The overnight culture was washed twice with fresh M9 minimal media (47.8 mM Na2HP04, 22 mM KH2P04, 8.55 mM NaCI, 18.69 mM NH4CI, 2 mM MgS04, 0.1 mM CaCI2 and 2% (w/v) glucose) and resuspended in the same media to an initial Oϋboo of ~0.1. The liquid cultures were spiked with different concentrations of 1-octanol (0-50 mM) and octyl acetate (0-200 mM) and transferred (200 pi) into a well in 96-well microtiter plate. The plate was incubated in Tecan Infinite M200 Pro Spectrophotometer (Tecan AG) at 37°C with continuous shaking at 432 rpm and Oϋboo was measured every hour for 15 hours. The specific growth rates were calculated for each treatment.
Production of 1-octanol, octyl acetate and octyl glucoside in engineered E. coli Overnight cultures were grown in LB media (10 ml) containing appropriate antibiotics with final concentrations as follows: kanamycin 50 pg/ml, spectinomycin 50 pg/ml, and carbenicillin 100 pg/ml. The overnight cultures were washed twice with fresh M9 minimal media and resuspended in 2 ml of M9 minimal media prior to inoculation in 25 ml of M9 minimal media with a starting Oϋboo of ~0.1 in 100-ml Erlenmeyer flasks supplied with appropriate antibiotics. For 1-octanol and octyl acetate production experiment, the liquid cultures were cultivated at 37°C, 180 rpm for 4 h and induced with various concentrations of IPTG (0.02, 0.05, 0.2, and 0.5 mM). After induction, 10% (v/v) hexadecane solvent overlay (Sigma Aldrich) was added and the liquid cultures cultivated at 30°C, 150 rpm. Oϋboo was measured every 24 h after inoculation for 48 h unless stated otherwise. Samples from the liquid cultures and hexadecane solvent overlay were collected every 24 h after inoculation for HPLC and GC-MS analysis, respectively. For octyl glucoside production, the cultures were cultivated at 37°C, 180 rpm for 4 h and induced with 0.5 mM isopropyl b-D-l-thiogalactopyranoside (IPTG) before continuing the incubation at 30°C, 150 rpm for 48 h. Samples were centrifuged and the supernatants were analyzed using HPLC. Sucrose (15 or 100 mM) was supplemented to the culture media at the time of induction when the function of AtSUSI was assessed. Solvent overlay was used when the production of 1-octanol and octyl glucoside was compared.
Production of 1-octanol and octyl acetate in engineered cyanobacteria
All cyanobacterial strains were cultivated in BG11 medium without cobalt (hereafter BG11-Co) as cobalt was used as an inducer. The preculture was grown in 5 ml BG11-Co containing appropriate antibiotic(s) (final concentration: kanamycin 10 pg/ml, spectinomycin 10 pg/ml, and erythromycin 20 pg/ml) in a 6-well plate at 30°C, 180 rpm, with continuous illumination (warm-white LED) at 60 pmol photons/m2. s and 1% (v/v) C02 in an Algaetron AG 230 (Photon Systems Instruments (PSI)). When the OD730 reached 3-4, the liquid preculture was transferred into an autoclaved 100-ml Erlenmeyer flask covered with aluminium foil. The OD730 was adjusted to 0.2 by adding BG11-Co to a final volume of 25 ml and antibiotics were added accordingly. The liquid culture was induced on day 2 with 15 pM nickel and 625 nM cobalt, and the OD730 was monitored for 20 days in the presence or absence of a 30% (v/v) hexadecane solvent overlay. On day 20, the liquid culture was transferred into fresh BG11-Co media containing antibiotics with initial OD730 -0.2. When the OD730 reached 1-1.5, the liquid culture was induced with 15 pM nickel and 625 nM cobalt and hexadecane solvent overlay 30% (v/v) added. The hexadecane solvent overlay was sampled 16 days after induction for GC-MS analysis. In a separate experiment, production cultures were inoculated directly from the initial pre-culture and cultured for 10 days in the presence of 30% (v/v) hexadecane solvent overlay, with induction of protein expression on day 2, as described above. The toxicity of the different products to Synechocystis sp. PCC 6803 were evaluated as described in Figure 3.
Preparation of BG11-Co liquid medium
The standard 1X BG11-Co liquid medium was prepared by mixing 10 ml 100X BG11-Co, 1 ml 1000X ferric ammonium citrate, 1 ml 1000X Na2C03, and 1 ml 1000X K2HPO4 in 1 L of ultrapure water (PURELAB flex 2). The concentrated BG11-Co stock solution (100X BG11- Co) containing 149.6 g NaN03, 7.49 g MgS04.7H20, 3.6 g CaCI2.2H20, 0.89 g Na- citrate.2H20, 1.12 ml 0.25 M NaEDTA pH 8.0, and 100 ml 1000X trace mineral solution in 1 L ultrapure water was prepared. The 1000X trace mineral solution was made by dissolving 2.86 g H3B03, 1.81 g MnCI2.4H20, 0.22 g ZnS04.7H20, 0.39 g Na2Mo04.2H20, and 0.079 g CuS04.5H20 in 1 L ultrapure water and stored at 4°C until being used. The 1000X ferric ammonium citrate, 1000X Na2C03, and 1000X K2HPO4 solutions were prepared by dissolving 0.6 g ferric ammonium citrate, 2 g Na2C03, and 3.05 g K2HPO4 in 100 ml ultrapure water, respectively. The 1 L standard 1X BG11-Co solution in a 1-L Duran bottle was sterilized by autoclaving for 15 min at 15 psi (121°C) and cooled down to room temperature prior to use.
Quantification and analysis of 1-octanol, octyl acetate, octyl glucoside, and other metabolites
GC-MS
Hexadecane solvent overlay was used to capture 1-octanol and octyl acetate from the liquid culture. One hundred microliters of solvent overlay were transferred into an insert in a 2-ml screw top GC vial (Agilent Technologies). Samples were analyzed using an Agilent 7890B GC with HP-5ms column, a 7693 autosampler and 5977B MSD. One pi of samples was injected using pulsed split ratio 10:1 and split flow at 10 min/ml. The GC method was programmed with an initial temperature of 70°C for 30 s, followed by a first ramp at 30°C/min to 250°C before ramping up to 300°C with a final hold for 2 min at 40°C/min. Target products were identified by comparing mass spectra and retention times with external standards. Serial dilutions of 1-octanol (³99%, Sigma Aldrich) and octyl acetate (³99%, ACROS organics) standards were used to quantify the concentration of 1-octanol and octyl acetate in the sample.
HPLC
An Agilent 1200 series HPLC equipped with different columns and Reflective Index detector (RID) was used to determine the concentration of octyl glucoside, glucose, fructose, sucrose and fermentation products in the E. coli sample every 24 h. One milliliter of liquid cultures was sampled at 24 h and 48 h and centrifuged at 17,000 x g for 15 min to separate the aqueous and hexadecane layer. The supernatant was transferred into a 2-ml HPLC vial. For octyl glucoside detection, samples (20 mI) were analysed with Zorbax XDB-C18 column. The flow rate was set at 1 ml/min with the column temperature at 30°C21. For the analysis of sugars (glucose, sucrose, and fructose) from samples supplemented with sucrose, Aminex HPX-87P column (Bio-Rad) was used to analyse the samples (20 mI) with the flow rate of 0.6 ml/min and the column temperature was set at 85°C. For glucose and other fermentation products when sucrose was not supplemented, samples (100 mI) were analyzed with Aminex HPX-87H column (Bio-Rad) and the flow rate and column temperature were set at 0.6 ml/min and 60°C, respectively. Serial dilutions of glucose (Sigma Aldrich), sucrose (Sigma Aldrich), fructose (Sigma Aldrich) sodium acetate (Sigma Aldrich), sodium lactate (Sigma Aldrich), and absolute ethanol (VWR) were used to determine the amount of these compounds in the sample.
Statistical treatment and data
Three biological replicates were employed for every treatment and/or condition. As all samples were collected from cultures that most likely were reasonably homogenous, normality was assumed in all cases, as discussed in Fay and Gerow (2013). With selected data, indicated in the text or figure legends with P-values obtained, a two-sided Student's t-test was employed. The data supporting the findings of this study are available within the paper and its supplementary information files. All other data that support the findings of this study are available from the corresponding author upon reasonable request.
E. coli toxicity assay
To investigate the toxic effects of different chemicals to cell growth, E. coli strains were used to inoculate LC media in 24 well-plates and incubated in a Tecan microplate reader at 37°C, 432 rpm, over 24h. A range of different concentrations (0-30 mM) of chemicals were added to E. coli culture with the same starting OD600 at the beginning of the cultivation. Where a fully soluble stock solution was not possible to prepare, each compound was spiked directly to each well. The specific growth rates were calculated for each treatment.
Results and Discussion
Comparing the toxicity of 1-octanol and octyl acetate
In previous studies (15, 16), we observed that 1-octanol was toxic, resulting in reduced growth and also genetic instability in cyanobacteria (16). Alcohols are known to compromise the integrity of cell membranes (22), thereby causing cellular toxicity that is most commonly observed as a growth defect. With 1-octanol having only a single hydroxyl-group as a functional group, we initially considered two different types of derivatization: (1) O- glucosylation and (2) esterification. For ester synthesis, the most valuable organic acid moiety (that could be recovered following hydrolysis of the bioderivative) would economically make most sense. However, although acetate is not a particularly valuable end-product, the biosynthesis of acetate esters is easiest to implement and therefore served as the starting point. Before commencing with metabolic engineering, we evaluated the tolerance of the E. coli host strain to 1-octanol and its two proposed derivatives (octyl glucoside and octyl acetate) in 96-well microtiter plates. The cells were unable to grow when the concentration of 1-octanol in the liquid culture was above 0.75 mM (Figure 2 and Figure 6A). In contrast, growth was observed at all tested concentrations of octyl acetate (0-50 mM) (Figure 2 and Figure 6B) and above (Figure 9), and up to 2.5 mM for octyl glucoside (Figure 2 and Figure 6C). In previous studies, alcohols displayed varying (sometimes more, sometimes less) toxicity relative to its corresponding esters, depending on the specific product and derivative in question (12). For example, butyl acetate was found to be more toxic than n-butanol. To further complicate matters, the apparent toxicity of externally applied substances is likely influenced by environmental factors including varying types of solvent overlay, and it remains unknown what the toxicity of internally accumulated 1-octanol, octanal and octanoic acid is. Nevertheless, at least in the case of externally applied chemicals, both the corresponding acetyl ester and glucoside were less toxic than 1-octanol under the tested conditions. Based on this experiment, both derivatives were pursued in vivo as a model system although clearly a 'one size fits all' generalization is not possible.
Selection of Alcohol acyltransferase (AAT) for octyl acetate biosynthesis
Octyl acetate is naturally found in wild strawberry ( Fragaria vesca) (23). For use as a food flavor additive, it is also synthesized by a direct esterification reaction between acetic acid and octyl alcohol, catalyzed by either acids, ion exchange resins or ionic liquids (24). Until now, we have found no reports of microbial in vivo production of octyl acetate using a renewable substrate.
The TPC3 pathway was extended with an alcohol acetyltransferase (AAT) under the assumption that native acetyl-CoA was not limiting (Figure 4A). Three AAT enzymes (CAT (25), SAAT (19), and ATF1 (26)) were selected based on the literature. CAT has previously been used in E. coli for ester biosynthesis (27), whilst both SAAT and ATF1 have been reported to utilize 1-octanol as a substrate (19, 27, 28). Prior to in vivo production of 1-octanol and octyl acetate, spiked experiment of 1-octanol and octyl acetate (1 mM) was carried out in M9 minimal media to investigate whether 1-octanol or octyl acetate still remained in aqueous phase after 24h-incubation with solvent overlay. The result suggested that either of the compounds was detected in the aqueous phase (Figure 13). Strains with all genetic combinations and controls (Table 1) were prepared and cultivated in M9 minimal media with 2% (w/v) glucose and 10% (v/v) hexadecane overlay. Analysis of the solvent overlay indicated that all the tested AAT enzymes were able to convert 1-octanol and acetyl-CoA into octyl acetate. However, SAAT and ATF1 were more effective than CAT (Figure 4C, P-values = 0.003 and 0.0002, respectively). The highest octyl acetate titer (0.54 ± 0.01 mM (93.82 mg/L) 48 h after inoculation) and yield (12.54 mmol/mol glucose) was found in cultures of the strain expressing ATF1 (Strain No. 5, Table 1). The introduction of AAT did not result in marked changes in growth (Figure 4E) and esterification of 1-octanol only resulted in a small positive impact on the final product titer. This may be due to at least two possible explanations: (i) the hexadecane solvent overlay reduced the toxicity of the products by in situ product removal (27), (ii) the 1-octanol producing strain only reached a final titer of 0.32 ± 0.03 mM, which is lower than the concentration that affected the growth of E. coli as shown in Figure 2. In order to more comprehensively evaluate the impact of bioderivatization, we hypothesized that it was important to exceed the titer at which the underivatized product affected the health of the host, at least in the absence of solvent overlay. Hence, the next task became to improve the flux through the 1-octanol pathway.
Identification of limiting factor(s) for 1-octanol pathway flux
The titers of 1-octanol and octyl acetate in the previous experiment were low indicating that one or more reactions in the introduced pathway prevented efficient pathway flux. In cyanobacteria, the availability of octanoic acid was found to limit the biosynthesis of 1-octanol (16). To understand if the supply of octanoic acid was also limiting in E. coli, octanoic acid was added externally to the strains expressing the 1st generation 1-octanol pathway with co expression of SAAT (Strain No. 4, Table 1) or without co-expression of SAAT (Strain No. 2, Table 1). Substantially more of each product was observed in cultures to which octanoic acid was added, confirming that the supply of acid indeed was limiting the 1-octanol pathway (Figure 5A). As all of the 1-octanol was converted into the corresponding ester in SAAT expressing strains, this experiment also confirmed that both acetyl-CoA and the AAT activity were not limiting (Figure 5B).
A similar substrate feeding experiment was also carried out in the absence of the hexadecane solvent overlay. This, however, prevented the quantification of the products since both 1- octanol and octyl acetate are volatile. Hence, the growth and glucose consumption were instead evaluated as indicators of cellular and metabolic health. The experiment indicated that feeding octanoic acid alone somewhat reduced the growth of E. coli even without conversion to 1-octanol and octyl acetate (compare the negative control in Figure 5C and 5D at 48 h, P- value = 0.027). This is in line with previous reports by Wilbanks and Trinh (12). The 1-octanol- producing strain (Strain No. 2, Tes3-Sfp-CAR) showed noticeably lower cell density (Figure 5C, 5D, 5G, and 5H) and glucose consumption (Figure 5E and 5F), an effect that was largely alleviated by esterification (Strain No. 4, Tes3-Sfp-CAR + SAAT), thereby providing early insight towards answering the main hypothesis of the work. The differences between the alcohol and ester-forming strains were reduced in the absence of externally added octanoic acid (Figure 5C, 5D, 5G, and 5H) supporting the idea that further pathway optimization was essential in order to evaluate the effect of bioderivatization under conditions that were more likely to be relevant for application.
The first generation 1-octanol pathway was also evaluated in two different E. coli strain backgrounds; the E. coli B strain C43 (DE3) and K-12 strain BW25113. As the latter strain (0.74 mM, 96.47 mg/L, 27 mmol/mol glucose) displayed greater alcohol yield (P-value = 0.042) than the former (0.35 mM, 45.44 mg/L, 10.15 mmol/mol glucose) (Figure 14), the E. coli BW25113 strain background was used in the following work.
Thioesterase selection for 1-octanol production and growth and metabolism in response to bioderivatization
Since the exogenous addition of octanoic acid greatly enhanced octyl acetate productivity (Figure 5), this suggested that the thioesterase was the primary limitation for the pathway. In unpublished work with cyanobacteria in our laboratory, a number of different C8-preferring thioesterases were evaluated and found to improve 1-octanol yield (I. Yunus, manuscript in preparation). Four of these new thioesterases were therefore also evaluated in E. coli at varying levels of protein expression. The new thioesterases were based on Cuphea palustris (CpFatBI) (29) and Cuphea avigera pulcherrima (CaFatB3) (30) with varying mutations and truncations (Figure 7), two of which were designed according to recent work by Lozada et al. (31). Surprisingly, all new thioesterases showed an improvement in 1-octanol production compared to Tes3 (15) but the performance varied greatly in response to changes in the inducer concentration (Figure 8). ‘CpFatB1-4 was the best performer with the highest concentration of 1-octanol (4.29 mM, 558.2 mg/L, 59.48 mmol/mol glucose) after 48 h of inoculation with 0.05 mM IPTG, a concentration of 1-octanol approximately 8 times higher than that obtained with Tes3 under the same condition (Figure 8B, P-values = 0.03 when comparing ‘CpFatB1-4 with Tes3 and 0.044 when comparing ‘CpFatB1-4 with ‘CaFatB3-5, all at 48 h). ‘CaFatB3-5 was not as effective but reached the maximum titer of 1-octanol (2.90 mM, 377.37 mg/L, 44.20 mmol/mol glucose) at the higher IPTG concentration, 0.2 mM (Figure 8C). With these improvements, the internally produced 1-octanol now exceeded the titer (0.75 mM) at which the underivatized product affected the health of the strain in the absence of solvent overlay.
Lozada et al (31) reported that ‘CpFatB1-4 was most effective when the expression level was low as the strain expressing it showed a growth defect at higher expression levels. The same was also observed in our study (Figure 15A). Since the same promoter was also used in the second AAT-encoding plasmid, we speculated that an imbalance in pathway enzyme activities may compromise the final outcome. The second best thioesterase (‘CaFatB3-5) was therefore also used in further production experiments given that growth of the ‘CaFatB3-5 strain was not greatly influenced by the IPTG concentration (Figure 15B).
Following the optimization of the 1-octanol pathway the effect of esterification on the health of the biocatalytic host was evaluated. Strains harboring ‘CpFatB1-4, with or without ATF1, were induced at the IPTG concentration found optimal for ‘CpFatB1-4 (0.05 mM) and grown in the absence of a hexadecane overlay. The presence of ATF1 enhanced both growth and glucose consumption (Figure 8E and 8F), as was observed also in the octanoic acid feeding experiments previously (Figure 5) with P-values of 0.006 and 0.003 when comparing the growth and glucose consumption at 48 h, respectively.
The effect of bioderivatization on production with enhanced pathway flux
Would bioderivatization also influence C8 productivity now that pathway flux was up to 8 times greater? To answer this, the effect of ATF1 in the improved (high flux) ‘CpFatB1-4 and ‘CaFatB3-5 strains was evaluated in the presence of a hexadecane solvent overlay at three different IPTG levels. The results were complex depending on the conditions, but a number of interesting observations were made. Firstly, even with the higher pathway flux, cellular growth and glucose consumption was now not influenced by ATF1 except for at 72 h at some of the IPTG levels (Figure 10 and 11). Microtiter plate well growth at 0.2 mM IPTG also indicate clear but modest growth differences in the first 24 h (Figure 16). In contrast, no difference was observed in response to esterification for any strain or IPTG combination, as was also found with the low flux strains (Figure 4), despite the fact that a substantial impact on growth was observed in the absence of solvent overlay (Figure 8E and F). Hence, the solvent overlay mitigated the 'health' defect caused by the pathway and/or its product, at least when compared to the same strain cultured in the absence of solvent overlay, i.e. 0.05 mM IPTG with the ‘CpFatB1-4 strain. At the lowest protein expression inducer level (0.05 mM IPTG), esterification had no impact on yield (Figure 12) or titer (Figure 19) for both strains. In contrast, at the higher IPTG levels, almost all IPTG/strain/time combinations showed both improved titer and yield when ATF1 was co-expressed. Interestingly, this means that all combinations of IPTG dosage and strain sampled at 24 and 48 hours, except for one (‘CpFatB3-5, 0.2 mM, 48 h), had increased C8 productivity even though there was no impact on growth or glucose consumption. In other words, the effect of esterification was under these conditions independent of any effect on cellular health, hence the effect of esterification on product toxicity was not the cause for improved productivity. Most likely, this is explained by esterification enhancing product removal, either cellular efflux and/or transfer from the water to the solvent overlay. The lack of an effect of ATF1 on C8 productivity at the low (0.05 mM) IPTG induction level may be explained by an imbalance between pathway catalysts complicated by the contrasting impact of IPTG on the two different thioesterases. This difference is illustrated by the change in the ratio between product titers between strains with and without ATF1 in response to the concentration of IPTG used for induction (Figures 21 and 20).
Transfer of the bioderivatization concept into a different organism
Recently, we reported the introduction of 1-octanol biosynthesis also into cyanobacteria (16). The use of solvent overlay was found to be important as the cyanobacterium ( Synechocystis sp. PCC 6803) was even more sensitive to 1-octanol than E. coli. Given the positive impact of esterification on C8 biosynthesis in E. coli we were therefore interested to see if similar benefits from bioderivatization could be observed also in Synechocystis sp. PCC 6803. External addition of 1-octanol and octyl acetate indicated reduced sensitivity to the ester (Figure 3), though Synechocystis sp. PCC 6803 was clearly more sensitive to octyl acetate than E. coli.
Heterologous expression of Strawberry AAT (SAAT) in combination with the ‘CpFatB1-4 thioesterase, Sfp and CAR enabled complete conversion of 1-octanol into octyl acetate also in this species. Strains expressing ATF1 could not be obtained despite repeated transformation attempts. Cultivation of the two strains in the absence of a solvent overlay resulted in marked growth differences by day 10 (Figure 17A and 17B), but by day 20 the octyl acetate strain had caught up and both cultures were vibrant green. The day 20 culture was then used as a pre-culture for a new culture with fresh media that contained hexadecane solvent overlay.
In the presence of solvent overlay, the addition of SAAT resulted in enhanced growth (Figure 17C and 17E), C8 product titer (Figure 17D), and yield (Figure 17F) after 16 days of cultivation and induction of protein expression on day 2. Surprisingly, none of the 1-octanol producing strains lost the ability to accumulate its product in this study, in contrast to what we observed earlier (16). The initial no induction pre-cultures of the same strains were also used to inoculate cultures that immediately were provided a solvent overlay and then induced for protein expression and cultured for 10 days. Such cultures accumulated 1.6 ± 0.4 (1-octanol) and 2.4 ± 0.5 mM (octyl acetate) of each respective product.
The effect of conjugate type - hydrophilic instead of hydrophobic
Also other conjugation types are possible, for example glycosylation. This requires changes in the metabolic engineering and has implications for both cellular efflux and product separation. The simplest glycosylation to implement is O-glucosylation, thereby resulting in the formation of octyl glucoside, a non-ionic alkyl glucoside that is used as a surfactant (32). Despite its detergent nature, octyl glucoside was also shown to be less toxic than 1-octanol (Figure 2). Glycosides are also highly hydrophilic which will require a different choice of downstream processing for separation, compared to esters. We are not aware of any prior reports of biotechnological production of octyl glucoside with a microbial host.
The ‘CaFatB3-5 thioesterase 1-octanol pathway was extended by overexpressing a glycosyltransferase (GT) (Figure 4A). Five candidates were selected based on their reported activity towards longer-chain alcohols (33, 34). By combining a glycosyltransferase from Medicago iruncaiula (MtH2) (35) with the 1-octanol pathway, 0.73 mM (214mg/L) octyl glucoside was produced after 48 h (Figure 18B). MtH2 was therefore selected for further investigations. The octyl glucoside titers were low if the amount of available 1-octanol was taken into consideration, in comparison with the octyl acetate pathway (Figure 4A and 19). A possible explanation may be inadequate GT activity, as reported previously (36).
Insufficient glucosylation may be due to a limitation in the supply of substrates, i.e. UDP- glucose or inhibition by UDP (36), one of the products of the glucosylation reaction. Another possible limiting factor is UDP-glucose availability. Naturally, UDP-glucose is involved in bacterial cell wall synthesis and is produced at a basal level in E. coli (37). Given that the UDP- glucose pool is relatively low, regeneration is important in order to archive a high production of glucosides. Sucrose synthase (SUS) is an enzyme catalyzing a reversible reaction that converts sucrose and UDP to UDP-glucose which has been shown to enhance glucoside production (38). In this work, sucrose synthase from Arabidopsis thaliana, namely AtSUSI was co-expressed with GT and 1-octanol pathway (Figure 4A) to improve octyl glucoside production. It was shown that the overexpression of AtSUSI improved the production of octyl glucoside (Figure 18C) and highly excessive amount of sucrose (100 mM) supplied resulted in more octyl glucoside produced (Figure 18D). Notably, SUS will not only help maintain the supply of UDP-glucose, but it is expected that it also will lower the intracellular concentration of UDP, thereby lowering any inhibitory effect that it may have on MtH2. Without further investigation, it is difficult to fully elucidate the cause of inefficiency in the octyl glucoside pathway, however, sufficient production was achieved allowing the question of whether glucosylation enhances net 1-octanol production or not to be answered.
Even though octyl glucoside is secreted and soluble in the aqueous culture media (Figure 22), hexadecane was used as an overlay of the cultures in order to quantify any 1-octanol that was excreted too quickly and thereby evaded glucosylation. Surprisingly, in the presence of solvent overlay, more octyl glucoside was produced than in its absence (P-value of 0.012 at 48 h, compare Figure 18C and Figure 23). As the solvent overlay had no impact on the growth of these strains, this could not be explained by any differential toxicity. An alternative possibility is that the solvent overlay captures 1-octanol and enables it to be reutilised by the cells and converted into glucosides in the stationary phase. The yield of 1-octanol was improved, with the P-value of 0.010 and 0.016 at 24 h and 48 h, respectively (Figure 18F and 18G), in strains combining the 1-octanol pathway with both MtH2 and AtSUSI, compared with the strains carrying only 1-octanol pathway. Similarly, the product titers were also improved by bioderivatization (Figure 23, P-value of 0.003 and 0.006 at 24 h and 48 h, respectively).
The combined analysis suggests that bioderivatization has the potential to enhance at least some biotechnological production systems where product toxicity places excessive limits on bio-based production of valuable chemicals. This outcome provides support for the natural evolution of similar strategies, as seen in most plants and fungi, but does not rule out alternative mechanisms of selective pressure. In future studies, it would be interesting to evaluate also how this approach may influence product: process separation as illustrated in Figure 1. For example, esters are attractive in this respect as they have lower water solubility than their corresponding alcohols which is likely to reduce the energetic cost of the product separation process from the aqueous media (39). The post-biology separation strategy and capability for cellular excretion will also influence whether to opt for a more hydrophobic or hydrophilic derivative. At least in the case of 1-octanol, the host cells evaluated in this work were able to excrete both conjugate types, but that may not apply in the case of other target chemicals.
Conclusions
Two different strategies for bioderivatization were implemented and the effect on bio production of the toxic chemical 1-octanol was thereafter systematically evaluated for the first time. In general, the conversion of 1-octanol into octyl acetate resulted in most cases in enhanced product titer and yield as well as enhanced growth and glucose consumption by the production host. A similar effect was observed with both of the tested biotechnological hosts. The impact of esterifcation on bioproduction was influenced by the presence or absence of solvent overlay, but the positive effect of conjugation could be observed under both environmental conditions. We assumed that any improvements in productivity that were accompanied by improvements in cellular health were inter-related. Interestingly, closer inspection of the data suggested independent effects of bioderivatization on either (1) cellular health (i.e. growth and glucose consumption) with or without an effect on productivity or (2) productivity without any effect on cellular health. The latter effect is likely related to product removal, but further experiments are needed in order to understand the exact cause. Although octyl glucoside has a completely different water solubility compared to the ester, also this form of bioderivatization resulted in enhanced bioproduction, supporting this concept as a generic and applicable strategy. The results presented in this article support the idea that engineered bioderivatization that mimics the evolved metabolism of many specialized metabolite accumulating species may offer benefits for biotechnology, even with entirely synthetic metabolism.
4. SUMMARY
Bio-based production technologies may complement or even replace petroleum-based production of chemicals. The development of such biotechnological production systems faces a number of technical challenges, incl. toxicity to the host caused by the chemical it has produced. Many plants and microorganisms are naturally capable of biosynthesizing toxic molecules but they often convert them into derivatives before storage or excretion. Inspired by this principle, we propose here a bioderivatization strategy for biotechnological chemicals production, defined as purposeful biochemical derivatization of intended target molecules. As a proof-of-principle, we evaluate the effect of hydrophobic (e.g. esterification) or hydrophilic (e.g. glycosylation) bioderivatization strategies on the biosynthesis of a relatively toxic chemical, 1-octanol, in two different microbial hosts. In order to evaluate the effect of bioderivatization we first optimized the 1-octanol pathway in order to reach product titers that exceeded the concentration at which the host displayed symptoms of toxicity. Optimal choice of genes and induction strength of thioesterases enhanced 1-octanol biosynthesis by a factor of 8 relative to the first-generation pathway reported previously. Solvent overlay was used to capture volatile products, but the presence of the overlay masked product toxicity. Hence, the experiments were also carried out in the absence of solvent overlay to more comprehensively understand the effect of bioderivatization. Regardless of whether solvent overlay was used or not, most strains with bioderivatization reached higher molar product titer and product yield, as well as improved cellular growth and glucose consumption than without it. The positive effect on bioproduction was observed with both the hydrophobic and hydrophilic strategies. The most productive heterotrophic strain displayed approximately two-fold greater productivity as a result of bioderivatization, reaching a titer of 4.34 mM (747 mg/L) after 72 h of batch cultivation. When the improved 1-octanol pathway was implemented in the cyanobacterium Synechocystis sp. PCC 6803, with or without bioderivatization, a similar pattern was observed as also seen in the heterotrophic system. With some metabolic engineering designs, bioderivatization affected (1) cellular health (i.e. growth and glucose consumption), independently of an effect on productivity, whilst in other designs (2) productivity was influenced without any apparent effect on cellular health. Overall, under most conditions, bioderivatization was found to benefit production of the toxic model bioproduct selected in this study and could be considered also for other products that are toxic to the production host.
5. Novel thioesterases
5. 1 Materials and methods
Each thioesterase-encoding gene was incorporated into an operon containing also CAR and Sfp in a pET-plasmid under the control of the Plac promoter, resulting in a series of pET-Plac- Tes-Sfp-CAR-KanR plasmids. When co-expressed together with pCDF-Plac-Yjgb-SpecR, this will result in the biosynthesis and excretion of fatty alcohols into the media.
Both plasmids were used to transform Escerichia coli strain JW1994-1. Pre-cultures were grown overnight in 5 ml_ LB, in a 50 mL falcon tube, supplemented with the appropriate antibiotics, at 37°C and 180 rpm. Overnight cultures were washed with fresh M9 and then used to inoculate 25 mL M9 with appropriate antibiotics, 2% (v/v) glucose and 10% (v/v) isopropyl myristate overlay in a 100 mL Erlenmeyer flask, at a starting OD600 of 0.1. Cultures were then incubated at 37°C, 180 rpm for four hours before induction with 0.5 mM IPTG. Cultures were then incubated at 30°C, 150 rpm. At 24 and 48 hours, cultures were sampled for growth (OD600) and the overlay sampled for GCMS analysis.
The sample was analysed using GC-MS Agilent Technologies G1530A series Gas Chromatograph (GC) equipped with DB-WAXetr column coupled with 5973 Mass Selective Detector. The oven temperature was initially held at 100°C for 50s, then ramped at 30°C/min until 250°C, where it was held for 1min.
5.2 Results
Variant thioesterase sequences were created and assayed for fatty acid derived products in Escherichia coli in some cases in combination with the CAR enzyme (hence the products were in this case alcohols) as described above. The amino acid encoded by the wild type CvFatBI gene (Jing et al (2011) BMC Biochemistry 12:44) produced no products. Truncated variant sequences surprisingly generated measurable alcohols. Variant T7 was found to be particularly good for producing octanol. The variant T4 was also of interest as it showed good C10 productivity, relative to C8 (Figure 25).
The second gene of interest that was investigated is ChoFatB2 (Dehesh et al (1996) Plant Journal 9(2), 167-172). Truncated variants of this gene were also created and evaluated as described above. The truncated variants showed improved productivity. ChoFatB2.2 was found to be particularly good (Figure 25).
6. Conversion of 1 -octanol into octyl glucoside enhances growth and productivity when photosynthetic bacteria is used as a biotechnology host
The entire octyl glucoside biosynthesis pathway was previously implemented in E. coli (Sattayawat et al., 2020), and demonstrated herein in figures 22-24. Here, we asked whether it could also function in Synechocystis sp. PCC 6803 with similar outcomes as observed in E. coli. Employing the same methods as used previously for construction of strains, cultivation and analysis of products, the octyl glucoside pathway was implemented in Synechocystis and cultured in the presence of solvent overlay in order to monitor both 1 -octanol (quantified in the solvent overlay) and octyl glucoside (quantified in the aqueous phase). We found that both growth of the biocatalyst (Fig. 26A), and the chemicals productivity (i.e. titer divided by time and by volume; Fig. 26B) was greater with glucosylation (i.e. bioderivatization (BD)), than without, both contributing to an overall higher product titer (Fig. 26B).
Subsequently, two strains with another C8-specific thioesterase ('CpFatB1-4; with or without the MtH2 glucosyltransferase) were cultured in the absence of solvent overlay. The bioderivatization afforded by MtH2 had a striking effect on the growth of the strains (Fig. 26C, left). Without glucosylation, strain growth suffered badly. The effect is most strikingly represented by the substantial difference in the density of the cyanobacteria cultures (Fig. 26C, right). The dark colour gives an indication of how much more richly populated the culture is with bioderivatization is (i.e. +Mth2).
In summary, glucosylation of 1-octanol had a major impact on the health of the cyanobacteria and also improved metabolic flux, resulting in enhanced productivity, similar to what was already observed with glucosylation in E. coli and with esterification in both E. coli and Synechocystis sp. PCC 6803.
The construction of strains was done according to 'Construction of cyanobacterial strains for 1-octanol and octyl acetate production' hereinabove, except by replacing the AAT-encoding gene with the Mth2-encoding gene as described in 'Construction of E. coli strains for octyl glucoside production' hereinabove. The evaluation of the strains combined cultivation methods from 'Production of 1-octanol and octyl acetate in engineered cyanobacteria' hereinabove and analytical methods from 'Quantification and analysis of 1-octanol, octyl acetate, octyl glucoside, and other metabolites' hereinabove.
7. Could the bioderivatization strategy also work for other compounds?
In order to understand how broad the utility of the bioderivatization concept is, we evaluated the toxicity of a range of compounds different to 1-octanol, and their corresponding acetate esters. The results are summarized in Figure 27. The associated methodology is also summarized hereinabove in the methods section.
Briefly, to evaluate the toxicity of the selected alcohols, different concentrations of alcohol or its corresponding acetyl (and in one case butyl) esters were added into the E. coli culture at a range of different concentrations (0-30 mM). For any molecule that was too hydrophobic for addition to water, it was instead added directly to the well before the media was added. After 24h growth the cell density was measured and used as an indicator of cellular health.
For all tested alcohols, the corresponding acetyl and butyryl esters were less toxic suggesting that there is potential to utilise bioderivatization concept also for these types of molecules.
Bioderivatization of other chemicals, from terpenoids to diverse aromatic molecules, also affected the toxicity to bacteria. In all cases tested, bioderivatization reduced the toxicity of the compounds. As none of the derivatives were more toxic than the precursor alcohols, the bioderivatization strategy described herein should be possible to implement also for bioproduction of a wider range of chemicals.
The evaluation of product toxicity was according to 'Evaluating the effect of 1-octanol, octyl acetate and octyl glucoside on growth' herinabove, except the molecules that were tested were replaced i.e. instead of 1-octanol and octyl acetate, the new alcohols (menthol, linalool, eugenol and cinnamyl alcohol) and corresponding esters (menthol acetate, linalyl acetate, linalyl butyrate, eugenyl acetate and cinnamyl acetate) were used, all in the range of 0-30 mM except for cinnamyl alcohol and cinnamyl acetate which were only tested in the range of 0-5 mM.
8. Extension of Bioderivatization strategy to a different chain-length product
We also evaluated the effect of bioderivatization (BD) on in vivo bioproduction of another molecule; n-butanol. The transformation from 1-octanol to n-butanol biosynthesis requires only a single change to the pathway shown in Fig. 4A. The C-8 specific thioesterase is replaced with a C-4 specific thioesterase, as earlier reported by us (Pasztor et al., 2014) (SEQ ID NO:102). With this system, we evaluated 16 different AAT-encoding genes in vivo: Atf1 ( Saccharomyces cerevisiae Alcohol O-acetyltransferase 1), CAT ( Escherichia coli Chloramphenicol acetyltransferase), SAAT ( Fragaria ananassa cv. Elesanta Alcohol acyltransferase), VAAT (, Fragaria vesca, Alcohol acyltransferase), VpAAH ( Vasconcellea pubescens, Alcohol acyltransferase 1), AtHPFT ( Arabidopsis thaliana Omega-hydroxypalmitate O-feruloyl transferase), CcVs ( Citrus Clementina Vinorine synthase), CsVs ( Citrus sinensis Vinorine synthase-like), LaAAH ( Lavandula angustifolia Putative alcohol acyltransferase 1), LaAAt2 (, Lavandula angustifolia Putative alcohol acyltransferase 2), LaAT1 ( Lavandula angustifolia Rosmarinic acid synthase), UAAT4 (Lavandula x intermedia clone AAT-4 alcohol acetyltransferase), NtBBT ( Nicotiana tabacum Benzyl alcohol O-benzoyltransferase), NtSHCT ( Nicotiana tabacum Shikimate O-hydroxycinnamoyltransferase), RsVs ( Rauvolfia serpentina Vinorine synthase), SsMMT ( Salvia splendens Malonyl-coenzyme:anthocyanin 5- 0-glucoside-6"'-0-malonyltransferase) (SEQ ID NO: 1 , 2, 3, 82, and 103-114), but most did not work (Fig.28A). It is important to keep in mind that at these concentration of n-butanol (<1 g/L) there is no toxicity to the biocatalyst. E. coli has been reported to show only weak toxicity to n-butanol at a concentration approx. 10x that which were obtained here (Fletcher et al., 2016), i.e. 7g/L. Four AAT-encoding genes displayed activity resulting in the biosynthesis of butyl acetate, Atf1 (SEQ ID NO: 3), SAAT (SEQ ID NO:2) , VAAT (SEQ ID NO: 103) and UAAT4 (SEQ ID NO:82).
Interestingly, the growth of the different strains was unaffected (Fig. 28C), whilst the UAAT4 (SEQ ID NO:82) containing strain had enhanced molar flux through the pathway (Fig.28B), supporting the idea that esterification did not influence strain toxicity and that the positive enhancement of pathway flux was independent of toxicity.
The construction of strains was described according to 'Construction of E. coli strains for 1- octanol and octyl acetate production' hereinabove except the AAT-encoding genes (Atf1 , CAT, SAAT) were replaced with the alternative AATs and the C-8 specific thioesterase was replaced with that described by Pasztor et al. (2014); GenBank ID CAH09236, originating from Bacteroides fragilis (SEQ ID NO: 102) . The evaluation of the strains combined cultivation methods from 'Production of 1-octanol and octyl acetate in engineered cyanobacteria' hereinabove and analytical methods from 'Quantification and analysis of 1-octanol, octyl acetate, octyl glucoside, and other metabolites' hereinabove, i.e. using 10% hexadecane as the solvent overlay to capture butyl acetate, whilst n-butanol was measured in the aqueous fraction by HPLC using the same method as for other fermentation products.
9. Refinement of thioesterases to enable a C10-preferring pathway In order to extend the evaluation of bioderivatization to a broader range of chemicals, we wanted to replace the C8 octyl-ACP thioesterase, which determines the chain-length of all products in the 1-octanol pathway and its corresponding derivatives, with one that prefers C10 decyl-ACP. In the literature, there have been no reports, as far as we are aware, of a clearly C10-preferring acyl-ACP thioesterase. Below we report the first discovery of a C10-preferring thioesterase. There are two steps to this achievement:
(1) Truncation of plant acyl-ACP thioesterase
(2) Choice of species for biocatalyst
Firstly, we selected a thioesterase from Cuphea viscosissima (CvFatB1)(Jing et al., 2011) (SEQ ID NO: 15) and systematically evaluated 21 different N-terminal truncations thereof (SEQ ID NO:17-23 and 86-99). A selection of these (T3-T8) shown in SEQ ID NO:19-23, were studied further in E. coli (Fig. 29A). The CvT3 and CvT4 truncations almost, but not quite, displayed a preference for C10 - in fact, the preference was still for C8, at a molar level. Subsequently, we also tested these truncated variants in a cyanobacteria host (Fig. 29B).
Three of these displayed a clear preference for C10 chain length acyl-ACP, T4-T6. Hence, by selecting optimal truncations (T5) and strain choice ( Synechocystis sp. PCC 6803), C10 products can be made to dominate the product profile.
The construction of strains was described according to 'Construction of E. coli strains for 1- octanol and octyl acetate production' as above except the thioesterase was Cuphea viscosissima (CvFatB1)(Jing et al., 2011) and different N-terminal truncations were prepared by shifting the location of the primer-recognizing sequence, against the CvFatBI encoding gene, prior to BASIC-assembly of the synthetic operons. The same constructs were then also used to prepare cyanobacteria strains according to 'Construction of cyanobacterial strains for 1-octanol and octyl acetate production' hereinabove. The evaluation of the strains combined cultivation methods from 'Production of 1-octanol and octyl acetate in engineered cyanobacteria' hereinabove, and analytical methods from 'Quantification and analysis of 1- octanol, octyl acetate, octyl glucoside, and other metabolites' hereinabove.
10. Enhancement of pathways through over-expression of acyl-carrier protein
We further over-expressed the acyl-carrier protein (ACP) in order to modify the chain-length specificity of the introduced metabolic pathway. Disappointingly, the chain-length specificity was not modified, however, unexpectedly, it resulted in an overall enhancement of pathway flux. This discovery presents an important addition to our ability to enhance the overall pathway flux. We would expect to be able to combine bioderivatization, optimal choice of thioesterase and strain, and ACP over-expression to deliver an optimal production process. The construction of strains was described according to 'Construction of E. coli strains for 1- octanol and octyl acetate production' as above, except an additional genetic part, encoding one out of three different ACP-encoding genes, was added to the operon: EcACP from Escherichia coli (SEQ ID NO: 83), 6803ACP from Synechocystis Sp. PCC 6803 (SEQ ID NO:84), and CIACP2 from Cuphea lanceolata (SEQ ID NO:85). Hence, the negative control strain (NA) combined pET-Plac-Tes-KanR + pCDF-Sfp-CAR, whilst the ACP strains combined pET-Plac- Tes-KanR + pCDF-PrhaBAD-ACP-Sfp-CAR. In all strains used in this experiment Tes = CvFatb1-T5. The evaluation of the strains combined cultivation methods from Production of 1-octanol, octyl acetate and octyl glucoside in engineered E. coli' hereinabove and analytical methods from 'Quantification and analysis of 1-octanol, octyl acetate, octyl glucoside, and other metabolites' hereinabove.
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Rottig, A., & Steinbuchela, A. (2013). Acyltransferases in Bacteria. Microbiology and Molecular Biology Reviews, 77(2), p. 277-321.
Shao, H., He, X., Achnine, L., Blount, J. W., Dixon, R. A., & Wang, X. (2005). Crystal structures of a multifunctional triterpene/flavonoid glycosyltransferase from Medicago truncatula. Plant Cell, 17(11), 3141-3154. doi: 10.1105/tpc.105.035055
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SEQUENCES
SEQ ID NO: 1 Amino acid sequence of CAT
MSSGMEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFLKTVKKNKHKFYP
AFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSSLWSEYHDDFRQFLHI
YSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANMDNFFAPVFTMGKYYTQ
GDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA
SEQ ID NO: 2 Amino acid sequence of SAAT
MGSSEKI EVSINSKHTIKPSTSSTPLQPYKLTLLDQLTPPAYVPIVFFYPITDHDFNLPQTLADL
RQALSETLTLYYPLSGRVKNNLYIDDFEEGVPYLEARVNCDMTDFLRLRKIECLNEFVPIKPF
SMEAISDERYPLLGVQVNVFDSGIAIGVSVSHKLIDGGTADCFLKSWGAVFRGCRENIIHPSL
SEAALLFPPRDDLPEKYVDQMEALWFAGKKVATRRFVFGVKAISSIQDEAKSESVPKPSRV
HAVTGFLWKHLIAASRALTSGTTSTRLSIAAQAVNLRTRMNMETVLDNATGNLFWWAQAIL
ELSHTTPEISDLKLCDLVNLLNGSVKQCNGDYFETFKGKEGYGRMCEYLDFQRTMSSMEP
APDIYLFSSWTNFFNPLDFGWGRTSWIGVAGKI ESASCKFIILVPTQCGSGIEAWVNLEEEK
MAMLEQDPHFLALASPKTLI
SEQ ID NO: 3 Amino acid sequence of ATF1
MNEIDEKNQAPVQQECLKEMIQNGHARRMGSVEDLYVALNRQNLYRNFCTYGELSDYCTR
DQLTLALREICLKNPTLLHIVLPTRWPNHENYYRSSEYYSRPHPVHDYISVLQELKLSGVVLN
EQPEYSAVM KQI LEEFKNSKGSYTAKI FKLTTTLTI PYFGPTGPSWRLICLPEEHTEKWKKFIF
VSNHCMSDGRSSIHFFHDLRDELNNIKTPPKKLDYIFKYEEDYQLLRKLPEPIEKVIDFRPPYL
FIPKSLLSGFIYNHLRFSSKGVCMRMDDVEKTDDVVTEIINISPTEFQAIKANIKSNIQGKCTIT
PFLH VCWFVSLH KWGKFFKPLN FEWLTDI FI PADCRSQLPDDDEM RQMYRYGAN VGFI DFT
PWISEFDMNDNKENFWPLIEHYHEVISEALRNKKHLHGLGFNIQGFVQKYVNIDKVMCDRAI
GKRRGGTLLSNVGLFNQLEEPDAKYSICDLAFGQFQGSWHQAFSLGVCSTNVKGMNIVVA
STKNVVGSQESLEELCSIYKALLLGP
SEQ ID NO: 4 Amino acid sequence of AdGT4
MGSAGMPEKPHAVCLPYPAQGHITPMLKLAKLLHSKGFHVTFVNTEFNHKRLLKSRGPDSL
TGLSSFRFETIPDGLPESDLDATQFIPSLCESTRKNCLGPFRQLLGKLNNTVSSGVPPVSCV
VSDGVMSFSLDAAEELGIPQVLFWTTSVCGFMAYVHYRNLIEKGYTPLKDVSYVTNGYLDT
VIDWI PGMEGIRLKDLPSFLRTTDPNDIMLDFVLSETKNTHRSSAIIFNTFDKLEHQVLEPLAS
MFPPIYTIGPLNLLMNQIKEESLKMIGSNLWKEEPMCIEWLNSKEPKSVVYVNFGSITVMTPN
QLVEFAWGLANSNQSFLWIIRPDLVVGESAVLPPEFVAVTKERGMLASWAPQEEVLAHSSV
GGFLTHCGWNSTLESISSGVAVVCWPFFAEQQTNCWYCCGELGIGMEIDSDVKREEVERL
VRELMVGEKGKEMKERAMGWKRLAEEATQSSSGSSFLNLDKLVHQVLLSPRP SEQ ID NO: 5 Amino acid sequence of VvGT 1
MSQTTTNPHVAVLAFPFSTHAAPLLAVVRRLAAAAPHAVFSFFSTSQSNASIFHDSMHTMQ
CNIKSYDVSDGVAEGYVFAGRPQEDIELFMRAAPESFRQGMVMAVAETGRPVSCLVADAFI
WFAADMAAEMGVAWLPFWTAGPNSLSTHVYTDEIREKIGVSGIQGREDELLNFIPGMSKVR
FRDLQEGIVFGNLNSLFSRMLHRMGQVLPKATAVFINSFEELDDSLTNDLKSKLKTYLNIGPF
NLITPPPVIPNTTGCLQWLKERKPTSVVYISFGTVTTPPPAELVALAEALEASRVPFIWSLRD
KARVHLPEGFLEKTRGYGMVVPWAPQAEVLAHEAVGAFVTHCGWNSLWESVAGGVPLIC
RPFYGDQRLNGRMVEDALEIGVRIEGGVFTESGLMSCFDQILSQEKGKKLRENLGALRETA
DRAVGPKGSSTENFKTLVDLVSKPKDV
SEQ ID NO: 6 Amino acid sequence of MtGT 1
MSMSDINKNSELIFIPAPGIGHLASALEFAKLLTNHDKNLYITVFCIKFPGMPFADSYIKSVLAS
QPQIQLI DLPEVEPPPQELLKSPEFYI LTFLESLI PH VKATI KTI LSN KVVGLVLDFFCVSM I DVG
NEFGIPSYLFLTSNVGFLSLMLSLKNRQIEEVFDDSDRDHQLLNIPGISNQVPSNVLPDACFN
KDGGYIAYYKLAERFRDTKGIIVNTFSDLEQSSIDALYDHDEKIPPIYAVGPLLDLKGQPNPKL
DQAQHDLILKWLDEQPDKSVVFLCFGSMGVSFGPSQIREIALGLKHSGVRFLWSNSAEKKV
FPEGFLEWMELEGKGMICGWAPQVEVLAHKAIGGFVSHCGWNSILESMWFGVPILTWPIY
AEQQLNAFRLVKEWGVGLGLRVDYRKGSDVVAAEEIEKGLKDLMDKDSIVHKKVQEMKEM
SRNAVVDGGSSLISVGKLIDDITGSN
SEQ ID NO: 7 Amino acid sequence of AtGT1
MEESKTPHVAIIPSPGMGHLIPLVEFAKRLVHLHGLTVTFVIAGEGPPSKAQRTVLDSLPSSIS
SVFLPPVDLTDLSSSTRIESRISLTVTRSNPELRKVFDSFVEGGRLPTALVVDLFGTDAFDVA
VEFHVPPYIFYPTTANVLSFFLHLPKLDETVSCEFRELTEPLMLPGCVPVAGKDFLDPAQDR
KDDAYKWLLHNTKRYKEAEGILVNTFFELEPNAIKALQEPGLDKPPVYPVGPLVNIGKQEAK
QTEESECLKWLDNQPLGSVLYVSFGSGGTLTCEQLNELALGLADSEQRFLWVIRSPSGIAN
SSYFDSHSQTDPLTFLPPGFLERTKKRVRAKWQPLNI
SEQ ID NO: 8 Amino acid sequence of MtH2
MGNFANRKPHVVMIPYPVQGHINPLFKLAKLLHLRGFHITFVNTEYNHKRLLKSRGPKAFDG
FTDFNFESIPDGLTPMEGDGDVSQDVPTLCQSVRKNFLKPYCELLTRLNHSTNVPPVTCLV
SDCCMSFTIQAAEEFELPNVLYFSSSACSLLNVMHFRSFVERGIIPFKDESYLTNGCLETKV
DWIPGLKNFRLKDIVDFIRTTNPNDIMLEFFIEVADRVNKDTTILLNTFNELESDVINALSSTIPS
IYPIGPLPSLLKQTPQIHQLDSLDSNLWKEDTECLDWLESKEPGSVVYVNFGSITVMTPEQLL
EFAWGLANCKKSFLWIIRPDLVIGGSVIFSSEFTNEIADRGLIASWCPQDKVLNHPSIGGFLT
HCGWNSTTESICAGVPMLCWPFFADQPTDCRFICNEWEIGMEIDTNVKREELAKLINEVIAG
DKGKKMKQKAMELKKKAEENTRPGGCSYMNLNKVIKDVLLKQN SEQ ID NO: 9 Amino acid sequence of CpFatBI
MRPNMLMDSFGLERVVQDGLVFRQSFSIRSYEICADRTASIETVMNHVQETSLNQCKSIGLL DDGFGRSPEMCKRDLIWVVTRMKIMVNRYPTWGDTIEVSTWLSQSGKIGMGRDWLISDCN TGEILVRATSVYAMMNQKTRRFSKLPHEVRQEFAPHFLDSPPAIEDNDGKLQKFDVKTGDSI RKGLTPGWYDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLTLEYRRECGRDSVLESVT SMDPSKVGDRFQYRHLLRLEDGADIMKGRTEWRPKNAGTNGAISTGKT
SEQ ID NO: 10 Amino acid sequence of CaFatB3
MHHHHHHKPGKFRIWPSSLSPSFKPKPIPNGGLQVKANSRAHPKANGSAVSLKSGSLNTQ EDTSSSPPPRTFLHQLPDWSRLLTAITTVFVKSKRPDMHDRKSKRPDMLMDSFGLESIVQE GLEFRQSFSIRSYEIGTDRTASIETLMNYLQETSLNHCKSTGILLDGFGRTPEMCKRDLIWVV TKM KIKVNRYPAWGDTVEINTWFSRLGKIGKGRDWLISDCNTGEILIRATSAYATMNQKTRR LCKLPYEVHQEIAPLFVDSPPVIEDNDLKLHKFEVKTGDSIHKGLTPGWNDLDVNQHVSNVK YIGWILESMPTEVLETQELCSLALEYRRECGRDSVLESVTAMDPTKVGGRSQYQHLLRLED GTDIVKCRTEWRPKNPGANGAISTGKTSNGNSVSS SEQ ID NO: 11 Amino acid sequence of Tes3
MKFKKKFKIGRMHVDPFNYISMRYLVALMNEVAFDQAEILEKDIDMKNLRWIIYSWDIQIENNI RLGEEIEITTIPTHMDKFYAYRDFIVESRGNILARAKATFLLMDITRLRPIKI PQNLSLAYGKEN PIFDIYDMEIRNDLAFIRDIQLRRADLDNNFHINNAVYFDLIKETVDIYDKDISYIKLIYRNEIRDK KQIQAFARREDKSIDFALRGEDGRDYCLGKIKTNV SEQ ID NO: 12 Amino acid sequence of CpFatBI -4
MFDRKSKRPSMLMDSFGLERVVQDGLVFRQSFSIRSYEICADRTASMETVMNHVQETSLN QCKSIGLLDDGFGRSPEMCKRDLIWVVTRMKIMVNRYPTWGDTIEVSTWLSQSGKIGMGR DWLISDCNTGEILVRATSVYAMMNQKTRRFSKLPHEVRQEFAPHFLDSPPAIEDNDGKLQK FDVKTGDSIRKGLTPGWYDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLTLEYRRECG RDSVLESVTSMDPSKVGDRFQYRHLLRLEDGADIMKGRTEWRPKNAGTNGAISTGKT
SEQ ID NO: 13 Amino acid sequence of CaFatB3-5
MHDRKSKRPSMLMDSFGLESIVQEGLEFRQSFSIRSYEIGTDRTASMETLMNYLQETSLNH CKSTGILLDGFGRTPEMCKRDLIWVVTKMKIKVNRYPAWGDTVEINTWFSRLGKIGKGRDW LISDCNTGEILIRATSAYATMNQKTRRLSKLPYEVHQEIAPLFVDSPPVIEDNDLKLHKFEVKT GDSIHKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRRECGRDSVL ESVTAMDPTKVGGRSQYQHLLRLEDGTDIVKCRTEWRPKNPGANGAISTGKTSNGNSVS
SEQ ID NO: 14 Amino acid sequence of AtSUSI MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTRK
KLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEELQPAEFLHFKEEL
VDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLH
SHQGKNLM LSEKIQNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAER
VLDM I RLLLDLLEAPDPCTLETFLGRVPM VFN VVI LSPHGYFAQDN VLGYPDTGGQVVYI LD
QVRALEI EM LQRI KQQGLN I KPRI LI LTRLLPDAVGTTCGERLERVYDSEYCDI LRVPFRTEKG
IVRKWISRFEVWPYLETYTEDAAVELSKELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIA
HALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHT
AFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENKEH
LCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELANLVVVGGDRRKESKDNEEKA
EMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAM
TCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADTLADFFTKCKEDPSHWDEISKGGL
QRIEEKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEARRYLEMFYALKYRPLAQAVPLAQD
D
SEQ ID NO: 15 Amino acid sequence of wild type CvFatBI
MVAAAATSAFFPVPAPGTSPKPGKSGNWPSSLSPTFKPKSIPNGGFQVKANASAHPKANG
SAVNLKSGSLNTQEDTSSSPPPRAFLNQLPDWSMLLTAITTVFVAAEKQWTMLDRKSKRPD
MLVDSVGLKSIVRDGLVSRHSFSIRSYEIGADRTASIETLMNHLQETTINHCKSLGLHNDGFG
RTPGMCKNDLIWVLTKMQIMVNRYPTWGDTVEINTWFSQSGKIGMASDWLISDCNTGEILI
RATSVWAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLRKFDVKTGDSIRKGL
TPRWN DLDVNQH VSN VKYIGWI LESM PI EVLETQELCSLTVEYRRECGM DSVLESVTAVDP
SENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNAGTNGAISTSTAKTSNGNSVS
SEQ ID NO: 16 Amino acid sequence of wild type ChoFatB2
MHHHHHHVAAAASSAFFPVPAPGASPKPGKFGNWPSSLSPSFKPKSIPNGGFQVKANDSA
HPKANGSAVSLKSGSLNTQEDTSSSPPPRTFLHQLPDWSRLLTAITTVFVKSKRPDMHDRK
SKRPDMLVDSFGLESTVQDGLVFRQSFSIRSYEIGTDRTASIETLMNHLQETSLNHCKSTGIL
LDGFGRTLEMCKRDLIWVVIKMQIKVNRYPAWGDTVEINTRFSRLGKIGMGRDWLISDCNT
GEILVRATSAYAMMNQKTRRLSKLPYEVHQEIVPLFVDSPVIEDSDLKVHKFKVKTGDSIQK
GLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRRECGRDSVLESVTAM
DPSKVGVRSQYQHLLRLEDGTAIVNGATEWRPKNAGANGAISTGKTSNGNSVS
SEQ ID NO: 17 Amino acid sequence of CvFatBI T 1
MANGSAVNLKSGSLNTQEDTSSSPPPRAFLNQLPDWSMLLTAITTVFVAAEKQWTMLDRK
SKRPDMLVDSVGLKSIVRDGLVSRHSFSIRSYEIGADRTASIETLMNHLQETTINHCKSLGLH
NDGFGRTPGMCKNDLIWVLTKMQIMVNRYPTWGDTVEINTWFSQSGKIGMASDWLISDCN
TGEILIRATSVWAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLRKFDVKTGDSI RKGLTPRWNDLDVNQHVSNVKYIGWILESMPIEVLETQELCSLTVEYRRECGMDSVLESVT
AVDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNAGTNGAISTSTAKTSNGNSVS
SEQ ID NO: 18 Amino acid sequence of CvFatBI T2
MLPDWSMLLTAITTVFVAAEKQWTMLDRKSKRPDMLVDSVGLKSIVRDGLVSRHSFSIRSY
EIGADRTASIETLMNHLQETTINHCKSLGLHNDGFGRTPGMCKNDLIWVLTKMQIMVNRYPT
WGDTVEINTWFSQSGKIGMASDWLISDCNTGEILIRATSVWAMMNQKTRRFSRLPYEVRQ
ELTPHFVDSPHVIEDNDQKLRKFDVKTGDSIRKGLTPRWNDLDVNQHVSNVKYIGWILESM
PIEVLETQELCSLTVEYRRECGMDSVLESVTAVDPSENGGRSQYKHLLRLEDGTDIVKSRT
EWRPKNAGTNGAISTSTAKTSNGNSVS
SEQ ID NO:19 Amino acid sequence of CvFatBI T3
MLLTAITTVFVAAEKQWTMLDRKSKRPDMLVDSVGLKSIVRDGLVSRHSFSIRSYEIGADRT
ASIETLMNHLQETTINHCKSLGLHNDGFGRTPGMCKNDLIWVLTKMQIMVNRYPTWGDTVE
INTWFSQSGKIGMASDWLISDCNTGEILIRATSVWAMMNQKTRRFSRLPYEVRQELTPHFV
DSPHVIEDNDQKLRKFDVKTGDSIRKGLTPRWNDLDVNQHVSNVKYIGWILESMPIEVLETQ
ELCSLTVEYRRECGMDSVLESVTAVDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNA
GTNGAISTSTAKTSNGNSVS
SEQ ID NO: 20 Amino acid sequence of CvFatBI T4
MEKQWTM LDRKSKRPDM LVDSVGLKSI VRDGLVSRHSFSI RSYEIGADRTASI ETLM NHL
QETTINHCKSLGLHNDGFGRTPGMCKNDLIWVLTKMQIMVNRYPTWGDTVEINTWFSQSG
KIGMASDWLISDCNTGEILIRATSVWAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIED
NDQKLRKFDVKTGDSIRKGLTPRWNDLDVNQHVSNVKYIGWILESMPIEVLETQELCSLT
VEYRRECGMDSVLESVTAVDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNAGTNGAI
STSTAKTSNGNSVS
SEQ ID NO: 21 Amino acid sequence of CvFatBI T5
MLDRKSKRPDMLVDSVGLKSIVRDGLVSRHSFSIRSYEIGADRTASIETLMNHLQETTINHCK
SLGLHNDGFGRTPGMCKNDLIWVLTKMQIMVNRYPTWGDTVEINTWFSQSGKIGMASDWL
ISDCNTGEILIRATSVWAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLRKFDVK
TGDSIRKGLTPRWNDLDVNQHVSNVKYIGWILESMPIEVLETQELCSLTVEYRRECGMDSV
LESVTAVDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNAGTNGAISTSTAKTSNGNSV
S
SEQ ID NO: 22 Amino acid sequence of CvFatBI T6
MRPDMLVDSVGLKSIVRDGLVSRHSFSIRSYEIGADRTASIETLMNHLQETTINHCKSLGLHN
DGFGRTPGMCKNDLIWVLTKMQIMVNRYPTWGDTVEINTWFSQSGKIGMASDWLISDCNT GEILIRATSVWAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLRKFDVKTGDSIR
KGLTPRWNDLDVNQHVSNVKYIGWILESMPIEVLETQELCSLTVEYRRECGMDSVLESVTA
VDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNAGTNGAISTSTAKTSNGNSVS
SEQ ID NO: 23 Amino acid sequence of CvFatBI T7
MKSIVRDGLVSRHSFSIRSYEIGADRTASIETLMNHLQETTINHCKSLGLHNDGFGRTPG MCKNDLIWVLTKMQIMVNRYPTWGDTVEINTWFSQSGKIGMASDWLISDCNTGEILIRAT SVWAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLRKFDVKTGDSIRKGLTPR WN DLDVNQH VSN VKYIGWI LESM PI EVLETQELCSLTVEYRRECGM DSVLESVTAVDPSE NGGRSQYKHLLRLEDGTDIVKSRTEWRPKNAGTNGAISTSTAKTSNGNSVS
SEQ ID NO: 24 Amino acid sequence of ChoFatB2.1
MANGSAVSLKSGSLNTQEDTSSSPPPRTFLHQLPDWSRLLTAITTVFVKSKRPDMHDRKSK
RPDMLVDSFGLESTVQDGLVFRQSFSIRSYEIGTDRTASIETLMNHLQETSLNHCKSTGILLD
GFGRTLEMCKRDLIWVVIKMQIKVNRYPAWGDTVEINTRFSRLGKIGMGRDWLISDCNTGEI
LVRATSAYAMMNQKTRRLSKLPYEVHQEIVPLFVDSPVIEDSDLKVHKFKVKTGDSIQKGLT
PGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRRECGRDSVLESVTAMDP
SKVGVRSQYQHLLRLEDGTAIVNGATEWRPKNAGANGAISTGKTSNGNSVS
SEQ ID NO: 25 Amino acid sequence of ChoFatB2.2
MLPDWSRLLTAITTVFVKSKRPDMHDRKSKRPDMLVDSFGLESTVQDGLVFRQSFSIRSYEI
GTDRTASIETLMNHLQETSLNHCKSTGILLDGFGRTLEMCKRDLIWVVIKMQIKVNRYPAWG
DTVEINTRFSRLGKIGMGRDWLISDCNTGEILVRATSAYAMMNQKTRRLSKLPYEVHQEIVP
LFVDSPVIEDSDLKVHKFKVKTGDSIQKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLE
TQELCSLALEYRRECGRDSVLESVTAMDPSKVGVRSQYQHLLRLEDGTAIVNGATEWRPK
NAGANGAISTGKTSNGNSVS
SEQ ID NO: 26 Amino acid sequence of ChoFatB2.3
MRPDMLVDSFGLESTVQDGLVFRQSFSIRSYEIGTDRTASIETLMNHLQETSLNHCKSTGIL
LDGFGRTLEMCKRDLIWVVIKMQIKVNRYPAWGDTVEINTRFSRLGKIGMGRDWLISDCNT
GEILVRATSAYAMMNQKTRRLSKLPYEVHQEIVPLFVDSPVIEDSDLKVHKFKVKTGDSIQK
GLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRRECGRDSVLESVTAM
DPSKVGVRSQYQHLLRLEDGTAIVNGATEWRPKNAGANGAISTGKTSNGNSVS
SEQ ID NO: 27 DNA sequence of [SEQ ID NO: 1]
AT GAGCAGCGGCAT GGAGAAAAAAATCACTGGAT AT ACCACCGTT GAT AT ATCCCAAT G GCATCGT AAAG AACATTTT G AGGCATTT CAGTCAGTTGCT CAAT GT ACCT AT AACCAG AC CGTT CAGCT GG AT ATTACGGCCTTTTT AAAG ACCGT AAAG AAAAAT AAGCACAAGTTTT A TCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGG CAAT G AAAG ACGGTG AGCTGGT GAT ATGGG AT AGTGTT CACCCTT GTT ACACCGTTTT C CATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCA GTTTCT ACACAT AT ATTCGCAAGAT GT GGCGT GTT ACGGT GAAAACCTGGCCT ATTTCC CTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCA GTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACTATGGGC AAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATG CCGTCT GT GAT GGCTTCCAT GTCGGCAGAATGCTT AAT GAATT ACAACAGT ACTGCGAT GAGTGGCAGGGCGGGGCGTAA
SEQ ID NO: 28 DNA sequence of [SEQ ID NO: 2]
AT G AG AAG ATT G AAGTGTCG ATT AATT CG AAACACACCAT CAAGCCG AGCACCTCCTCC
ACCCCTCTTCAACCGTACAAACTCACGTTACTGGACCAATTAACGCCGCCAGCATATGT
TCCAATT GT GTTTTTTT ATCCG ATT ACAG AT CACG ACTTT AACCTTCCGCAG ACTTTGGC
CGATCTGCGTCAAGCGCT GAGCGAAACACT GACGCTGT ATT ATCCACT GAGCGGCCGC
GTCAAGAACAACCT GT ACATT GACGATTTTGAAGAGGGGGTGCCGT ACCTGGAAGCTC
GTGTGAATTGCGACATGACGGATTTCCTCCGTCTGCGTAAGATCGAGTGCTTAAACGAA
TTTGTCCCTATTAAGCCGTTTAGTATGGAAGCAATTTCGGATGAGCGCTATCCGCTGCT
TGGGGTTCAAGTAAACGTATTTGATTCCGGTATTGCGATCGGGGTCAGTGTCTCCCATA
AACTGATCGACGGCGGGACCGCTGATTGCTTCCTGAAGTCGTGGGGAGCGGTGTTCC
GCGGATGCCGCGAGAACATTATCCATCCGTCCCTCTCGGAAGCAGCATTGCTTTTTCC
GCCACGT GACGACCTCCCGGAAAAAT AT GT AGATCAAATGGAAGCGTT ATGGTTCGCG
GGTAAAAAGGTTGCGACCCGTCGTTTCGTGTTTGGTGTGAAAGCTATCTCATCTATTCA
GGATGAAGCAAAATCTGAGAGCGTACCAAAGCCGAGTCGCGTGCACGCCGTCACGGG
TTTTTT GT GGAAGCATCT GATCGCGGCGAGTCGTGCACT GACCTCAGGTACT ACTTCT A
CTCGCCTGAGTATCGCCGCCCAAGCGGTTAATTTGCGCACGCGCATGAATATGGAAAC
CGTACTGGATAACGCGACGGGTAATCTGTTTTGGTGGGCGCAGGCGATCCTCGAGTTA
AGCCAT ACCACTCCAG AG AT CAGCG AT CT GAAACT GTGT GAT CTGGT G AACCT GTT G AA
CGGCAGCGTTAAGCAGTGCAATGGGGATTATTTTGAAACCTTTAAAGGTAAAGAAGGTT
ATGGCCGT ATGTGCGAGT ACCTT GATTTTCAGCGGACCAT GAGTTCCATGGAACCTGCA
CCGGAT ATTT ACCT GTTTTCTTCTTGGACGAACTTTTTCAACCCCTTGGATTTTGGCT GG
GGCCGCACCTCTTGGATTGGGGTTGCGGGCAAGATTGAAAGCGCATCTTGCAAATTTA
TTATCCTTGTCCCGACACAGTGTGGCTCAGGCATCGAAGCGTGGGTAAATCTGGAGGA
AGAAAAAATGGCGATGCTT GAACAGGACCCCCATTTCCTGGCTCTTGCGTCTCCCAAAA
CGTTGATTTAA
SEQ ID NO: 29 DNA sequence of [SEQ ID NO: 3] ATGAACGAGATCGATGAGAAGAATCAAGCACCGGTCCAGCAAGAGTGTTTGAAGGAGA TGATTCAAAATGGGCATGCACGTCGCATGGGTTCTGTCGAAGATTTGTATGTAGCACTG AACCGTCAGAACTT AT ACCGT AATTTTTGCACCT ACGGGGAGTT ATCT GACT ATT GT ACC CGT GAT CAATT G ACT CTTGCCTT ACGT GAAATCTGCCT G AAG AATCCCACATT GTT ACAC ATCGT ACTGCCAACCCGTTGGCCAAAT CACG AG AATT ACT ATCGCTCCAGT G AGT ACTA CT CACGCCCTCATCCCGTGCACG ATT AT AT CT CT GTGCTT CAAG AACT G AAATT AT CAG GCGT AGTCCTT AAT GAACAACCGGAGT ATTCTGCGGTT AT GAAGCAGATTTTGGAGGAG TTT AAG AAT AGT AAGGGCT CTT AT ACAGCGAAAAT CTTT AAATT AACT ACT ACTTT AACAA TTCCGTATTTCGGTCCTACGGGGCCCTCATGGCGTCTGATTTGCTTACCGGAAGAGCA CACT G AAAAATGG AAAAAGTT CAT CTTCGTTT CT AAT CATT GTAT GTCCG AT GGCCGCTC AAGT ATCCATTT CTTCCACG ACCTGCGT GAT G AATT G AAT AAT ATT AAG ACACCACCT AA AAAGTTGGATT ACATCTTT AAGT AT GAGGAAGACT ACCAGTT ACTGCGT AAATTGCCCG AGCCT ATCG AG AAAGTAATT G ATTTTCGTCCCCCGT ACCTTTT CATCCCG AAAT CT CTTT T AAGCGGTTTT ATCT AT AATCACCTGCGTTTCAGTTCGAAGGGAGT GT GTATGCGT AT G GACGATGTGGAGAAGACGGACGATGTTGTCACCGAAATTATCAACATCAGCCCTACGG AATTTCAAGCTATCAAGGCCAATATTAAATCTAATATTCAAGGCAAATGCACAATCACTC CGTTTTTGCAT GTTT GCTGGTTT GTCTCACT GCACAAATGGGGAAAATTCTTT AAACCAT T AAATTTCG AGT GGCTT ACT G ACATTTT CATTCCAGCCG ACTGCCGTT CTCAGTT ACCTG ACGAT GAT GAGATGCGTCAGAT GT ACCGTT ATGGAGCAAACGTTGGATTCATCGACTTC ACACCGTGGATTTCCGAGTTCGACAT GAAT GACAACAAGGAAAATTTTTGGCCCCTT AT CGAACATTACCATGAAGTAATTTCGGAAGCACTGCGCAATAAGAAGCACCTGCATGGGT TAGGCTTCAATATTCAGGGGTTCGTCCAGAAATATGTAAATATCGATAAGGTGATGTGT GATCGTGCCATTGGTAAGCGTCGTGGCGGAACACTGCTGTCCAACGTCGGTTTGTTCA ACCAGTTAGAAGAACCTGATGCGAAATATTCAATCTGTGACTTAGCTTTCGGGCAATTTC AGGGATCGTGGCATCAGGCTTTTAGCTTGGGCGTATGTTCAACCAATGTAAAAGGCATG AACATCGTT GTTGCT AGT ACAAAAAAT GTGGTTGGCTCTCAAGAAAGTCTT GAGGAACT TTGCTCT ATTTAT AAAGCCCT GTT GTT GGGGCCCT AA
SEQ ID NO: 30 DNA sequence of [SEQ ID NO: 4]
ATGGGCTCGGCGGGCATGCCTGAAAAGCCCCACGCAGTGTGCCTGCCGTATCCGGCG CAGGGCCACATCACGCCGATGTTAAAACTCGCGAAATTGCTGCACTCAAAAGGTTTTCA CGTT ACATTT GT AAACACGG AATT CAAT CAT AAACGTCT GTT G AAAT CTCGTGGCCCT G A TTCATTGACCGGCCTGTCGAGCTTCCGTTTCGAGACAATTCCAGACGGTCTGCCTGAAT CT GAT CTGG ATGCAACT CAGTT CATCCCT AGTCT CTGCG AAT CAACCCGT AAAAATT GT CTGGGCCCGTTCCGTCAACTGCTGGGCAAACT GAACAAT ACAGT GAGTTCT GGGGTGC CGCCAGTCTCCTGCGTTGTTAGTGATGGCGTGATGTCCTTTTCCCTGGATGCCGCAGA GGAACTGGGCATCCCGCAGGTCTTATTTTGGACGACGAGTGTTTGTGGGTTTATGGCC T ACGTGCACT ATCGT AATCT GATCGAAAAAGGAT AT ACGCCGCT GAAGGACGTTTCGT A
CGTGACAAATGGCTATTTAGATACAGTTATCGACTGGATTCCGGGTATGGAGGGTATTC
GGCTTAAAGATCTGCCGTCGTTTCTGCGTACCACTGACCCTAACGATATCATGTTAGAT
TTT GT ACT G AGCG AAACCAAG AACACCCACCGTTCGTCTGCT ATT AT CTT CAAT ACCTT C
GAT AAACTCG AACAT CAAGT ACTGG AACCT CTTGCGTCT AT GTTTCCGCCT ATTT ACACC
ATTGGCCCTCTGAACTTGCTGATGAATCAGATCAAGGAAGAAAGCCTTAAAATGATTGG
CAGCAATTT GTGG AAAG AGG AACCAAT GT GTATCG AATGGTT G AATT CAAAAG AACCT A
AAAGT GTT GT GT ACGTGAACTTTGGATCAATT ACGGTCAT GACACCGAACCAGCTGGT A
GAATTCGCATGGGGTCTGGCGAACAGCAATCAGTCATTTCTGTGGATTATTCGCCCAGA
CCTGGTGGTCGGCGAGTCGGCGGTTCTGCCGCCTGAATTTGTTGCGGTTACCAAAGAA
CGTGGTATGCTGGCAAGCTGGGCCCCGCAGGAAGAGGTGCTGGCGCACAGCTCCGTT
GGCGGTTTTCTGACCCATTGTGGTTGGAACTCTACCTTAGAAAGCATTAGCTCTGGTGT
TGCCGTGGTGTGTTGGCCGTTTTTCGCCGAGCAGCAGACAAATTGCTGGTACTGTTGC
GGGGAACT GGGGATTGGT AT GGAAATCGACAGCGATGT GAAGCGCGAAGAAGTT GAG
CGGCTCGTCCGGGAATTGATGGTTGGTGAAAAAGGCAAAGAAATGAAAGAACGCGCCA
TGGGCTGGAAACGCCTGGCAGAAGAAGCGACCCAAAGCTCCTCCGGCAGCTCGTTTTT
AAATCTGGACAAACTGGTCCACCAGGTATTGCTGTCCCCGCGTCCGTAA
SEQ ID NO: 31 DNA sequence of [SEQ ID NO: 5]
AT GTCACAGACGACAACCAATCCTCAT GTTGCCGTGCTGGCTTTCCCGTTT AGCACCCA
TGCCGCACCGTTACTGGCCGTCGTGCGTCGTTTGGCGGCGGCGGCCCCGCACGCCGT
TTTTTCATTCTTT AGCACGAGCCAGAGCAAT GCT AGCATCTTCCAT GACAGT ATGCAT AC
AATGCAGT GT AACATTAAGTCCT ACGACGTTTCAGATGGCGTCGCAGAAGGAT AT GT GT
TCGCAGGACGTCCGCAAGAAGACATTGAACTGTTCATGCGCGCGGCTCCAGAATCGTT
CCGTCAAGGCATGGTTATGGCGGTGGCGGAGACGGGCCGTCCAGTGTCTTGCTTAGT
AGCAGATGCGTTTATCTGGTTCGCCGCTGATATGGCCGCGGAGATGGGCGTGGCCTG
GCTTCCGTTTT GGACGGCAGGCCCT AACTCCTT GAGCACCCAT GT GTACACCGAT GAA
ATTCGCGAAAAAATTGGTGTGTCGGGAATTCAGGGGCGCGAAGATGAACTTTTGAATTT
CATTCCGGGAATGTCGAAAGTACGCTTTCGTGACCTGCAGGAAGGAATTGTCTTCGGG
AATTTAAATTCCTTATTCTCTCGCATGCTGCATCGCATGGGTCAAGTTTTGCCAAAAGCG
ACAGCGGT ATTT ATT AACAGTTTCG AAGAATTGG ACG ATTCCCT G ACG AACG AT CT GAA
AAGCAAACT CAAAACTT AT CT G AACATCGG ACCGTTT AACTT AAT CACCCCTCCCCCTGT
GATTCCGAACACCACAGGCTGCCTGCAATGGTTAAAAGAACGCAAACCGACCTCGGTA
GTCTATATTTCATTTGGCACAGTTACGACTCCACCGCCGGCCGAGCTGGTGGCGCTCG
CCGAAGCGCTCGAGGCGTCCCGTGTGCCGTTTATTTGGAGCTTGCGCGATAAAGCCCG
CGTCCATCTGCCGGAAGGATTCTTAGAAAAAACCCGCGGCTATGGTATGGTTGTGCCC
TGGGCGCCGCAGGCGGAAGTACTTGCACACGAGGCGGTGGGTGCGTTTGTGACGCAT TGTGGTTGGAACTCTCTGTGGGAATCGGTGGCTGGCGGCGTGCCGCTGATTTGTCGCC
CATTCTACGGCGATCAACGCTTAAATGGGCGCATGGTGGAAGATGCCTTGGAGATCGG
GGTGCGCATTGAAGGTGGCGTCTTTACCGAAAGTGGACTGATGTCATGTTTCGATCAG
ATTTTATCTCAAGAAAAGGGTAAAAAATTGCGTGAAAATTTAGGCGCCCTCCGTGAGAC
TGCCGATCGTGCGGTCGGTCCCAAAGGCAGTAGCACCGAGAATTTTAAGACCCTGGTG
GACCTT GT GTCCAAACCGAAAGACGTTTAA
SEQ ID NO: 32 DNA sequence of [SEQ ID NO: 6]
ATGTCTATGAGCGATATCAACAAAAACTCGGAACTGATCTTTATCCCGGCCCCGGGTAT TGGTCATCTGGCGAGCGCGCTGGAATTTGCGAAACTTTTAACAAACCACGACAAGAATC T GT AT ATT ACCGT GTTTTGCATCAAGTTCCCGGGT ATGCCTTTTGCT GAT AGCT ACATT A AGTCAGTCCTGGCGTCCCAACCTCAGATTCAGCTGATTGACCTGCCTGAAGTGGAACC TCCGCCGCAGGAACTGCTT AAGAGCCCGGAATTTT ACATCTT GACCTTCCTGGAGAGTT T AATCCCGCACGT GAAAGCAACGATT AAAACAATCCT GTCT AACAAGGT AGTTGGCCT G GTTTTGGATTTTTTCTGCGTTTCCATGATCGATGTCGGCAACGAGTTTGGCATTCCGTC CT ATCT GTTTTT AACGTCGAACGTGGGCTTTCT GTCACT GATGCT GTCGCTCAAAAACC GTCAGATTGAGGAGGTCTTTGATGACAGCGACCGTGATCACCAGCTTCTGAATATCCCT GGAATCAGCAACCAGGTTCCCAGT AAT GT GTTGCCT GACGCGT GTTTCAACAAGGAT G GTGGTTATATTGCCTATTATAAATTGGCAGAACGTTTTCGCGATACAAAAGGCATCATTG TT AACACCTTTT CT G ACCTGG AACAG AGTAGCATT G ATGCGTT GTAT GAT CAT GAT G AAA AAATCCCTCCGATCTATGCCGTGGGTCCGCTGCTGGATTTGAAAGGCCAACCCAACCC GAAACTGGACCAAGCCCAGCAT GACCT GATTCT GAAATGGTT GGAT GAGCAACCCGAC AAATCTGTAGTGTTTCTGTGCTTCGGATCGATGGGCGTCAGCTTTGGCCCTTCTCAGAT TCGTGAGATTGCGCTGGGCCTGAAACACTCTGGTGTCCGTTTTCTCTGGTCAAACAGTG CTGAGAAAAAAGTATTCCCGGAAGGTTTTCTGGAGTGGATGGAACTGGAAGGCAAAGG GATGATTTGTGGCTGGGCACCGCAGGTTGAAGTACTGGCACATAAGGCGATCGGCGGT TTCGTTAGCCACTGTGGTTGGAACAGTATCCTGGAATCGATGTGGTTTGGGGTCCCGAT CCT GACTTGGCCT ATCT ACGCGGAACAGCAGCT GAATGCGTTTCGGTT AGTT AAAGAAT GGGGAGTGGGCCTGGGTTTGCGTGTGGACTACCGCAAAGGAAGCGACGTCGTCGCTG CGGAAGAAATT GAAAAAGGCCT GAAAGATCT GAT GGAT AAAGACAGT ATT GTCCAT AAA AAAGT ACAGGAAAT GAAGGAGAT GAGTCGGAACGCAGT GGT AGAT GGTGGTTCGAGTC T GATT AGTGT AGGCAAACT CATT GAT GAT ATT ACCGGCT CT AATT AA
SEQ ID NO: 33 DNA sequence of [SEQ ID NO: 7]
ATGGAAGAATCTAAAACCCCGCACGTTGCTATCATCCCGTCTCCGGGTATGGGTCACCT
GATCCCGCTGGTTGAATTCGCTAAACGTCTGGTTCACCTGCACGGTCTGACCGTTACCT
TCGTTATCGCTGGTGAAGGTCCGCCGTCTAAAGCTCAGCGTACCGTTCTGGACTCTCT GCCGTCTTCTATCTCTTCTGTTTTCCTGCCGCCGGTTGACCTGACCGACCTGTCTTCTT
CTACCCGTATCGAATCTCGTATCTCTCTGACCGTTACCCGTTCTAACCCGGAACTGCGT
AAAGTTTTCGACTCTTTCGTTGAAGGTGGTCGTCTGCCGACCGCTCTGGTTGTTGACCT
GTTCGGTACCGACGCTTTCGACGTTGCTGTTGAATTCCACGTTCCGCCGTACATCTTCT
ACCCGACCACCGCT AACGTTCT GTCTTTCTTCCTGCACCTGCCGAAACTGGACGAAACC
GTTTCTTGCGAATTCCGT GAACT GACCGAACCGCT GATGCTGCCGGGTTGCGTTCCGG
TTGCT GGT AAAGACTTCCTGGACCCGGCTCAGGACCGT AAAGACGACGCTT ACAAAT G
GCTGCTGCACAACACCAAACGTTACAAAGAAGCTGAAGGTATCCTGGTTAACACCTTCT
TCGAACTGGAACCGAACGCTATCAAAGCTCTGCAGGAACCGGGTCTGGACAAACCGCC
GGTTTACCCGGTTGGTCCGCTGGTTAACATCGGTAAACAGGAAGCTAAACAGACCGAA
GAATCTGAATGCCTGAAATGGCTGGACAACCAGCCGCTGGGTTCTGTTCTGTACGTTTC
TTTCGGTTCTGGTGGTACCCTGACCTGCGAACAGCTGAACGAACTGGCTCTGGGTCTG
GCTGACTCTGAACAGCGTTTCCTGTGGGTTATCCGTTCTCCGTCTGGTATCGCTAACTC
TTCTTACTTCGACTCTCACTCTCAGACCGACCCGCTGACCTTCCTGCCACCCGGCTTCC
TGGAACGTACTAAAAAACGTGTTCGTGCTAAATGGCAGCCGCTGAACATCTAA
SEQ ID NO: 34 DNA sequence of [SEQ ID NO: 8]
ATGGGT AACTTTGCCAATCGCAAACCGCAT GT AGT GATGATCCCAT ATCCGGT ACAGGG CCATATTAATCCGCTGTTCAAATTGGCGAAGCTGCTGCATCTGCGCGGTTTTCACATTA CATTT GT AAACACCGAGT AT AACCAT AAGCGCTTGCTCAAGTCCCGTGGTCCAAAAGCA TTT GATGGTTTCACCGATTTCAATTTT GAGAGCATCCCGGATGGTTT AACGCCGAT GGA AGGCGACGGCGAT GTGAGCCAGGAT GTTCCT ACCCT GT GTCAGTCT GT ACGCAAAAAC TTTCTCAAGCCGTATTGCGAGTTACTCACTCGCCTGAACCATTCCACCAACGTGCCACC GGTCACTTGTCTCGTTAGCGACTGCTGTATGTCATTCACGATCCAGGCGGCTGAAGAAT TT GAGCTGCCGAACGTGCTTT ATTTT AGCAGT AGTGCCTGCTCACTGCT GAACGT AAT G CATTTTCGTT CATTCGTGG AACGTGG AAT CATTCCGTT CAAAG ACG AAAGCT ACTT G AC CAATGGCTGCCTGGAAACCAAAGTCGATTGGATCCCTGGGCTTAAAAACTTTCGCCTGA AAG AT ATT GTCG ACTTT ATTCGT ACCACT AATCCG AAT GAT ATT ATGCTGG AGTTTTTT AT TGAGGTAGCGGATCGGGTGAACAAAGATACCACCATTCTCCTCAATACCTTTAACGAGC TGGAAAGCGACGTGATCAACGCACTGTCGAGTACTATTCCTTCGATTTATCCGATCGGT CCGTT ACCGTCATT GTT AAAACAGACCCCGCAAATCCACCAGTT AGACAGCTTGGAT AG CAATCTCTGGAAGGAGGATACCGAATGTCTTGACTGGCTGGAGTCAAAGGAGCCGGGG TCGGTGGTTTATGTGAATTTCGGCTCTATTACGGTGATGACGCCAGAACAGCTGTTAGA ATTCGCCTGGGGGCTGGCAAATTGTAAAAAATCTTTTCTGTGGATTATCCGCCCGGATC TGGTG ATTGGT GGCT CAGTCAT CTT CAGT AGT G AATT CACCAACG AAATTGCAG ATCGT GGTCTGATTGCGTCTTGGTGCCCCCAGGACAAAGTCCTGAATCATCCGTCAATCGGTG GGTTTCTGACTCATTGCGGTTGGAACAGTACAACCGAGTCGATTTGTGCGGGCGTCCC AAT GTT GTGTTGGCCTTTTTTTGCGG ACCAACCG ACAG ATT GTCGTTTT ATTT GT AACG A ATGGGAAATTGGCATGGAAATTGATACGAACGTGAAACGTGAAGAGCTGGCCAAACTG ATTAACGAAGTCATTGCCGGCGATAAGGGCAAAAAAATGAAACAGAAAGCCATGGAGC TGAAAAAAAAGGCAGAAGAGAATACGCGCCCAGGGGGGTGCAGCTACATGAATCTGAA CAAAGTT AT CAAAG AT GTGTTGTT G AAGCAAAACT AA
SEQ ID NO: 35 DNA sequence of [SEQ ID NO: 9]
ATGCGGCCAAACATGTTAATGGATAGCTTTGGGCTGGAACGGGTGGTTCAGGACGGCC TGGTGTTTCGGCAATCATTCAGCATTCGCAGTTATGAAATCTGTGCGGATCGCACCGCT TCCATCG AAACT GT GAT G AACCACGTT CAGG AAACCAGCCT G AAT CAAT GT AAAAGT AT TGGCTTACTGGATGACGGGTTTGGCCGTTCCCCCGAAATGTGCAAACGGGATCTGATT TGGGTGGTT ACCCGCAT GAAAATCATGGT GAACCGTT ATCCGACCTGGGGCGAT ACT A TTGAAGTTAGCACCTGGCTGAGTCAGTCCGGTAAAATTGGTATGGGGCGGGATTGGCT GATCTCTGACTGCAACACCGGTGAAATTTTGGTGCGCGCAACTAGCGTTTACGCCATGA T G AAT CAG AAAACCCGCCGTTTT AGT AAATT GCCCCAT G AAGTGCGTCAAG AATTTGCG CCGCACTTCTT AGATTCCCCACCCGCT ATCGAAGAT AAT GACGGT AAACTGCAAAAATT CGAT GT GAAAACCGGGGACTCT ATTCGCAAAGGGTT GACTCCTGGCTGGT AT GATTT A GACGTTAATCAGCATGTGTCTAACGTTAAATACATTGGCTGGATTCTGGAATCAATGCC CACCGAAGTGCTGGAAACTCAAGAATT GT GT AGCCT GACCTT GGAAT ATCGTCGCGAAT GCGGGCGTGATAGTGTGTTGGAAAGCGTGACCTCAATGGACCCCTCCAAAGTGGGTGA CCGCTTTCAATACCGTCACTTGTTACGGCTGGAAGATGGTGCGGACATCATGAAAGGG CGCACCGAATGGCGTCCCAAAAATGCGGGCACTAACGGTGCTATTAGTACCGGTAAAA CTTGA
SEQ ID NO: 36 DNA sequence of [SEQ ID NO: 10]
ATGCATCATCACCACCACCATAAGCCAGGCAAATTCCGTATTTGGCCGTCGAGTCTGTC
GCCTTCATTTAAGCCGAAGCCGATCCCCAACGGTGGACTTCAAGTAAAGGCAAATTCG
CGCGCTCACCCGAAGGCCAACGGCTCTGCCGTCTCACTGAAAAGTGGTTCGCTTAATA
CACAAGAAGAT ACATCATCT AGTCCCCCGCCACGCACTTTTTT ACACCAGTTGCCT GAT
TGGTCCCGTTT ATT AACCGCT ATT ACGACCGTCTTCGT AAAGAGCAAACGCCCT GAT AT
GCAT GACCGCAAATCGAAGCGTCCGGACATGCT GATGGACAGTTTTGGGCTGGAGTCC
ATCGTCCAAGAAGGATTAGAGTTCCGTCAGTCCTTTAGCATCCGTTCATACGAGATCGG
CACGGATCGTACCGCTTCCATCGAGACGCTTATGAACTATTTACAGGAAACGTCCTTAA
ATCATTGCAAAAGTACCGGCATTCTGTTAGATGGATTCGGACGTACGCCTGAGATGTGC
AAGCGTGATTTAATCTGGGTCGTGACTAAAATGAAGATCAAAGTGAATCGCTACCCGGC
GTGGGGGGACACGGTGGAAATTAATACGTGGTTTAGCCGCTTGGGAAAAATTGGCAAA
GGTCGCGATTGGCTTATCAGCGACTGTAATACGGGTGAAATCCTGATCCGTGCAACAA GCGCAT ACGCT ACT AT GAACCAGAAGACCCGTCGTTT ATGCAAGTT GCCAT ACGAAGT A CACCAGGAAATCGCGCCGTTGTTCGTTGACAGCCCCCCTGTTATTGAGGACAATGATCT T AAGTT ACAT AAGTTT GAGGT AAAAACAGGCGACTCCATTCACAAAGGGCT GACCCCCG GATGGAATGATCTGGACGTCAATCAACATGTAAGCAACGTGAAGTATATTGGGTGGATC TT AGAATCAATGCCT ACGGAAGTTTT AGAAACCCAAGAATT GT GTTCCTT AGCGCTGGA GTATCGTCGTGAATGCGGGCGCGACTCCGTACTGGAATCAGTCACGGCCATGGACCC CACT AAGGT GGGTGGACGTTCGCAAT ATCAACACTTGCTTCGTCTT GAAGACGGT ACC GACATT GT AAAAT GTCGT ACCGAGT GGCGTCCT AAAAACCCTGGTGCAAACGGTGCAAT TTCGACAGGCAAGACCTCGAACGGCAATTCAGTTTCTT
SEQ ID NO: 37 DNA sequence of [SEQ ID NO: 11]
AT GAAATTT AAAAAAAAATTT AAAATT GGGCGGATGCACGTT GACCCCTTT AACT ACATT AGT ATGCGCT ATCTGGTTGCCTT GATGAAT GAAGTGGCTTTT GATCAAGCCGAAATTTT GGAAAAAGAT ATT GACAT GAAAAACCTGCGTTGGATT ATTT AT AGTT GGGAT ATTCAGAT T GAAAAT AACATTCGCCTGGGGGAAGAAATT GAAATT ACCACT ATTCCCACCCACATGG ATAAATTTTATGCTTACCGGGACTTTATTGTTGAAAGCCGCGGAAATATTTTAGCCCGTG CT AAAGCCACCTTTTT GTT AATGG AT ATTACTCGCCTGCGTCCCATT AAAATTCCCCAAA ATCT GTCTTTGGCCT ATGGCAAAGAAAACCCCATTTTT GAT ATTT ACGACATGGAAATTC GGAACGATCTGGCTTTTATTCGCGACATTCAGTTACGTCGGGCCGATCTGGACAATAAC TTT CACATT AACAACGCCGT GT ACTTT GATTT GATT AAAG AAACCGTT GAT ATTT ACG AT A AAG ACATTTCCT ACATT AAATT G ATTT ACCGG AACG AAATTCGCG AT AAAAAACAAATT C AGGCTTTTGCCCGTCGGGAAGATAAAAGTATTGACTTTGCCCTGCGTGGCGAAGATGG TCGGG ACT ACT GTTT GGGG AAAATT AAAACT AACGT GTAA
SEQ ID NO: 38 DNA sequence of [SEQ ID NO: 12]
ATGTTCGATCGTAAATCAAAACGGCCATCCATGTTAATGGATAGCTTTGGGCTGGAACG GGTGGTTCAGGACGGCCTGGTGTTTCGGCAATCATTCAGCATTCGCAGTTATGAAATCT GTGCGGATCGCACCGCTTCCATGGAAACTGTGATGAACCACGTTCAGGAAACCAGCCT GAATCAATGTAAAAGTATTGGCTTACTGGATGACGGGTTTGGCCGTTCCCCCGAAATGT GCAAACGGGATCTGATTTGGGTGGTTACCCGCATGAAAATCATGGTGAACCGTTATCC GACCTGGGGCGAT ACT ATT GAAGTT AGCACCTGGCT GAGTCAGTCCGGT AAAATTGGT ATGGGGCGGGATTGGCTGATCTCTGACTGCAACACCGGTGAAATTTTGGTGCGCGCAA CT AGCGTTT ACGCCAT GAT GAATCAGAAAACCCGCCGTTTT AGT AAATTGCCCCAT GAA GTGCGTCAAGAATTTGCGCCGCACTTCTT AGATTCCCCACCCGCT ATCGAAGAT AAT GA CGGTAAACTGCAAAAATTCGATGTGAAAACCGGGGACTCTATTCGCAAAGGGTTGACTC CTGGCTGGTATGATTTAGACGTTAATCAGCATGTGTCTAACGTTAAATACATTGGCTGG ATTCTGGAATCAATGCCCACCGAAGTGCT GGAAACTCAAGAATT GT GT AGCCT GACCTT GGAATATCGTCGCGAATGCGGGCGTGATAGTGTGTTGGAAAGCGTGACCTCAATGGAC CCCTCCAAAGTGGGTGACCGCTTTCAATACCGTCACTTGTTACGGCTGGAAGATGGTG CGGACATCATGAAAGGGCGCACCGAATGGCGTCCCAAAAATGCGGGCACTAACGGTG CT ATT AGT ACCGGT AAAACTT G A
SEQ ID NO: 39 DNA sequence of [SEQ ID NO: 13]
ATGCATGACCGCAAATCGAAGCGTCCGTCCATGCTGATGGACAGTTTTGGGCTGGAGT CCATCGTCCAAGAAGGATTAGAGTTCCGTCAGTCCTTTAGCATCCGTTCATACGAGATC GGCACGGATCGT ACCGCTTCCATGGAGACGCTT AT GAACT ATTT ACAGGAAACGTCCTT AAATCATTGCAAAAGT ACCGGCATTCT GTT AGATGGATTCGGACGT ACGCCT GAGATGT GCAAGCGTGATTTAATCTGGGTCGTGACTAAAATGAAGATCAAAGTGAATCGCTACCCG GCGTGGGGGGACACGGTGGAAATTAATACGTGGTTTAGCCGCTTGGGAAAAATTGGCA AAGGTCGCGATTGGCTTATCAGCGACTGTAATACGGGTGAAATCCTGATCCGTGCAAC AAGCGCAT ACGCT ACT AT GAACCAGAAGACCCGTCGTTT AAGCAAGTT GCCAT ACGAAG T ACACCAGGAAATCGCGCCGTT GTTCGTT GACAGCCCCCCT GTT ATT GAGGACAAT GAT CTTAAGTTACATAAGTTTGAGGTAAAAACAGGCGACTCCATTCACAAAGGGCTGACCCC CGGATGGAATGATCTGGACGTCAATCAACAT GT AAGCAACGT GAAGT AT ATTGGGT GGA T CTT AG AAT CAATGCCT ACGG AAGTTTTAGAAACCCAAG AATT GT GTTCCTT AGCGCT G GAGTATCGTCGTGAATGCGGGCGCGACTCCGTACTGGAATCAGTCACGGCCATGGAC CCCACTAAGGTGGGTGGACGTTCGCAATATCAACACTTGCTTCGTCTTGAAGACGGTAC CGACATTGTAAAATGTCGTACCGAGTGGCGTCCTAAAAACCCTGGTGCAAACGGTGCA ATTTCGACAGGCAAGACCTCGAACGGCAATTCAGTTTCTTAA
SEQ ID NO: 40 DNA sequence of [SEQ ID NO: 14]
ATGGCTAACGCTGAACGTATGATCACCCGTGTTCACTCTCAGCGTGAACGTCTGAACGA
AACCCTGGTTTCTGAACGTAACGAAGTTCTGGCTCTGCTGTCTCGTGTTGAAGCTAAAG
GTAAAGGTATCCTGCAGCAGAACCAGATCATCGCTGAATTCGAAGCTCTGCCGGAACA
G ACCCGT AAAAAACTGG AAGGT GGTCCGTT CTTCG ACCTGCT G AAAT CT ACCCAGG AA
GCTATCGTTCTGCCGCCGTGGGTTGCTCTGGCTGTTCGTCCGCGTCCAGGTGTGTGGG
AGTACCTGCGTGTTAACCTGCACGCTCTGGTTGTTGAAGAACTGCAGCCGGCTGAATT
CCTGCACTTCAAAGAAGAACTGGTT GACGGT GTT AAAAACGGT AACTTCACCCTGGAAC
TGGACTTCGAACCGTTCAACGCTTCTATCCCGCGTCCGACCCTGCACAAATACATCGGT
AACGGTGTTGACTTCCTGAACCGTCACCTGTCTGCTAAACTGTTCCACGACAAAGAATC
TCTGCTGCCGCTGCTGAAATTCCTGCGTCTGCACTCTCACCAGGGTAAAAACCTGATGC
T GTCT GAAAAAATCCAGAACCT GAACACCCT GCAGCACACCCTGCGT AAAGCT GAAGAA
T ACCTGGCT GAACT G AAAT CT G AAACCCTGT ACG AAG AATTCG AAGCT AAATTCG AAG A
AATCGGTCTGGAACGTGGTTGGGGTGACAACGCTGAACGTGTTCTGGACATGATCCGT CTGCTGCTGGACCTGCTGGAAGCTCCGGACCCGTGCACCCTGGAAACCTTCCTGGGT
CGTGTTCCGATGGTTTTCAACGTTGTTATCCTGTCTCCGCACGGTTACTTCGCTCAGGA
CAACGTTCTGGGTT ACCCGGACACCGGT GGTCAGGTTGTTT ACATCCTGGACCAGGTT
CGTGCTCT GGAAATCGAAATGCTGCAGCGT ATCAAACAGCAGGGTCT GAACATCAAAC
CGCGTATCCTGATCCTGACCCGTCTGCTGCCGGACGCTGTTGGTACCACCTGCGGTGA
ACGTCTGGAACGTGTTTACGACTCTGAATACTGCGACATCCTGCGTGTTCCGTTCCGTA
CCGAAAAAGGTATCGTTCGTAAATGGATCTCTCGTTTCGAAGTTTGGCCGTACCTGGAA
ACCT ACACCGAAGACGCTGCT GTT GAACTGTCT AAAGAACT GAACGGT AAACCGGACC
TGATCATCGGTAACTACTCTGACGGTAACCTGGTTGCTTCTCTGCTGGCTCACAAACTG
GGTGTTACCCAGTGCACCATCGCTCACGCTCTGGAAAAAACCAAATACCCGGACTCTG
ACAT CT ACTGG AAAAAACTGG ACG ACAAATACCACTT CT CTTGCCAGTT CACCGCT G AC
ATCTTCGCTATGAACCACACCGACTTCATCATCACCTCTACCTTCCAGGAAATCGCTGG
TTCTAAAGAAACCGTTGGTCAGTACGAATCTCACACCGCTTTCACCCTGCCGGGTCTGT
ACCGTGTTGTTCATGGCATCGACGTATTCGACCCAAAATTCAACATCGTTTCTCCGGGT
GCTGACATGTCTATCTACTTCCCGTACACCGAAGAAAAACGTCGTCTGACCAAATTCCA
CT CT G AAATCG AAG AACTGCT GTACTCT GACGTT G AAAACAAAG AACACCT GTGCGTTC
T G AAAG ACAAAAAAAAACCG ATCCT GTT CACCATGGCT CGTCTGG ACCGT GTT AAAAAC
CTGTCTGGTCTGGTTGAATGGTACGGTAAAAACACCCGTCTGCGTGAACTGGCTAACCT
GGTTGTTGTTGGTGGT G ACCGTCGT AAAG AAT CT AAAG ACAACG AAG AAAAAGCT G AAA
T G AAAAAAAT GT ACG ACCT G ATCG AAG AAT ACAAACT G AACGGTCAGTTCCGTT GG AT C
TCTTCTCAGATGGACCGTGTTCGTAACGGTGAACTGTACCGTTACATCTGCGACACAAA
GGGTGCGTTCGTTCAGCCGGCGCTGTACGAGGCGTTCGGTCTGACCGTTGTTGAAGCT
ATGACCTGCGGTCTGCCGACCTTCGCTACCTGCAAAGGTGGTCCGGCTGAAATCATCG
TT CACGGT AAAT CT GGTTTCCACATCG ACCCGT ACCACGGT G ACCAGGCTGCT G ACAC
CCTGGCTGACTTCTTCACCAAATGCAAAGAAGACCCGTCTCACTGGGACGAAATCTCTA
AAGGTGGTCTGCAGCGTATCGAAGAAAAATACACCTGGCAGATCTACTCTCAGCGTCT
GCT GACCCT GACCGGT GTTT ATGGTTTTTGGAAACACGT AAGCAACCTGGACCGTCT G
GAAGCTCGTCGTTACCTGGAAATGTTCTACGCTCTGAAATACCGTCCGCTGGCTCAGG
CTGTTCCGCTGGCTCAGGACGACTAA
SEQ ID NO:82 UAAT4 (Lavandula x intermedia clone, AAT-4 alcohol acetyltransferase)
MAMIITKQILRPSSPTPQAFKNHKLSYLDQIQA
PIYIPLLFFYKNEESKYPDQISQRFKQSLSEILTI
FYPLAGTMRHNSFVDCNDRGVEFVEVRVHA
RLAQFIQDPKMEELKQLIPVDCISHTDDDFLLL
VKISYFDCGEWVGVCMSHKIGDGISLAAFMN
AWAATCRGESSSEIIHPSFDLALHFPPKDHLS
SASSFRVAIAQENIMTKRLVFDREKLEKLRKRI AASSDGVRDPSRVEAVSVFIWKSLIEAHKAES
HMTETPAVSIASHAVNLRPRTVPQMDQTFGN
CYAPASAVVSWDEDYVHHSRLRAALREIDDD
YINKVLKADNNYLTQDQIGDLFKPENSVLSSW
WRFPVYKVDFGWGKPVWVSTTTIQYMNLIIFT
STPSEDGIEAWVTTTHNFFQVLQANYNKLDT
SEQ ID NO:83 EcACP {E.coli Acyl Carrier Protein)
MSTI EERVKKI IGEQLGVKQEEVTN NASFVEDLGADSLDTVELVM ALEEEFDTEI PDEEAEKI TTVQAAIDYINGHQA
SEQ ID NO: 846803ACP ( Synechocystis Sp. PCC 6803 Acyl Carrier Protein)
M NQEI FEKVKKI VVEQLEVDPDKVTPDATFAEDLGADSLDTVELVM ALEEEFDI El PDEVAETI DTVGKAVEHIESK
SEQ ID NO:85 CIACP2 ( Cuphea lanceolata Acyl Carrier Protein)
MAAKPETVKKVCEIVKKQLALPDDSAVTGASKFSALGADSLDTVEIVMGLEEEFGISVEEES AQSIQTVQDAADLI E
SEQ ID NO:86 Amino acid sequence of CvFatBI T8
MRHSFSIRSYEIGADRTASIETLMNHLQETTINHCKSLGLHNDGFGRTPGMCKNDLIWVLTK MQIMVNRYPTWGDTVEINTWFSQSGKIGMASDWLISDCNTGEILIRATSVWAMMNQKTRR FSRLPYEVRQELTPHFVDSPHVIEDNDQKLRKFDVKTGDSIRKGLTPRWNDLDVNQHVSNV KYIGWI LESM PI EVLETQELCSLTVEYRRECGM DSVLESVTAVDPSENGGRSQYKH LLRLED GTDIVKSRTEWRPKNAGTNGAISTSTAKTSNGNSVS
SEQ ID NO:87 Amino acid sequence of CvFatBI T9 MIGADRTASIETLMNHLQETTINHCKSLGLHNDGFGRTPGMCKNDLIWVLTKMQIMVNRYP TWGDTVEINTWFSQSGKIGMASDWLISDCNTGEILIRATSVWAMMNQKTRRFSRLPYEVRQ ELTPHFVDSPHVIEDNDQKLRKFDVKTGDSIRKGLTPRWNDLDVNQHVSNVKYIGWILESM PI EVLETQELCSLTVEYRRECGM DSVLESVTAVDPSENGGRSQYKH LLRLEDGTDIVKSRT EWRPKNAGTNGAISTSTAKTSNGNSVS
SEQ ID NO:88 Amino acid sequence of CvFatBI T10
MTLMNHLQETTINHCKSLGLHNDGFGRTPGMCKNDLIWVLTKMQIMVNRYPTWGDTVEINT WFSQSGKIGMASDWLISDCNTGEILIRATSVWAMMNQKTRRFSRLPYEVRQELTPHFVDSP HVIEDNDQKLRKFDVKTGDSIRKGLTPRWNDLDVNQHVSNVKYIGWILESMPIEVLETQELC SLTVEYRRECGMDSVLESVTAVDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNAGTN GAISTSTAKTSNGNSVS
SEQ ID NO:89 Amino acid sequence of CvFatBI T11 MINHCKSLGLHNDGFGRTPGMCKNDLIWVLTKMQIMVNRYPTWGDTVEINTWFSQSGKIG MASDWLISDCNTGEILIRATSVWAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQK LRKFDVKTGDSIRKGLTPRWNDLDVNQHVSNVKYIGWILESMPIEVLETQELCSLTVEYRRE CGMDSVLESVTAVDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNAGTNGAISTSTAKT SNGNSVS
SEQ ID NO:90 Amino acid sequence of CvFatBI T12
MNDGFGRTPGMCKNDLIWVLTKMQIMVNRYPTWGDTVEINTWFSQSGKIGMASDWLISDC NTGEILIRATSVWAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLRKFDVKTGD SI RKGLTPRWN DLDVNQH VSN VKYIGWI LESM PI EVLETQELCSLTVEYRRECGM DSVLESV TAVDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNAGTNGAISTSTAKTSNGNSVS
SEQ ID NO:91 Amino acid sequence of CvFatBI T13
MCKNDLIWVLTKMQIMVNRYPTWGDTVEINTWFSQSGKIGMASDWLISDCNTGEILIRATSV WAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLRKFDVKTGDSI RKGLTPRWN DLDVNQHVSNVKYIGWILESMPIEVLETQELCSLTVEYRRECGMDSVLESVTAVDPSENGG RSQYKHLLRLEDGTDIVKSRTEWRPKNAGTNGAISTSTAKTSNGNSVS
SEQ ID NO:92 Amino acid sequence of CvFatBI T14 MKMQIMVNRYPTWGDTVEINTWFSQSGKIGMASDWLISDCNTGEILIRATSVWAMMNQKT RRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLRKFDVKTGDSIRKGLTPRWNDLDVNQHVS NVKYIGWI LESMPI EVLETQELCSLTVEYRRECGM DSVLESVTAVDPSENGGRSQYKHLLRL EDGTDIVKSRTEWRPKNAGTNGAISTSTAKTSNGNSVS SEQ ID NO:93 Amino acid sequence of CvFatBI T15
MTWGDTVEINTWFSQSGKIGMASDWLISDCNTGEILIRATSVWAMMNQKTRRFSRLPYEV RQELTPHFVDSPHVIEDNDQKLRKFDVKTGDSIRKGLTPRWNDLDVNQHVSNVKYIGWILE SMPIEVLETQELCSLTVEYRRECGMDSVLESVTAVDPSENGGRSQYKHLLRLEDGTDIVKS RTEWRPKNAGTNGAISTSTAKTSNGNSVS
SEQ ID NO:94 Amino acid sequence of CvFatBI T16
MWFSQSGKIGMASDWLISDCNTGEILIRATSVWAMMNQKTRRFSRLPYEVRQELTPHFVD SPHVIEDNDQKLRKFDVKTGDSIRKGLTPRWNDLDVNQHVSNVKYIGWILESMPIEVLETQE LCSLTVEYRRECGMDSVLESVTAVDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNAG TNGAISTSTAKTSNGNSVS
SEQ ID NO:95 Amino acid sequence of CvFatBI T17
MASDWLISDCNTGEILIRATSVWAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQK LRKFDVKTGDSIRKGLTPRWNDLDVNQHVSNVKYIGWILESMPIEVLETQELCSLTVEYRRE CGMDSVLESVTAVDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNAGTNGAISTSTAKT SNGNSVS
SEQ ID NO:96 Amino acid sequence of CvFatBI T18 MTGEILIRATSVWAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLRKFDVKTGD SI RKGLTPRWN DLDVNQH VSN VKYIGWI LESM PI EVLETQELCSLTVEYRRECGM DSVLESV TAVDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNAGTNGAISTSTAKTSNGNSVS SEQ ID NO:97 Amino acid sequence of CvFatBI T19
MVWAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLRKFDVKTGDSIRKGLTPR WN DLDVNQH VSN VKYIGWI LESM PI EVLETQELCSLTVEYRRECGM DSVLESVTAVDPSEN GGRSQYKHLLRLEDGTDIVKSRTEWRPKNAGTNGAISTSTAKTSNGNSVS SEQ ID NO:98 Amino acid sequence of CvFatBI T20
MRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLRKFDVKTGDSIRKGLTPRWNDLDVNQHV
SNVKYIGWILESMPIEVLETQELCSLTVEYRRECGMDSVLESVTAVDPSENGGRSQYKHLL
RLEDGTDIVKSRTEWRPKNAGTNGAISTSTAKTSNGNSVS SEQ ID NO:99 Amino acid sequence of CvFatBI T21
MQELTPHFVDSPHVIEDNDQKLRKFDVKTGDSIRKGLTPRWNDLDVNQHVSNVKYIGWILE
SMPIEVLETQELCSLTVEYRRECGMDSVLESVTAVDPSENGGRSQYKHLLRLEDGTDIVKS
RTEWRPKNAGTNGAISTSTAKTSNGNSVS SEQ ID NQ:100 Amino acid sequence of CpFatB1_Wild Type
MVAAAASSACFPVPSPGASPKPGKLGNWSSSLSPSLKPKSIPNGGFQVKANASAHPKANG SAVTLKSGSLNTQEDTLSSSPPPRAFFNQLPDWSM LLTAITTVFVAPEKRWTM FDRKSKR PNMLMDSFGLERVVQDGLVFRQSFSIRSYEICADRTASIETVMNHVQETSLNQCKSIGLL DDGFGRSPEMCKRDLIWVVTRMKIMVNRYPTWGDTIEVSTWLSQSGKIGMGRDWLISDCN TGEILVRATSVYAMMNQKTRRFSKLPHEVRQEFAPHFLDSPPAIEDNDGKLQKFDVKTGD SIRKGLTPGWYDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLTLEYRRECGRDSVLE SVTSMDPSKVGDRFQYRHLLRLEDGADIMKGRTEWRPKNAGTNGAISTGKT SEQ ID NQ:101 Amino acid sequence of CaFatB3_Wilde Type
MVAAAASSAFFSVPVPGTSPKPGKFRIWPSSLSPSFKPKPIPNGGLQVKANSRAHPKANG SAVSLKSGSLNTQEDTSSSPPPRTFLHQLPDWSRLLTAITTVFVKSKRPDMHDRKSKRPD MLMDSFGLESIVQEGLEFRQSFSIRSYEIGTDRTASIETLMNYLQETSLNHCKSTGILLD GFGRTPEMCKRDLIWVVTKM KIKVNRYPAWGDTVEINTWFSRLGKIGKGRDWLISDCNTG El LI RATSAYATM NQKTRRLSKLPYEVHQEI APLFVDSPPVI EDN DLKLH KFEVKTGDSI HKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRRECGRDSVLESV TAMDPTKVGGRSQYQHLLRLEDGTDIVKCRTEWRPKNPGANGAISTGKTSNGNSVS SEQ ID NO:102 Bacillus fragilis CAH09236.1 MSDDKKIGSYKFIAEPFHVDFNGRLTMGVLGNHLLNCAGFHASERGFGIATLNEDNYTWVL SRLAI DLEEM PYQYEEFTVQTWVEN VYRLFTDRN FAI I DKDGKKIGYARSVWAM I N LNTRKP ADLLTLHGGSI VDYVCDEPCPI EKPSRI KVATDQPCAKLTAKYSDI Dl NGH VNSI RYI EH I LDLF PIDLYKSKRIQRFEMAYVAESYYGDELSFFEEEVSENEYHVEIKKNGSEVVCRAKVKFV
SEQ ID NO:103 VAAT (Fragaria vesca Alcohol acyltransferase)
MEKI EVSIISKHTIKPSTSSSPLQPYKLTLLDQLTPPSYVPMVFFYPITGPAVFNLQTLADLRH
ALSETLTLYYPLSGRVKNNLYIDDFEEGVPYLEARVNCDMNDFLRLPKIECLNEFVPIKPFSM
EAISDERYPLLGVQVNIFNSGIAIGVSVSHKLIDGRTSDCFLKSWCAVFRGSRDKIIHPNLSQ
AALLFPPRDDLPEKYARQMEGLWFVGKKVATRRFVFGAKAISVIQDEAKSESVPKPSRVQA
VTSFLWKHLIATSRALTSGTTSTRLSIATQVVNIRSRRNMETVWDNAIGNLIWFAPAILELSHT
TLEISDLKLCDLVNLLNGSVKQCNGDYFETFMGKEGYGSMCEYLDFQRTMSSMEPAPEIYL
FTSWTNFFNQLDFGWGRTSWIGVAGKIESAFCNLTTLVPTPCDTGIEAWVNLEEEKMAMLE
QDPQFLALASPKTLISRY
SEQ ID NO:104 VpAATI ( Vasconcellea pubescens Alcohol acyltransferase 1)
MAEKASSLMFNVRRHEPELITPAKPTPREIKLLSDIDDQDGLRFQVPIIQFYKNNSSMQGKN
PAKIIKSALAETLVHYYPLAGRLREGFGRKLMVECTGEGILFIEADADVTLHEFGDDLPPPFP
CLVELLYDVPGSSGIIDTPLLLIQVTRLKCGGFIFALRLNHTMSDASGLVQFMTAVGEMARG
QRSLSIQPVWERHLLNARDPPRVTHIHHEYDDLEDTKGTIIPLDDMVHRSFFFGPSEMAAIR
RLVPAHFHRSTTSEVLTAYLWRCYTIALQPDPEEEMRVICVVNSRTKLNPPLPTGFYGNGIA
FPAAISQAKKICENPFGYTLQLVKQTKVDVTEEYMRSAADLMAMKGRPHFTWRRYMVSDV
TRAGFGLVDFGWGRPEPVYGGPAKGGVGPIPGVTSFFVPFKNRKGEKGIVVPTCLPTPAM
ERFAKLMNEILQNQLLVSAEENKSVFIVSAI
SEQ ID NO:105 AtHPFT ( Arabidopsis thaliana Omega-hydroxypalmitate O-feruloyl transferase)
LVSPLHPTPKRSFFLSNIDRMLNYNIPTVYFFAANPEYPPPVAAANLKLALQKLLVPYDFMAG
RLKMNADSGRLEIDCNGAGAGFVVASSALSLEQIGDLVRPNLGFRDLAVQTMIKKDDHDPM
FILQLTSFACGGFAIGLCVNHILLDGMSAKAFNQNLASQAFHDDRPLAVVPCFDRRLMAARS
PPRPAFDHPEFFKPDLWGPAVFDCEREELEYRVFQLDPTHITLLKEKALHLQESGSRISSLT
VAAALIWKCKALSKEYKDDKDKVSTLLNVMDLRARLKPPLPADYCGNALLVAYATGRCGEL
EFWEVAKMVGEGPRRVTDEYAKSAIDWLEINKGKGVPRGDYMVSSWLRLGFEDVVFPWG
KALHSGPLVSHRKDICWLFPTPHG
SEQ ID NO:106 CcVS ( Citrus Clementina Vinorine synthase)
MEIGIVSREVVRPSSLNVHLLKPFRISLLDQLTPTTFAPLVLFYPMKSTHLKGTQISTQLKESL
SKTLEHFYPLAGRVRDNLIINDYDEGVPYIETRVNTHLFEFLQNPPMELLNQCLPYPPFRYQ
PNPDRVPQVAVQLNTFDCGGIALGLSFSHKINDGATTSAFLRSWAANSRGACHKAVKYKNL
SEASMIFPPQNPSPNHHLSVMEKIWFREAKYKTRRFVFDAKAIATLRSECKGERVPNPTRIE
ALSAFILKSAMLASRSTASSRFVLHQAVNLRRLTEPRLSPCSVGNLFLWATAAYNMEHAAE
MELHGLVARMKQAVGKINSEYLKTLHGDEGFPKVCEYIKRIEEVSAHKNLEAFTFSSWVKF
GFNEVDFGWGNPIWSGIFGEVGSNSFRNLTFFKETRSANYDNAVEAWVTLDEKIMSLLEHD
PQFLAFASPNPSILLFDVCRF SEQ ID NO:107 CsVS ( Citrus sinensis Vinorine synthase-like)
MEIGIVSREVVRPSSLNVHLLKPFKISLLDQLTPTTFAPLVLFYPMKSTHLKGTQISTQLKESL SKTLEHFYPLAGRVRDNLIINDYDEGVPYIETRVNTHLFEFLQNPPMELLNQCLPYPPFSYQ PNPDRVPQVAVQLNTFDCGGIALGLCFSHKINDGATTSAFLRSWAAFSRGAYHKAVKYKNI SEASMIFPPQNPSPNHRLSVMEKIWFREAKYKTRRFVFDAKAIATLRSECKGERVPNPTRIE ALSAFILKSAMLASRSTASSRFVLHQAVNLRRLTEPRLSPCSVGNLFLWATAAYNMEHAAE MELHGLVARMKQAVGKINSEYLKTLHGDEGFPKVCEYIKRIEEVSAHKNLEAFTFSSWVKF GFNEVDFGWGNPIWSGIFGEVGSNSFRNLTFFKETRSANYDNAVEAWVTLDEKIMSVLEH DPQFLAFASPNPSILLFDVCGF
SEQ ID NO:108 LaAATI ( Lavandula angustifolia Putative alcohol acyltransferase 1)
MKIEIKESTMVRPAAETPSGSLWLSNLDLLSPANYHTLSVHFYSHDGSANFFDATALKEALS RALVDFYPYAGRLKLNKENRLEIECNGEGILLVEAECSGALDELGDFTPRPELNLIPKVDYSK GMSTYPLMLFQITRFKCGGVALGVANEHHLSDGVAALHFINTWAHYSRGVPAPSPPPHFD RTALSARNPPQPQFSHAEYQPPPTLENPLPATDIAHSKFKLTRAQLNSLKAKCAAGDSDGH TNGTANGKSDANGTADGKSDANGTANGKSAAKRYSTFEVLAGHIWRSVCTARGLPAEQET KLHIPFDGRSRLNLPPGYFGNAIFFATPIATCGEIESNSLSYAVRRVGDGIARLDEEYLKSSL DFLELQPDISKLAQGAHSFRCPNLWVISWVWLPIYEPDFGWGKAVHMGPWAAPFEGKSYL LPNPENDGSLFVSITLHKQHMERFQKLFYEI
SEQ ID NO:109 LaAAT2 ( Lavandula angustifolia Putative alcohol acyltransferase 2) MGEVANDEKKVGSVKTFNPTYVKPKKPIGRKECQLVTFDLPYLAFYYNQKLLIYNGGDDFY GAVEKLKDGLAVVLEEFHQLAGKLEKDEDGVFKVVYDDDMEGVEVVEAAAEGVEVADITAE EGFSKFKELLPYNGVLNLEGLQRPLLAVQLTKLKDGMVMGCAFNHAILDGTSTWHFMSSW AAICSGATSVSVPPFLERTKARNTRVKLDLSQPSDAPEHANTASNGDTPVNPLLRGKVFKF SESVIDQIKSKVNAGSDSSKPFSTFQSLSAHVWQAVTRARELGPTDYTVFTVFADCRKRVD PPMPESYFGNLIQAIFTVTGAGLILANPVEFGAGLIRGAIESHNAEAINKRNEEWESKPVIFQY KDAGVNCVAVGSSPRFQVYGVDFGWGSPESVRSGLNNRFDGMVYLYPGKSGGRSIDVEL SLEAKCM EKLEKDKEFLM EA
SEQ ID NO:110 LaAT1 ( Lavandula angustifolia Rosmarinic acid synthase (LaAT1)
MKIEIKESTMVRPAAETPSGSLWLSNLDLLSPANYHTLSVHFYSHDGSANFFGAAALKEALS RALVDFYPYAGRLKLNKENRLEIECNGEGILLVEAECGGALDELGDFTPRPQLNLIPKVDYS KGMSAYPLM LFQITRFKCGGVALGVANEHHLSDGVAALHFINTWAHYSRGVPAPSPPPHF DRTALSARNPSKPQFSHAEYQPPPTMENPLPYTDIAHSKFKLTRAQLNSLKAKCAAADSNA HTNSIANGKSDANGTANGKSDANRTANSAAAAKRYSTFEVLAGHIWRCVCTARGLPAEQE TKLHIPFDGRSRLNLPPGYFGNAIFFATPIATCGEIESNSLSYAVRRVGDGIARLDEEYLKSSL DFLELQPDISKLAQGAHSFRCPNLWVISWVWLPIYEPDFGWGKAVHMGPWAAPFEGKSYL LPNPENDGSLFVSITLHKQHMERFQKLFYEI SEQ ID NO:111 NtBBT ( Nicotiana tabacum Benzyl alcohol O-benzoyltransferase)
MLIPPAKPTPHEFKFLSDIDDQQSLRFQIPLIQFYRRNPSVEGKDQVKVIRDAIAKALVFYYPF AGRLRERATSKLVVECTGEGVVFIEADADVALEQFGDELYPPFPCLGELLYDVPGTSGIINC PLLLIQVTRLRCGGFVFAIRFNHAMSDAAGMFQFMSAVAELARGAEAPTVLPVWRRDLLSA RDPPRVTCTHHEFGVVPVTTFTPPANMVERGFFFSPADIAALRSTLPPHLRRRTSAFQIAVA CAWRCRVIALSPDPSEEIRISCMVNCLNRFDPPLPEGYYGNAVVYPAAVAAAGRLCASPLE
YAVELVRSAKSQATEEYVKSVADLMVMRGRPLFKAAGTFLASDVTRAGFEQVDFGWGE
SEQ ID NO:112 NtSHCT (Nicotians tabacum Shikimate O hydroxycinnamoyltransferase)
MLTSALARTLSEFYPVAGRLKKDGNGRVEIDCSGEGAVFVEAEADGEIDDLGDFSPNPNICL
APKVDYSQGISSFPLLLVQVTRFKCGGVCLGVAMDHQVKDGISALHIIHTWCDIARGLDIAVP
PYMDRRVLAAREPPQPKFEHVEFHPPPPLKNSEAHTNGSETKFAVLKLTREQLIILKGNSQE
DDGKRSPYSSFEALTGHVWRCICKARRLPEDQETKLTIIIDGRSRLRPPLPPGYFGNAVFKA
THIALSREVESNPLKYAVCKVHEALTRMDDEYLRSAIDYLEVEGGLGPNARGTGLYKSPNL
GITSWARLPFYEADFGWGRPFHVGLGAIPAEGHLIVM PSPTNDGGLSMAIALPEEQMKMFE
K
SEQ ID NO:113 RsVS ( Rauvolfia serpentina Vinorine synthase)
MAQILASHLIKPSSPTPNTFKKHKLSVLDQISPPAYLTLIFFYQDLESNQHEEISRRLKQSLSEI
LTIFYPLAGTVHRNSFVDCNDRGAEFVEARVHGGLSKFVQNPKMEELEQLLPADFSSHTDN
PILSVRISYFDCGGIAVGVCFPHKIGDTSSFATFMNAWAATCRGEASRITPPSFDLALRFPPR
ESLASGFYLGISGEKIVTRRLVFEREKVEKIRKEASRNHEVKDPSRVEAISSLLWRSFIEAHK
KVDKETTSFPTSHMVSMRRRAVPPVPDHGFGNCFTLAPAMASNEEEAEEDGVLVSRLRAT
IRGVDEDYIKAISDDEFIKEVLGQIGDFFKPGNCIFTSWLRFPLYEVDFGWGKPARVCTATMP
CMNLVILMPTPPSRDDGGVEAWVNVADEQ
SEQ ID NO:114 SsMMT ( Salvia splendens Malonyl-coenzyme:anthocyanin 5-0- glucoside-6'"-0-malonyltransferase)
MLVFFDMPFYNYATDSLLFFEFPYSKPHFLNAIVPHLKKSLSLALSHFLPLAGNIIHPLNPQD
MPLLRYVSTDSVPFTVSESEADFSRLTGNQPRDCDEYYAFAPLLPRAAPSGTSISCPVLALQ
VTLFPGKGLSIGIVTRHAVADGSTTVSFIRAWAMISEIGPDEGSVEARLTEAGLLPFLDRKTV
TN I NGLDSVH WEKKVKSSCSAEPLPM KFPI NKVRATFVLRRDEI EKLKN LVVAKSDGGVAH V
SSFTVVCALVWACSARAAASGGEEVAEEEEEYFMFVADFRGRLSPALPANYFGNCVAPAK
AVLKHGQVKGDDGFVTAAEAIGAAVKGLIMSSDKKGILDVAEDWLEEYGNLAGKRRHGVA
GSPRFDFYEADFGWGKP

Claims

1. A method of producing a derivative of 1-octanol, wherein the method comprises: providing a first enzyme to a microorganism that produces 1-octanol, wherein the first enzyme modifies the hydroxyl group of the 1-octanol to form a derivative of 1-octanol; and harvesting the derivative of 1-octanol from the microorganism.
2. The method of claim 1, wherein the microorganism is a bacterium.
3. The method of claim 2, wherein the bacterium is a heterotrophic bacterium.
4. The method of claim 2 or 3, wherein the bacterium is selected from the group consisting of Escherichia, Halomonas and Cyanobacterium.
5. The method of claim 4, wherein the Escherichia is Escherichia coli (E. coli), for example B strain C43 (DE3) or K-12 strain BW25113.
6. The method of claim 4, wherein the cyanobacterium is Synechocystis sp (for example PCC 6803).
7. The method of any preceding claim, wherein two or more first enzymes are provided.
8. The method of any preceding claim, wherein the first enzyme is heterologous or homologous.
9. The method of claim 8, wherein the first heterologous enzyme is selected from the group consisting of an alcohol acetyltransferase and a glycosyltransferase.
10. The method of claim 9, wherein the alcohol acetyltransferase is selected from the group consisting of CAT (SEQ ID NO: 1), SAAT (SEQ ID NO: 2) and ATF1 (SEQ ID NO: 3), or a variant thereof, and/or wherein the glycosyltransferase is selected from the group consisting of AdGT4 (SEQ ID NO: 4), VvGT1 (SEQ ID NO: 5), MtGT1 (SEQ ID NO: 6), AtGT1 (SEQ ID NO: 7) and MtH2 (SEQ ID NO: 8), or a variant thereof.
11. The method of any preceding claim, wherein the derivative of 1-octanol is octyl acetate and/or octyl glucoside.
12. The method of any preceding claim, wherein the method further comprises providing a second enzyme to the microorganism.
13. The method of claim 12, wherein the second enzyme is heterologous.
14. The method of claim 13, wherein the second enzyme is selected from the group consisting of thioesterase, carboxylic acid reductase, and phosphopantetheinyl.
15. The method of claim 14, wherein the thioesterase is a C8-preferring thioesterase.
16. The method of claim 15, wherein the C8-preferring thioesterase is selected from the group consisting of CpFatBI (SEQ ID NO: 9), CaFatB3 (SEQ ID NO: 10), Tes3 (SEQ ID NO: 11), or a variant thereof.
17. The method of claim 16, wherein the variant is CpFatB1-4 (SEQ ID NO: 12) or CaFatB3-5 (SEQ ID NO: 13), or a variant thereof.
18. The method of claims 14 or 15, wherein the thioesterase enzyme comprises or consists of a truncated SEQ ID NO: 15 or SEQ ID NO: 16, or a variant thereof.
19. The method of claim 18, wherein the thioesterase enzyme comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO: 90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 or a variant thereof.
20. The method of claim 12, wherein the second enzyme is homologous.
21. The method of claim 20, wherein the second homologous enzyme is aldehyde reductase.
22. The method of any preceding claim, wherein the method further comprises providing a third enzyme to the microorganism.
23. The method of claim 22, wherein the third enzyme is heterologous or homologous.
24. The method of claim 23, wherein the third heterologous enzyme is sucrose synthase.
25. The method of claim 24, wherein the sucrose synthase is Arabidopsis thaliana sucrose synthase, optionally wherein the Arabidopsis thaliana sucrose synthase is according to SEQ ID NO: 14, or a fragment or variant thereof.
26. The method of any preceding claim, wherein the first, second, and/or third enzyme is provided to the microorganism directly or indirectly.
27. The method of claim 26, wherein the first, second, and/or third enzyme is provided to the microorganism indirectly by an expression vector comprising a nucleic acid encoding the enzyme.
28. The method of any preceding claim, wherein the method further comprises supplying the microorganism with a precursor of 1-octanol.
29. The method of claim 28, wherein the precursor is selected from the group consisting of octanoic acid, glucose, octanoyl-ACP, and octanal.
30. A vector for use in a method according to any one of claims 1 to 29.
31. The vector for use according to claim 30, wherein the vector comprises a nucleic acid encoding a first enzyme, and optionally encoding a second and/or third enzyme, wherein the first enzyme modifies the hydroxyl group of the 1-octanol to form a derivative of 1-octanol.
32. A microorganism for use in a method according to any one of claims 1 to 29.
33. The microorganism of claim 32, wherein the microorganism comprises:
(i) a first enzyme, and optionally a second and/or third enzyme, wherein the first enzyme modifies the hydroxyl group of the 1-octanol to form a derivative of 1-octanol; or
(ii) an expression vector comprising a nucleic acid encoding a first enzyme, and optionally a nucleic acid encoding a second and/or third enzyme, wherein the first enzyme modifies the hydroxyl group of the 1-octanol to form a derivative of 1-octanol; or
(iii) a combination of (i) and (ii).
34. A thioesterase enzyme, wherein the thioesterase enzyme comprises or consists of a truncated SEQ ID NO: 15 or SEQ ID NO: 16, or a variant thereof.
35. The thioesterase enzyme of claim 34, wherein the enzyme comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO: 90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 or a variant thereof.
36. Use of the thioesterase enzyme of claims 34 or 35 in the method according to any one of claims 1 to 29.
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