Yeast cells and methods for producing E8,E10-dodecadienyl coenzyme A, cyproterone acetate and its derivatives

Abstract

This invention relates to yeast cells engineered for the production of E8,E10-dodecadienyl coenzyme A, codlemone (E8,E10-dodecadien-1-ol), and optionally its derivatives E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienal. Methods for producing E8,E10-dodecadienyl coenzyme A, codlemone (E8,E10-dodecadien-1-ol), and optionally its derivatives E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienal are also provided. Nucleic acid constructs for obtaining such yeast cells are also provided.

Description

Technical Field

[0001] This invention relates to yeast cells engineered for the production of E8,E10-dodecadienyl coenzyme A, codlemone (E8,E10-dodecadien-1-ol), and optionally its derivatives E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienal. Methods for producing E8,E10-dodecadienyl coenzyme A, codlemone (E8,E10-dodecadien-1-ol), and optionally its derivatives E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienal are also provided. Nucleic acid constructs for obtaining such yeast cells are also provided. Background Technology

[0002] Integrated Pest Management (IPM) is expected to play a major role in increasing crop yields, minimizing environmental impact, and achieving organic food production. IPM uses alternative pest control methods, such as using pheromones to disrupt mating, using pheromones for mass trapping, and using beneficial insects.

[0003] Pheromones comprise a diverse group of chemicals that insects (like other organisms) use in various situations to communicate with individuals of the same species, including mate attraction, alarm, tracking tagging, and aggregation. Insect pheromones associated with finding distant mates are already being used in agricultural and forestry applications for pest monitoring and control, serving as a safe and environmentally friendly alternative to pesticides.

[0004] Pheromonees represent a healthy and environmentally friendly alternative to pesticides. Distributing sex pheromones in fields or orchards disrupts insect communication and prevents mating; therefore, no fertilized eggs are laid, and larval damage to crops is avoided. This method is known as "mating disruption." Pheromonees are an attractive alternative to pesticides because they are biodegradable, species-specific compounds that do not harm beneficial species or humans.

[0005] The use of insect pheromones for pest control only became possible after the industrial-scale synthesis of pheromones began several decades ago. However, the high price of chemically synthesized pheromones remains a major obstacle to their widespread use in agriculture and forestry. Another drawback of the chemical production of pheromones is the need for toxic chemicals as precursors, catalysts, and solvents, as well as the generation of significant amounts of organic waste during purification. Therefore, current production methods based on complex chemical synthesis make the products prohibitively expensive for widespread use in many potential applications in agriculture and forestry.

[0006] Compared to chemical production methods, bioproduction methods offer several advantages. First, all reactions are carried out in fermenters at ambient temperatures using engineered cells, rather than through multiple chemical reaction steps requiring different precursors, catalysts, and conditions (typically high temperature and high pressure). Furthermore, engineered cells use inexpensive, renewable materials such as sugars or vegetable oils, rather than multiple expensive specialty chemicals as precursors. While chemical reactions typically have low specificity and therefore require purification of intermediate compounds and large-scale purification of the final product, enzymatic bioreactions are generally highly specific, and byproduct formation is limited, reducing the use of organic solvents and other toxic chemicals for purification. Additionally, specific stereochemistry often important for pheromone activity can be very difficult to achieve chemically, while enzymatic methods can utilize enzymes specific to either the cis or trans isomers.

[0007] The specific pheromone of interest is cetame, a diunsaturated fatty alcohol with the formula E8,E10-dodecadien-1-ol (E8,E10-C12:OH, CAS nr. 33956-49-9). Cetame is a sex pheromone component in many species and is the primary sex pheromone of the codling moth (Cydia pomonella), a major pest of apples, pears, plums, and other fruits.

[0008] Ding et al. (2014) disclosed plant cells expressing desaturases and tested them to determine whether they could produce moth pheromones. Using degenerate PCR, three desaturases were identified in the codling moth (Ding et al., On the way of making plants smell like moths – a synthetic biology approach. Lund University, Faculty of Science, Department of Biology).

[0009] Therefore, there is a need for biological methods to produce insect pheromones, particularly chlorpheniramine. In addition to the benefit of low cost, fermentation methods are inherently less dangerous and more environmentally friendly than chemical synthesis. Summary of the Invention

[0010] As defined in the claims of this invention.

[0011] This article provides yeast cells capable of producing E8,E10-dodecadienyl-CoA and optionally E8,E10-dodecadien-1-ol, said yeast cells expressing at least one heterodesaturase capable of introducing one or more double bonds into a 12-carbon acyl-CoA fatty acid chain, thereby converting said fatty acyl-CoA into desaturated fatty acyl-CoA, wherein at least a portion of said desaturated fatty acyl-CoA is E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA).

[0012] This article provides yeast cells capable of producing E8,E10-dodecadien-1-ol, which express:

[0013] i) at least one heterologous desaturase capable of introducing one or more double bonds into a fatty acyl-CoA with a carbon chain length of 12, thereby converting said fatty acyl-CoA into desaturated fatty acyl-CoA, wherein at least a portion of said desaturated fatty acyl-CoA is E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA); and

[0014] ii) At least one heterologous fatty acyl-CoA reductase (EC 1.2.1.84) capable of converting at least a portion of the desaturated fatty acyl-CoA into desaturated fatty alcohol, wherein the fatty acyl-CoA reductase is capable of converting at least a portion of the E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA) into E8,E10-dodecadien-1-ol.

[0015] Also provided is a method for producing E8,E10-dodecadienyl coenzyme A and optionally E8,E10-dodecadien-1-ol in yeast cells, the method comprising the steps of providing yeast cells and incubating the yeast cells in a culture medium, wherein the yeast cells express:

[0016] i) at least one heterologous desaturase capable of introducing one or more double bonds into a fatty acyl-CoA with a carbon chain length of 12, thereby converting said fatty acyl-CoA into desaturated fatty acyl-CoA, wherein at least a portion of said desaturated fatty acyl-CoA is E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA); and

[0017] ii) Optionally, at least one heterologous fatty acyl-CoA reductase (EC 1.2.1.84) capable of converting at least a portion of the desaturated fatty acyl-CoA into a desaturated fatty alcohol, wherein the fatty acyl-CoA reductase is capable of converting at least a portion of the E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA) into E8,E10-dodecadien-1-ol.

[0018] This produces E8,E10-dodecadienyl coenzyme A and optionally E8,E10-dodecadien-1-ol.

[0019] Nucleic acid constructs for modifying yeast cells are also provided, the constructs comprising:

[0020] i) at least one first polynucleotide encoding at least one heterodesaturase capable of introducing one or more double bonds into a 12-carbon acyl-CoA fatty acid chain, thereby converting the fatty acid-CoA into desaturated fatty acid-CoA, wherein at least a portion of the desaturated fatty acid-CoA is E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA); and

[0021] ii) Optionally encoding a second polynucleotide of at least one heterologous fatty acyl-CoA reductase (EC 1.2.1.84), said at least one heterologous fatty acyl-CoA reductase being capable of converting at least a portion of said desaturated fatty acyl-CoA into desaturated fatty alcohol, said fatty acyl-CoA reductase being capable of converting at least a portion of said E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA) into E8,E10-dodecadien-1-ol.

[0022] A method for monitoring the presence of harmful organisms or interfering with their mating is also provided, the method comprising the following steps:

[0023] i) E8,E10-dodecadien-1-ol and optionally E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienal are produced by the methods described herein.

[0024] ii) Formulating the E8,E10-dodecadien-1-ol and optionally the E8,E10-dodecadienyl acetate and / or the E8,E10-dodecadienal into a pheromone composition; and

[0025] iii) Use the pheromone composition as an integrated pest management composition.

[0026] This article also provides E8,E10-dodecadienyl coenzyme A, E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienal that can be obtained by the methods described herein.

[0027] This article also provides a component kit, which includes instructions for use and:

[0028] a) the yeast cells described herein; and / or

[0029] b) The nucleic acid constructs for modifying yeast cells described herein and optionally the yeast cells to be modified, wherein, after expression of the polynucleotide contained in the nucleic acid construct, the modified yeast cells are able to produce E8,E10-dodecadienyl-CoA and optionally E8,E10-dodecadien-1-ol. Attached Figure Description

[0030] Figure 1. A biosynthetic pathway for the production of clodomyr (E8, E10-C12:OH) in yeast is proposed. ACC: Acetyl-CoA carboxylase; FA: Fatty acid; FAS: Fatty acid synthase; TE: Thioesterase; FAA: Fatty acyl-CoA synthase; L: Lipid; FAE: Fatty acid ester; FAD: Fatty acyl desaturase; FAR: Fatty acyl reductase; Comp.β-ox.: Complete β-oxidation.

[0031] Figure 2. GC-MS analysis of FAME extracts from yeast transformed with (A) empty plasmid or (B) vector containing Cpo_CPRQ (with added 12:Me), (C) mass spectra of E9-12:Me; GC-MS analysis of FAME extracts from yeast transformed with (D) empty plasmid or (E) Cpo_CPRQ (with added E9-12:Me), (F) mass spectra of E8, E10-12:Me. Detailed Implementation

[0032] definition

[0033] Biopesticides: The term "biopesticide" is an abbreviation for "biological pesticide" and refers to several types of pest control interventions: through predation, parasitism, or chemical relationships. In the European Union, a biopesticide is defined as "a pesticide based on microorganisms or natural products." In the United States, the EPA defines it as "a pesticide that includes naturally occurring substances that control pests (biochemical pesticides), microorganisms that control pests (microbial pesticides), and plant-produced pesticides containing added genetic material (plant-incorporated protectants) or PIPs." This publication specifically relates to biopesticides that contain natural products or naturally occurring substances. They are typically produced by growing and concentrating naturally occurring organisms and / or their metabolites (including bacteria and other microorganisms, fungi, nematodes, proteins, etc.). They are generally considered an important component of integrated pest management (IPM) programs and have received considerable practical attention as an alternative to synthetic chemical plant protection products (PPPs). The Handbook of Biological Control Agents (2009: formerly the Handbook of Biological Pesticides) provides an overview of available biological pesticides (and other biology-based control products).

[0034] Turbidity concentration: This term is used herein to refer to the concentration of a surfactant, particularly a nonionic or glycol solution, in a solution above which, at a given temperature, the mixture of the surfactant and the solution begins to separate into two phases, thus becoming turbid. For example, the turbidity concentration of a surfactant in an aqueous solution at a given temperature is the minimum concentration of the surfactant required to produce two phases when mixed with the aqueous solution. The turbidity concentration can be obtained from the surfactant manufacturer or determined experimentally by creating a dosing profile and measuring the concentration at which the mixture separates into phases.

[0035] Cloud point: The cloud point of a surfactant, particularly a nonionic solution or glycol solution, in a solution such as an aqueous solution, is the temperature at which the mixture of the surfactant and the solution (e.g., the aqueous solution) begins to separate into two phases and becomes turbid. This behavior is characteristic of nonionic surfactants containing polyoxyethylene chains, which exhibit solubility in water that is opposite to temperature behavior, thus becoming "turbid" at a certain point as temperature increases. Diols exhibiting this behavior are called "cloud point diols." The cloud point is affected by salinity and is generally lower in fluids with higher salinity.

[0036] Codmonol: The term refers to a diunsaturated alcohol having the formula E8,E10-dodecadien-1-ol (E8,E10-C12:OH). Codmonol is a major sex pheromone component in many species, particularly the codling moth (Cydia pomonella), which belongs to the order Lepidoptera and is a major pest of apples, pears, plums, and other fruits. The terms "codmonol," "E8,E10-dodecadien-1-ol," and "E8,E10-C12:OH" are used interchangeably herein.

[0037] Desaturated: The term “desaturated” will be used interchangeably with “unsaturated” in this document and refers to a compound containing one or more carbon-carbon double or triple bonds.

[0038] Ethoxylation and propoxylation C 16 -C 18 Alcohol-based defoamers: The term refers to a group of polyethoxylated nonionic surfactants that comprise or consist primarily of the following: C 16 -C 18 Ethoxylated and propoxylated alcohols, such as CAS number 68002-96-0, also known as C 16 -C 18 Alkyl alcohol ethoxylates, propoxylates, or C 16 -C 18 Alcohol-ethoxylated and propoxylated polymers.

[0039] Extractant: As used herein, the term "extractant" refers to a nonionic surfactant (such as an antifoaming agent) that promotes the recovery of hydrophobic compounds generated during fermentation, particularly polyethoxylated surfactants selected from: polyethylene polypropylene glycol, mixtures of polyether dispersions, antifoaming agents containing polyethylene glycol monostearate such as dimethicone, and ethoxylated and propoxylated C 16 -C 18 Alcohol-based defoamers and combinations thereof.

[0040] Fatty acids: The term "fatty acid" refers to a carboxylic acid with a long aliphatic chain (i.e., an aliphatic chain with between 4 and 28 carbon atoms, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 carbon atoms). Most naturally occurring fatty acids are unbranched. They can be saturated or desaturated.

[0041] Fatty alcohol acetate: The term will be used interchangeably with "fatty acetate" herein, referring to an acetate having an aliphatic carbon chain (i.e., an aliphatic chain with between 4 and 28 carbon atoms (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 carbon atoms)). Fatty alcohol acetates may be saturated or desaturated.

[0042] Fatty acyl-CoA: This term will be used interchangeably with "fatty acyl-CoA ester" herein, referring to compounds of the general formula R-CO-SCoA, where R is a fatty carbon chain. The fatty carbon chain is linked to the -SH group of coenzyme A via a thioester bond. Fatty acyl-CoA can be saturated or desaturated, depending on whether the fatty acid from which it is derived is saturated or desaturated.

[0043] Fatty alcohols: The term "fatty alcohol" in this document refers to alcohols derived from fatty acyl-CoA that have a carbon chain length of 4 to 28 carbon atoms (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 carbon atoms). Fatty alcohols can be saturated or desaturated.

[0044] Aliphatic aldehydes: The term referred to herein means aldehydes derived from fatty acyl-CoA that have a carbon chain length of 4 to 28 carbon atoms (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 carbon atoms). Aliphatic aldehydes can be saturated or desaturated.

[0045] Heterogeneous: When referring to polypeptides (such as proteins or enzymes) or polynucleotides, the term "heterogeneous" shall be interpreted herein as referring to polypeptides or polynucleotides that are not naturally present in wild-type cells. For example, when applied to *Yarrowia lipolytica*, the term "heterogeneous Δ9 desaturase" refers to a Δ9 desaturase that is not naturally present in wild-type *Yarrowia lipolytica* cells, such as a Δ9 desaturase derived from *Drosophila melanogaster*.

[0046] Mixture of polyether dispersions: The term refers to a group of polyethoxylated nonionic surfactants that comprise or consist primarily of mixtures of polyether dispersions, such as organic defoamer 204 from Sigma Aldrich (product numbers A6426 and A8311, MDL number MFCD00130523).

[0047] Natural: When referring to polypeptides (such as proteins or enzymes) or polynucleotides, the term "natural" shall be interpreted herein as referring to polypeptides or polynucleotides that are naturally present in wild-type cells. This term may be used interchangeably with the term "endogenous".

[0048] Pests: As used herein, the term “pest” should refer to organisms that are harmful to humans or human care (especially in the context of agricultural or livestock production), particularly animals. A pest is any living organism that is invasive or prolific, harmful, troublesome, toxic, destructive, or unpleasant to plants or animals, humans or human care, livestock, human structures, wild ecosystems, etc. The term often overlaps with related terms pests, weeds, plant and animal parasites, and pathogens. It is possible for an organism to be a pest in one environment while being beneficial, domesticated, or acceptable in another.

[0049] Pheromones: Pheromones are naturally occurring compounds defined as unbranched aliphatic chains (between 9 and 18 carbons) ending with an alcohol, aldehyde, or acetate functional group and containing up to three double bonds in an aliphatic backbone. Pheromonic compositions can be produced chemically or biochemically, for example as described herein. Thus, pheromones can comprise desaturated fatty alcohols, fatty aldehydes, or fatty alcohol acetates, for example, obtainable by the methods described herein and by cells.

[0050] Polyethoxylated surfactant: The term referred to herein means polyethoxylated surfactant, i.e., nonionic surfactant.

[0051] Polyoxyethylene polyoxypropylene ether: The term refers to a group of polyethoxylated nonionic surfactants that comprise or consist primarily of PEG-PPG-PEG block copolymer defoamers, such as... P407 (CAS No. 9003-11-6) is also known as poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol).

[0052] Reduced activity: The term "reduced activity" as used herein may refer to the complete or partial loss of activity of a given peptide (such as a protein or enzyme). In some cases, the peptide is encoded by an essential gene that cannot be deleted. In these cases, the activity of the peptide can be reduced by methods known in the art (e.g., downregulating transcription or translation, or repressing the peptide). In other cases, the peptide is encoded by a non-essential gene, and its activity may be reduced or completely lost, for example, due to the deletion of the gene encoding the peptide. Reduction of enzyme activity can also be achieved by repressing the transcription of the gene encoding the enzyme, as known in the art, for example, by using a repressive promoter, by inhibiting activity, or by silencing at the translational level.

[0053] Saturated: The term "saturated" refers to a compound that does not contain carbon-carbon double or triple bonds.

[0054] Dimethicone: The term refers to a group of polyethoxylated nonionic surfactants that comprise or consist primarily of dimethicone (also known as simethicone (CAS No. 8050-81-5)), dimethylpolysiloxane, or activated polymethylsiloxane. Dimethicone is a silicone-based emulsion that also contains 1.2%-1.6% polyethylene glycol monostearate.

[0055] Surfactant: The term refers to a compound that reduces the surface tension (or interfacial tension) between two liquids, between a gas and a liquid, or between a liquid and a solid. Surfactants can be used as detergents, wetting agents, emulsifiers, defoamers, and dispersants. Surfactants are typically amphiphilic organic compounds, meaning they contain both hydrophobic groups (their tails) and hydrophilic groups (their heads). Therefore, surfactants typically contain both water-insoluble (or oil-soluble) and water-soluble components. Most commonly, surfactants are classified according to the polar head group. Nonionic surfactants do not have charged groups at their heads.

[0056] Tit: The titer of a compound in this document refers to the concentration of the compound produced. When the compound is produced by cells, the term refers to the total concentration produced by the cells, i.e., the total amount of the compound divided by the volume of the culture medium. This means, especially for volatile compounds, that the titer includes the portion of the compound that may have evaporated from the culture medium, and is therefore determined by collecting the compound from the fermentation broth and from potential exhaust gases from the fermenter.

[0057] Kedemon (E8,E10-C12:OH)

[0058] The biosynthesis of codex is based on acetyl-CoA (CoA), which is carboxylated to malonyl-CoA; this reaction is catalyzed by acetyl-CoA carboxylase (ACC). Malonyl-CoA and acetyl-CoA are precursors used by fatty acid synthases (FAS) to synthesize C16 / C18 fatty acyl-CoA. It is hypothesized that peroxisome oxidase (POX) in the codling moth catalyzes the chain shortening (-2C) of C16:CoA via C14:CoA to C12:CoA (lauryl-CoA) (Ding, 2014). Evidence has previously been found of desaturases converting C12:CoA to E9-C12:CoA in the codling moth, but recently only the gene encoding this desaturase and two other genes encoding other desaturases (Cpo_SPTQ / Cpo_NPVE / Cpo_CPRQ) have been identified. The first desaturation step results in the conversion of C12:CoA to E / Z9-C12:CoA, which in the second desaturation step is converted to E8,E10-C12:CoA (E8,E10-dodecadienyl coenzyme A). Fatty acyl reductase (FAR) may then reduce the diene E8,E10-C12:CoA, ultimately forming cortisol (E8,E10-C12:OH). The gene encoding FAR in the codling moth has not yet been identified (Ding 2014). et al., 1988.

[0059] Figure 1 shows the proposed biosynthetic pathway of ketamine.

[0060] The production of can be obtained

[0061] This disclosure relates to yeast cells capable of producing E8,E10-dodecadienyl coenzyme A and optionally quercetin (E8,E10-C12:OH or E8,E10-dodecadien-1-ol) and methods for producing quercetin (E8,E10-C12:OH or E8,E10-dodecadien-1-ol) in yeast cells.

[0062] The inventors have devised a heterologous pathway (described by way of example in Figure 1) for the production of E8,E10-dodecadienyl coenzyme A and optionally E8,E10-dodecadien-1-ol in yeast.

[0063] Therefore, this article provides a method for producing E8,E10-dodecadienyl coenzyme A and optionally E8,E10-dodecadien-1-ol in yeast cells, the method comprising the steps of providing yeast cells and incubating the yeast cells in a culture medium, wherein the yeast cells express:

[0064] i) at least one heterologous desaturase capable of introducing one or more double bonds into a fatty acyl-CoA with a carbon chain length of 12, thereby converting at least a portion of the fatty acyl-CoA into E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA); and

[0065] ii) Optionally, at least one heterologous fatty acyl-CoA reductase (EC 1.2.1.84) capable of converting at least a portion of the E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA) into E8,E10-dodecadien-1-ol.

[0066] This produces E8,E10-dodecadienyl coenzyme A and optionally E8,E10-dodecadien-1-ol.

[0067] Therefore, the yeast cells and methods of the present invention can be used to produce ketamine by: producing E8,E10-dodecadienyl-CoA as described herein, and then converting it in vivo to E8,E10-dodecadien-1-ol by expressing a reductase in yeast cells; or E8,E10-dodecadienyl-CoA can be converted to lipids (such as triglycerides) or free fatty acids, and then said lipids (such as triglycerides) or free fatty acids are recovered and converted in vitro to E8,E10-dodecadien-1-ol, as is known in the art, for example, by contacting with a reductase. In both cases, E8,E10-dodecadien-1-ol is produced.

[0068] yeast cells

[0069] In the first step of the method, yeast cells are provided that can biosynthesize longer acyl-CoA using acetyl-CoA and malonyl-CoA. Any yeast cell capable of synthesizing acyl-CoA can be used to produce E8,E10-dodecadienyl-CoA and optionally E8,E10-dodecadien-1-ol, as described herein. Alternatively, suitable carbon sources known in the art can be provided to the yeast cells. The yeast cells can be non-naturally occurring yeast cells, for example, yeast cells engineered as described herein to produce E8,E10-dodecadienyl-CoA and optionally E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate, and / or E8,E10-dodecadienal.

[0070] Acetyl-CoA and malonyl-CoA can be converted into acyl-CoA, particularly acyl-CoA with a carbon chain length of 12. This can involve, for example, the conversion of dodecyl-CoA to dodecanoic acid (lauric acid) by the action of a natural or heterologous acyl-CoA thioesterase (EC 3.1.2.20). Lauric acid can then be converted to dodecanoyl-CoA by the action of a natural or heterologous fatty acyl-CoA synthase (FAA) (EC 6.2.1.3).

[0071] Therefore, yeast cells are also able to convert acetyl-CoA and malonyl-CoA into fatty acyl-CoA, particularly fatty acyl-CoA with a carbon chain length of 12. In some embodiments, yeast cells thus express one or more fatty acyl-CoA synthases (EC 6.2.1.3) and / or one or more acyl-CoA thioesterases (EC 3.1.2.20) capable of carrying out the aforementioned reaction.

[0072] In some implementations, the yeast cells are provided with lauric acid, methyl laurate, trilauroyl glycerol, or another fatty acid derivative in the culture medium. When the yeast cells have been engineered to shorten the carbon chain by β-oxidation as described in detail below, the cells may be provided with oils or fats or any fatty acid derivatives with a carbon chain length greater than 12.

[0073] In some implementations, the cells have already been modified at the genome level, for example, through gene editing within the genome. Cells can also be modified by inserting at least one nucleic acid construct (such as at least one vector). Vectors can be designed, as is known to those skilled in the art, to enable the integration of nucleic acid sequences into the genome, or to express polypeptides encoded by nucleic acid sequences contained within the vector without genome integration.

[0074] In some embodiments of this disclosure, yeasts or fungi of the following genera are used, including but not limited to *Blakeslea*, *Candida*, *Cryptococcus*, *Cunninghamella*, *Lipomyces*, *Mortierella*, *Mucor*, *Phycomyces*, *Pythium*, *Rhodosporidium*, *Rhodotorula*, *Trichosporon*, *Saccharomyces*, and *Yarrowia*. In certain specific embodiments, organisms of the following species are used, including but not limited to *Blakeslea trispora*, *Candida pulcherrima*, *C. revkaufi*, *C. tropicalalis*, *Cryptococcus curvatus*, *Cunninghamella echinulata*, *C. elegans*, *C. japonica*, *Lipomyces starkeyi*, *L. lipoferus*, *Mortierella alpina*, *M. isabellina*, *M. ramanniana*, *M. vinacea*, *Mucor circinelloides*, *Phycomyces blakesleanus*, and *Pythium*. The yeasts include *Rhodosporidium toruoides*, *Rhodotorula glutinis*, *R. gracilis*, *R. graminis*, *R. mucilaginosa*, *R. pinicola*, *Trichosporon pullans*, *T. cutaneum*, *Saccharomyces cerevisiae*, and *Yarrowia lipolytica*. In some embodiments, the yeast cells are *Yarrowia lipolytica* cells or *Saccharomyces cerevisiae* cells.

[0075] The yeast cells to be modified (also known as host cells) may express a natural enzyme that can negatively affect the titer of available E8,E10-dodecadien-1-ol; the natural enzyme can therefore be inactivated by methods known in the art, such as gene editing. For example, the gene encoding the natural enzyme that negatively affects the titer may be deleted or mutated, resulting in the complete or partial loss of the natural enzyme's activity, as described below.

[0076] Desaturase

[0077] The method of the present invention relies on yeast cells expressing enzymes essential for converting 12-carbon-chain fatty acyl-CoA into E8,E10-dodecadienyl-CoA and optionally E8,E10-dodecadien-1-ol. The first enzyme required for this purpose is a desaturase capable of introducing one or more double bonds into the 12-carbon-chain fatty acyl-CoA, thereby converting the fatty acyl-CoA into a 12-carbon-chain desaturated fatty acyl-CoA having one or more double bonds. The 12-carbon-chain desaturated fatty acyl-CoA can be a mixture of 12-carbon-chain desaturated fatty acyl-CoA; said mixture contains E8,E10-C12:CoA, but typically also includes monounsaturated fatty acyl-CoA E9-C12:CoA and Z9-C12:CoA. Therefore, in some embodiments, yeast cell expression can introduce one or more double bonds into a 12-carbon acyl-CoA fatty acid, thereby converting at least a portion of the fatty acyl-CoA into an E8,E10-C12:CoA (E8,E10-dodecadienyl-CoA) desaturase. Desaturases of EC category EC 1.14.19 are capable of performing such a reaction.

[0078] The production of clomazone depends on two desaturation steps. These can be performed by one desaturase as described herein, such as Cpo_CPRQ or Gmo_CPRQ, its mutants and functional variants, or by two different desaturases. In embodiments using two different desaturases, at least one desaturase is Cpo_CPRQ, its mutants or functional variants as described herein. In other embodiments using two different desaturases, at least one desaturase is Gmo_CPRQ, its mutants or functional variants as described herein. Another desaturase can introduce at least one double bond into a 12-carbon acyl-CoA fatty acid chain, or into a 14-carbon acyl-CoA fatty acid chain, and then shorten it to a 12-carbon desaturated acyl-CoA fatty acid chain, as described in the “Chain Shortening” section below. A 12- or 14-carbon acyl-CoA fatty acid chain with one double bond can then be further desaturated by, for example, Cpo_CPRQ, its mutants or functional variants.

[0079] The desaturase is preferably a heterologous desaturase. In some embodiments, the desaturase is Cpo_CPRQ (SEQ ID NO:2), a naturally occurring desaturase in the codling moth. As demonstrated in Example 16, expression of Cpo_CPRQ alone is sufficient to produce E8,E10-C12:CoA. Expression of Cpo_SPTQ or Cpo_NPVE alone does not result in the production of E8,E10-C12:CoA. This finding, according to Ding 2014, is unexpected, as functional assays of these three desaturases indicate that they act sequentially to form conjugated double bonds in the codling moth pheromone—a situation not observed in yeast.

[0080] Heteroenzymes can also be functional variants of heteroenzymes such as Cpo_CPRQ, i.e., variants that retain the ability to convert 12-carbon-chain fatty acyl-CoA to 12-carbon-chain-length desaturated fatty acyl-CoA such as E8, E10-C12:CoA. In some embodiments, the functional variant is similar to Cpo_CPRQ (SEQ ID NO: 1). NO:2) Possessing at least 60% homology or identity, such as at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, or at least 73%. Homology or identity of at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0081] Heteroenzymes can also be functional variants of heteroenzymes such as Cpo_CPRQ, i.e., variants that retain the ability to convert 12-carbon-chain fatty acyl-CoA to 12-carbon-chain-length desaturated fatty acyl-CoA such as E8, E10-C12:CoA. In some embodiments, the functional variant is similar to Cpo_CPRQ (SEQ ID NO: 1). NO:2) Possessing at least 60% homology or identity, such as at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, or at least 73%. Homology or identity of at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0082] The desaturase is preferably a heterologous desaturase. In some embodiments, the desaturase is Gmo_CPRQ (SEQ ID NO: 77), a desaturase naturally found in the pear fruit moth (Grapholita molesta), or a functional variant thereof retaining the ability to convert 12-carbon-chain fatty acyl-CoA to 12-carbon-chain-chain-length desaturated fatty acyl-CoA such as E8, E10-C12:CoA. In some embodiments, the functional variant is similar to Gmo_CPRQ (SEQ ID NO: 77). NO:78) possesses at least 60% homology or identity, such as at least 61% homology or identity, at least 62% homology or identity, at least 63% homology or identity, at least 64% homology or identity, at least 65% homology or identity, at least 66% homology or identity, at least 67% homology or identity, at least 68% homology or identity, at least 69% homology or identity, at least 70% homology or identity, at least 71% homology or identity, at least 72%, at least 73%. Homology or identity of at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0083] In some embodiments, as is known in the art, the desaturase is expressed by introducing a nucleic acid encoding the desaturase. As is known in the art, such nucleic acids can be codon-optimized. In a particular embodiment, the nucleic acid encoding the desaturase is as shown in SEQ ID NO:1, or has at least 60% homology or identity with it, or has at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, or so on, with SEQ ID NO:1. Homologous to or identical with at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%. In other embodiments, the nucleic acid encoding the desaturase is as shown in SEQ ID NO:78, or has at least 60% homology or identity with SEQ ID NO:78. NO:78 possesses at least 61% homology or identity, at least 62% homology or identity, at least 63% homology or identity, at least 64% homology or identity, at least 65% homology or identity, at least 66% homology or identity, at least 67% homology or identity, at least 68% homology or identity, at least 69% homology or identity, at least 70% homology or identity, at least 71% homology or identity, at least 72%, at least 73%, at least 74%, and so on. At least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its homologous products.

[0084] In some embodiments, yeast cells express several desaturases capable of introducing one or more double bonds into a 12-carbon acyl-CoA fatty acid. In such embodiments, preferably, at least one of the desaturases is Cpo_CPRQ, a mutant thereof, or a functional variant thereof, as detailed below. In other embodiments, preferably, at least one of the desaturases is Gmo_CPRQ, a mutant thereof, or a functional variant thereof. Other desaturases may be, for example, Cpo_NPVE (accession number: AHW98355, SEQ ID NO: 67) or Cpo_SPTQ (accession number: AHW98356, SEQ ID NO: 67). NO:69), or its functional variants having at least 65% homology or identity with it, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%. Such desaturases can be expressed in yeast cells after the introduction of nucleic acids, and the nucleic acids can be codon-optimized for yeast cells, such as the nucleic acids shown in SEQ ID NO:66 or SEQ ID NO:68, or their homologs having at least 65% homology or identity, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0085] Yeast cells can be engineered to express several copies of a heterologous desaturase. This can be done as is known in the art. One or more desaturases can also be expressed at high levels, as is known in the art, for example by using constitutive promoters that result in strong expression levels—such promoters are known in the art.

[0086] In some embodiments, the desaturase is a mutant Cpo_CPRQ, such as a Cpo_CPRQ mutant with a mutation at position 85. In some embodiments, the mutation is the S85A mutation. The desaturase can also be a functional variant of the mutant and has at least 65% homology or identity with a mutant Cpo_CPRQ (such as the S85A mutant) having a mutation at position 85, such as at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, the mutant is the S85T mutant.

[0087] In some embodiments, the desaturase is a mutant Cpo_CPRQ, such as a Cpo_CPRQ mutant with a mutation at position 82. In some embodiments, the mutation is the S82A mutation. The desaturase may also be a functional variant of the mutant and has at least 65% homology or identity with the mutant Cpo_CPRQ (such as the S82A mutant) having a mutation at position 82, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0088] In some embodiments, yeast cells express two or more heterologous desaturases. The two or more desaturases may be identical or different. In specific embodiments, yeast cells express Cpo_CPRQ as shown in SEQ ID NO:2 and mutant Cpo_CPRQ (e.g., with a mutation at position 85, such as the S85A mutant). In some embodiments, yeast cells express Cpo_CPRQ (SEQ ID NO:2), its mutant Cpo_CPRQ or functional variant having at least 65% homology or identity, and also express another desaturase capable of introducing at least one double bond into a 12-carbon acyl-CoA fatty acid. In some embodiments, yeast cells express Gmo_CPRQ as shown in SEQ ID NO:77 and Cpo_CPRQ as shown in SEQ ID NO:2, or its mutants or functional variants described herein.

[0089] In some embodiments, the other desaturase is Cpo_NPVE as shown in SEQ ID NO:67, a mutant or functional variant thereof having at least 65% homology or identity, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%. In some embodiments, yeast cells express Cpo_CPRQ, its mutants or functional variants, and Cpo_NPVE, its mutants or functional variants. In some embodiments, yeast cells express Gmo_CPRQ, its mutants or functional variants, and Cpo_NPVE or its mutants or functional variants.

[0090] In other embodiments, the other desaturase is Cpo_SPTQ as shown in SEQ ID NO:69, having at least 65% homology or identity with it, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with it, or its mutants or functional variants. In some embodiments, yeast cells express Cpo_CPRQ, its mutants or functional variants, and Cpo_SPTQ, its mutants or functional variants. In some embodiments, yeast cells express Gmo_CPRQ, its mutants or functional variants, and Cpo_SPTQ or its mutants or functional variants.

[0091] In a preferred embodiment, at least one heterologous desaturase is Cpo_CPRQ or a mutant or functional variant thereof, as described above herein.

[0092] In addition to the desaturases described herein that can introduce one or two double bonds into fatty acyl-CoA with a carbon chain length of 12, yeast cells expressing desaturases capable of introducing at least one double bond into fatty acyl-CoA with a carbon chain length >12, such as 14 or greater, must also express other enzymes capable of reducing the carbon chain length of desaturated fatty acyl-CoA with a carbon chain length >12. This is described in detail in the "Chain Shortening" section below.

[0093] Therefore, yeast cells can express desaturases capable of introducing one or more double bonds into fatty acyl-CoA with a carbon chain length of 12, thereby converting fatty acyl-CoA into desaturated fatty acyl-CoA with a carbon chain length of 12 and having one or more double bonds, such as any desaturases described above herein, or functional variants thereof retaining the ability to convert fatty acyl-CoA into desaturated fatty acyl-CoA with a carbon chain length of 12. Yeast cells can also express desaturases capable of introducing at least one double bond into fatty acyl-CoA with a carbon chain length >12, such as 14 or greater, or functional variants thereof retaining the ability to introduce at least one double bond into fatty acyl-CoA with a carbon chain length >12, such as 14 or greater.

[0094] To test whether a desaturase or its functional variants possess the desired activity, methods known in the art can be used. For example, the candidate enzyme to be tested can be introduced into yeast cells (e.g., on a vector or in the genome of the yeast cells), the yeast cells can be incubated in a suitable culture medium, fatty alcohols and / or fatty acid methyl esters can be extracted from the culture medium, and analyses such as GC-MS can be performed to determine whether desaturated compounds have been produced. Testing the activity in yeast cells lacking one or more natural elongase genes can be advantageous. Examples of such procedures are described in Example 4 or in Schneiter et al., 2000.

[0095] Fatty acyl-CoA reductase (EC) 1.2.1.84)

[0096] The terms "fatty acyl-CoA reductase," "reductase," and "FAR" are used interchangeably in this document. FAR catalyzes a two-step reaction:

[0097] Acyl-CoA + 2NADPH <=> CoA + alcohol + 2NADP (+)

[0098] In the first step, fatty acyl-CoA is reduced to fatty aldehydes, and then in the second step, the fatty aldehydes are further reduced to fatty alcohols. Fatty acyl-CoA can be desaturated fatty acyl-CoA, especially E8,E10-C12:CoA, which is then converted to E8,E10-dodecadien-1-ol.

[0099] The FAR capable of catalyzing this reaction is the alcohol-forming fatty acyl-CoA reductase, EC number 1.2.1.84. Therefore, yeast cells used in the method of this invention can express a heterologous FAR capable of catalyzing the above reaction. Alternatively, after recovering E8,E10-C12:CoA and contacting the E8,E10-C12:CoA with FAR in vitro, E8,E10-C12:CoA can be converted to E8,E10-dodecadien-1-ol.

[0100] The preferred FARs are insect FARs, such as natural FARs for insects of the genera *Helicoverpa*, *Helicoverpa*, *Helicoverpa virescens*, or *Helicoverpa sulta*. For example, FARs are natural for *Agrotis segetum*, *Agrotis ipsilon*, *Heliothis subflexa*, *Helicoverpa assulta*, *Helicoverpa virescens*, or *Helicoverpa sulta*.

[0101] In some embodiments, FAR is Ase_FAR (SEQ ID NO:10), which is the FAR naturally found in *Gnaphalium affine*. In some embodiments, heterologous FAR is a functional variant of Ase_FAR that retains the ability to convert E8,E10-C12:CoA to E8,E10-dodecadien-1-ol. For example, the functional variant has at least 65% homology or identity with Ase_FAR (SEQ ID NO:10), such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0102] In some embodiments, FAR is the mutant Ase_FAR, such as a mutant with a mutation at positions 198 or 413. In some embodiments, the Ase_FAR mutant is the T198A mutant. In other embodiments, the Ase_FAR mutant is the S413A mutant.

[0103] In some implementations, Ase_FAR or its functional variants are expressed by introducing a nucleic acid encoding Ase_FAR or a functional variant thereof into yeast cells. For example, the nucleic acid shown in SEQ ID NO:9, or having at least 60% homology or identity with it, or having at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or more homology or identity with SEQ ID NO:9. 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its homologs.

[0104] In other embodiments, FAR is Aip_FAR (SEQ ID NO:61), i.e., FAR naturally found in cutworms. In some embodiments, heterologous FAR is a functional variant of Aip_FAR that retains the ability to convert E8,E10-C12:CoA to E8,E10-dodecadien-1-ol. For example, the functional variant has at least 65% homology or identity with Aip_FAR (SEQ ID NO:61), such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0105] In some implementations, Aip_FAR or its functional variants are expressed by introducing a nucleic acid encoding Aip_FAR or a functional variant thereof into yeast cells. For example, the nucleic acid shown in SEQ ID NO:60, or having at least 60% homology or identity with it, or having at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or more homology or identity with SEQ ID NO:60. At least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its homologous products.

[0106] In other embodiments, the FAR is Hs_FAR (SEQ ID NO:71), which is the FAR naturally present in Heliothissubflexa. In some embodiments, the heterologous FAR is a functional variant of Hs_FAR that retains the ability to convert E8,E10-C12:CoA to E8,E10-dodecadien-1-ol. For example, the functional variant has at least 65% homology or identity with Hs_FAR (SEQ ID NO:71), such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0107] In some embodiments, Hs_FAR or its functional variants are expressed by introducing a nucleic acid encoding Hs_FAR or a functional variant thereof into yeast cells. For example, the nucleic acid shown in SEQ ID NO:70, or having at least 60% homology or identity with it, or having at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or more homology or identity with SEQ ID NO:70. At least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its homologous products.

[0108] In other embodiments, FAR is Has_FAR (SEQ ID NO:73), which is the FAR naturally found in the tobacco burrowing moth. In some embodiments, heterologous FAR is a functional variant of Has_FAR that retains the ability to convert E8,E10-C12:CoA to E8,E10-dodecadien-1-ol. For example, the functional variant has at least 65% homology or identity with Has_FAR (SEQ ID NO:73), such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0109] In some embodiments, Has_FAR or its functional variants are expressed by introducing a nucleic acid encoding Has_FAR or a functional variant thereof into yeast cells. For example, the nucleic acid shown in SEQ ID NO:72, or having at least 60% homology or identity with it, or having at least 61% homology or identity with SEQ ID NO:72, such as at least 62% homology or identity, at least 63% homology or identity, at least 64% homology or identity, at least 65% homology or identity, at least 66% homology or identity, at least 67% homology or identity, at least 68% homology or identity, at least 69% homology or identity, at least 70% homology or identity, at least 71% homology or identity, at least 72%, at least 73%, at least 74%, or so on. At least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its homologous products.

[0110] In other embodiments, the FAR is Hv_FAR (SEQ ID NO:75), which is the FAR naturally present in the tobacco shoot moth. In some embodiments, the heterologous FAR is a functional variant of Hv_FAR that retains the ability to convert E8,E10-C12:CoA to E8,E10-dodecadien-1-ol. For example, the functional variant has at least 65% homology or identity with Hv_FAR (SEQ ID NO:75), such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0111] In some embodiments, Hv_FAR or its functional variants are expressed by introducing a nucleic acid encoding Hv_FAR or a functional variant thereof into yeast cells. For example, the nucleic acid shown in SEQ ID NO:74, or having at least 60% homology or identity with it, or having at least 61% homology or identity with SEQ ID NO:74, such as at least 62% homology or identity, at least 63% homology or identity, at least 64% homology or identity, at least 65% homology or identity, at least 66% homology or identity, at least 67% homology or identity, at least 68% homology or identity, at least 69% homology or identity, at least 70% homology or identity, at least 71% homology or identity, at least 72%, at least 73%, at least 74%, or so on. At least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its homologous products.

[0112] In some embodiments, yeast cells express FAR derived from the codling moth. In some embodiments, the FAR is Cpo_FAR (SEQ ID NO:76), which is a naturally occurring FAR in the codling moth. In some embodiments, the heterologous FAR is a functional variant of Cpo_FAR that retains the ability to convert E8,E10-C12:CoA to E8,E10-dodecadien-1-ol. For example, the functional variant has at least 65% homology or identity with Cpo_FAR (SEQ ID NO:76), such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0113] In some implementations, Cpo_FAR or its functional variants are expressed by introducing a nucleic acid encoding Cpo_FAR or a functional variant thereof into yeast cells. For example, the nucleic acid shown in SEQ ID NO:76, or having at least 60% homology or identity with it, or having at least 61% homology or identity with SEQ ID NO:76, such as at least 62% homology or identity, at least 63% homology or identity, at least 64% homology or identity, at least 65% homology or identity, at least 66% homology or identity, at least 67% homology or identity, at least 68% homology or identity, at least 69% homology or identity, at least 70% homology or identity, at least 71% homology or identity, at least 72%, at least 73%, at least 74%, or so on. At least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its homologous products.

[0114] In some embodiments, FAR is Har_FAR (SEQ ID NO:12), which is the FAR naturally found in the cotton bollworm (Helicoverpa armigera). In some embodiments, heterologous FAR is a functional variant of Har_FAR that retains the ability to convert E8,E10-C12:CoA to E8,E10-dodecadien-1-ol. For example, the functional variant has at least 65% homology or identity with Har_FAR (SEQ ID NO:12), such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0115] In some implementations, Har_FAR or its functional variants are expressed by introducing a nucleic acid encoding Har_FAR or a functional variant thereof into yeast cells. For example, the nucleic acid shown in SEQ ID NO:11, or having at least 60% homology or identity with it, or having at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or so on, is introduced. At least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its homologous products.

[0116] In some implementations, yeast cells express several copies of FAR. For example, as is known in the art, FAR is expressed at high levels.

[0117] In some embodiments, yeast cells express the desaturase and FAR as described herein. In specific embodiments, yeast cells express Cpo_CPRQ (SEQ ID NO:2) or a functional variant thereof having at least 65% homology or identity with it, and Ase_FAR (SEQ ID NO:10) or a functional variant thereof having at least 65% homology or identity with it. In some embodiments, yeast cells express Cpo_CPRQ (SEQ ID NO:2) or a functional variant thereof having at least 65% homology or identity with it, and FAR is a mutant Ase_FAR, such as a mutant with a mutation at position 198 or 413, for example, the T198A mutant or the S413A mutant. In some embodiments, the desaturase is a mutant Cpo_CPRQ, such as a mutant with a mutation at position 85, for example, the S85A mutant, and FAR is Ase_FAR or a functional variant thereof. In other embodiments, the desaturase is an S85A Cpo_CPRQ mutant, and the FAR is a mutant Ase_FAR, such as a mutant with a mutation at position 198 or 413, for example, the T198A mutant or the S413A mutant. In some embodiments, the desaturase is two desaturases, such as two identical desaturases, such as Cpo_CPRQ or a mutant Cpo_CPRQ with a mutation at position 85, such as the S85A mutant, and the FAR is Ase_FAR or a functional variant thereof. In some embodiments, the desaturase is two desaturases, such as two identical desaturases, such as Cpo_CPRQ or a mutant Cpo_CPRQ with a mutation at position 85, such as the S85A mutant, and the FAR is a mutant Ase_FAR, such as a mutant with a mutation at position 198 or 413, for example, the T198A mutant or the S413A mutant. In other embodiments, the desaturase is two different desaturases, such as a Cpo_CPRQ desaturase and a mutant Cpo_CPRQ desaturase with a mutation at position 85, such as the S85A mutant, and FAR is Ase_FAR or a functional variant thereof. In other embodiments, the desaturase is two different desaturases, such as a Cpo_CPRQ desaturase and a mutant Cpo_CPRQ desaturase with a mutation at position 85, such as the S85A mutant, and FAR is a mutant Ase_FAR, such as a mutant with a mutation at positions 198 or 413, such as the T198A mutant or the S413A mutant. In other embodiments, yeast cells express Gmo_CPRQ (SEQ ID NO:77) and Ase_FAR or a mutant or functional variant thereof.

[0118] In specific embodiments, yeast cells express Cpo_CPRQ (SEQ ID NO:2) or a functional variant thereof having at least 65% homology or identity with it, and Aip_FAR (SEQ ID NO:61) or a functional variant thereof having at least 65% homology or identity with it. In some embodiments, the desaturase is a mutant Cpo_CPRQ, such as a mutant with a mutation at position 85, for example, the S85A mutant, and FAR is Aip_FAR or a functional variant thereof. In some embodiments, the desaturase is two desaturases, such as two identical desaturases, for example, two Cpo_CPRQ desaturases or two mutant Cpo_CPRQ desaturases with a mutation at position 85, for example, two S85A mutants, and FAR is Aip_FAR or a functional variant thereof. In other embodiments, the desaturase is two different desaturases, such as the Cpo_CPRQ desaturase and a mutant Cpo_CPRQ desaturase with a mutation at position 85, such as the S85A mutant, and FAR is Aip_FAR or a functional variant thereof. In other embodiments, yeast cells express Gmo_CPRQ (SEQ ID NO:77) and Aip_FAR or mutants or functional variants thereof.

[0119] In some embodiments, yeast cells express Cpo_CPRQ (SEQ ID NO:2) or a functional variant thereof having at least 65% homology or identity with it, and Hs_FAR (SEQ ID NO:71) or a functional variant thereof having at least 65% homology or identity with it. In some embodiments, the desaturase is a mutant Cpo_CPRQ, such as a mutant with a mutation at position 85, for example, the S85A mutant, and FAR is Hs_FAR or a functional variant thereof. In some embodiments, the desaturase is two desaturases, such as two identical desaturases, for example, two Cpo_CPRQ desaturases or two mutant Cpo_CPRQ desaturases with a mutation at position 85, for example, two S85A mutants, and FAR is Hs_FAR or a functional variant thereof. In other embodiments, the desaturase is two different desaturases, such as the Cpo_CPRQ desaturase and a mutant Cpo_CPRQ desaturase with a mutation at position 85, such as the S85A mutant, and FAR is Hs_FAR or a functional variant thereof. In other embodiments, yeast cells express Gmo_CPRQ (SEQ ID NO:77) and Hs_FAR or a mutant or functional variant thereof.

[0120] In specific embodiments, yeast cells express Cpo_CPRQ (SEQ ID NO:2) or a functional variant thereof having at least 65% homology or identity with it, and Has_FAR (SEQ ID NO:73) or a functional variant thereof having at least 65% homology or identity with it. In some embodiments, the desaturase is a mutant Cpo_CPRQ, such as a mutant with a mutation at position 85, for example, the S85A mutant, and FAR is Hs_FAR or a functional variant thereof. In some embodiments, the desaturase is two desaturases, such as two identical desaturases, for example, two Cpo_CPRQ desaturases or two mutant Cpo_CPRQ desaturases with a mutation at position 85, for example, two S85A mutants, and FAR is Hs_FAR or a functional variant thereof. In other embodiments, the desaturase is two different desaturases, such as the Cpo_CPRQ desaturase and a mutant Cpo_CPRQ desaturase with a mutation at position 85, such as the S85A mutant, and FAR is Hs_FAR or a functional variant thereof. In other embodiments, yeast cells express Gmo_CPRQ (SEQ ID NO:77) and Has_FAR or mutants or functional variants thereof.

[0121] In specific embodiments, yeast cells express Cpo_CPRQ (SEQ ID NO:2) or a functional variant thereof having at least 65% homology or identity with it, and Hv_FAR (SEQ ID NO:75) or a functional variant thereof having at least 65% homology or identity with it. In some embodiments, the desaturase is a mutant Cpo_CPRQ, such as a mutant with a mutation at position 85, for example, the S85A mutant, and FAR is Hv_FAR or a functional variant thereof. In some embodiments, the desaturase is two desaturases, such as two identical desaturases, for example, two Cpo_CPRQ desaturases or two mutant Cpo_CPRQ desaturases with a mutation at position 85, for example, two S85A mutants, and FAR is Hv_FAR or a functional variant thereof. In other embodiments, the desaturase is two different desaturases, such as the Cpo_CPRQ desaturase and a mutant Cpo_CPRQ desaturase with a mutation at position 85, such as the S85A mutant, and FAR is Hv_FAR or a functional variant thereof. In other embodiments, yeast cells express Gmo_CPRQ (SEQ ID NO:77) and Hv_FAR or mutants or functional variants thereof.

[0122] In specific embodiments, yeast cells express Cpo_CPRQ (SEQ ID NO:2) or a functional variant thereof having at least 65% homology or identity with it, and Cpo_FAR (SEQ ID NO:76) or a functional variant thereof having at least 65% homology or identity with it. In some embodiments, the desaturase is a mutant Cpo_CPRQ, such as a mutant with a mutation at position 85, for example, the S85A mutant, and FAR is Cpo_FAR or a functional variant thereof. In some embodiments, the desaturase is two desaturases, such as two identical desaturases, for example, two Cpo_CPRQ desaturases or two mutant Cpo_CPRQ desaturases with a mutation at position 85, for example, two S85A mutants, and FAR is Cpo_FAR or a functional variant thereof. In other embodiments, the desaturase is two different desaturases, such as the Cpo_CPRQ desaturase and a mutant Cpo_CPRQ desaturase with a mutation at position 85, such as the S85A mutant, and FAR is Cpo_FAR or a functional variant thereof. In other embodiments, yeast cells express Gmo_CPRQ (SEQ ID NO:77) and Cpo_FAR or a mutant or functional variant thereof.

[0123] In some embodiments, yeast cells express desaturases as described above, such as Cpo_CPRQ or Gmo_CPRQ, their mutants or functional variants, FARs as described above, particularly Ase_FAR, Aip_FAR, Hs_AR, Has_FAR or Hv_FAR, or their mutants or functional variants having at least 65% homology or identity with them, and also express another desaturase capable of introducing at least one double bond into a 12-carbon acyl-CoA fatty acid, such as Cpo_NPVE (SEQ ID NO:67) or Cpo_SPTQ (SEQ ID NO:69), having at least 65% homology or identity with SEQ ID NO:67 or SEQ ID NO:69, and with SEQ ID NO:67 or SEQ ID NO:69. NO:69 has mutants or functional variants of it that have at least 70% homology or identity, at least 71% homology or identity, at least 72%, at least 73%, at least 74%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity.

[0124] In some implementations, FAR is not Har_FAR (FAR from the cotton bollworm, SEQ ID NO:12). In some implementations, FAR is not Ta_FAR (FAR from the barn owl, SEQ ID NO:8).

[0125] Therefore, yeast cells can express desaturases capable of introducing one or more double bonds into fatty acyl-CoA with a carbon chain length of 12, thereby converting fatty acyl-CoA into desaturated fatty acyl-CoA with a carbon chain length of 12 and having one or more double bonds, such as any desaturases described above herein, or functional variants thereof that retain the ability to convert fatty acyl-CoA into desaturated fatty acyl-CoA with a carbon chain length of 12. Yeast cells can also express desaturases capable of introducing at least one double bond into fatty acyl-CoA with a carbon chain length >12, such as 14 or greater, or functional variants thereof that retain the ability to introduce at least one double bond into fatty acyl-CoA with a carbon chain length >12, such as 14 or greater, as described above. Any of these yeast cells can also express reductases as described above herein, or functional variants thereof that retain reductase activity.

[0126] To test whether a reductase or a functional variant thereof possesses the desired activity, methods known in the art can be used. For example, the candidate enzyme to be tested can be introduced into yeast cells (e.g., on a vector or in the genome of the yeast cells), the yeast cells can be incubated in a suitable culture medium, fatty alcohols can be extracted from the culture medium, and analyses such as GC-MS can be performed to determine whether desaturated fatty alcohols have been produced. Testing the activity in yeast cells lacking one or more natural elongase genes can be advantageous. Examples of such procedures are described in Example 4 or in Schneiter et al., 2000.

[0127] Increase the availability of precursors

[0128] To improve the production of E8,E10-dodecadienyl coenzyme A and optionally E8,E10-dodecadien-1-ol and its derivatives, introducing additional modifications into yeast cells to increase the availability of desired precursors, particularly E8,E10-C12:CoA, can be advantageous. Therefore, yeast cells can be further modified with any of the modifications detailed below, particularly:

[0129] - Expression of heterologous cytochrome b5

[0130] - Expression of heterologous cytochrome b5 reductase

[0131] - Hemoglobin expression

[0132] - Inactivation of one or more natural elongases, resulting in complete or partial loss of activity.

[0133] - Inactivation of one or more natural thioesterases, resulting in complete or partial loss of activity.

[0134] - Inactivation or activity modification of one or more natural fatty aldehyde dehydrogenases, one or more fatty alcohol oxidases, peroxisome biogenic factors and / or one or more fatty acyl synthases.

[0135] - Expression of heterodithioesterase genes

[0136] Expression of a fusion protein of fatty acyl synthase and thioesterase

[0137] Enzymes (e.g., elongases, thioesterases, fatty aldehyde dehydrogenases, fatty alcohol oxidases, peroxisome biogenetic factors, or fatty acyl synthases) can be inactivated, for example, by introducing one or more mutations (including complete or partial deletions, insertions, substitutions, or nonsense or missense mutations) into the gene (e.g., in the coding sequence, promoter, Kozak sequence, terminator, or other regulatory element). For example, a natural promoter or natural terminator can be replaced by another weaker promoter or another terminator, respectively. Other inactivation methods that result in partial or complete loss of activity include transcriptional repression and post-transcriptional inactivation (e.g., silencing), such as using RNAi systems or CRISPR / Cas systems to cause degradation of the relevant transcript, thereby preventing or at least reducing translation; and post-translational inactivation (e.g., protein repression). Enzyme activity can be otherwise modified using methods known in the art, for example, to modify enzyme properties (e.g., intracellular localization) or to increase activity.

[0138] Elongase activity can be tested by analyzing fatty acid profiles, as described, for example, by Schneiter et al., 2000.

[0139] Thioesterase activity can be tested by appropriate assays, such as the thioesterase activity assay described in Nancolas et al., 2017.

[0140] Fatty aldehyde dehydrogenase activity can be tested by appropriate assays, such as the fatty aldehyde degradation assay described in Iwama et al., 2014.

[0141] Fatty alcohol oxidase activity can be tested by appropriate assays, such as the fatty alcohol degradation assay described in Iwama et al., 2015.

[0142] Peroxisome biogenic factor activity can be tested by appropriate assays, such as growth assays of yeast cells expressing candidate fatty alcohol oxidases in a medium containing fatty acids as the sole carbon source.

[0143] Fatty acyl synthase activity can be tested by measuring cell growth, since fatty acyl synthase is an essential gene.

[0144] Any of the modifications can be combined, meaning that a yeast cell can contain several of the modifications.

[0145] Expression of heterologous cytochrome b5

[0146] The inventors have discovered that a beneficial modification for the production of clofibrate and its derivatives is the expression of heterologous cytochrome b5 in yeast cells. This membrane-bound heme protein acts as an electron carrier for several membrane-bound oxygenases. As shown in the examples (particularly Example 6), the expression of heterologous cytochrome b5 has been found to increase the availability of fatty acid methyl esters (particularly E8, E10-C12:Me and E9 / Z9-C12:Me). Therefore, such a modification is expected to increase the production of E8, E10-dodecadienyl coenzyme A and optionally desaturated fatty alcohols with a carbon chain length of 12 (such as clofibrate).

[0147] In some embodiments, cytochrome b5 is a naturally occurring cytochrome b5 for lepidopteran species. In a particular embodiment, cytochrome b5 is cytochrome b5 from a species of the genus *Bollworm*, preferably cytochrome b5 from *Bollworm* as shown in SEQ ID NO:4, or a functional variant thereof having at least 65% homology or identity, such as at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity.

[0148] Cytochrome b5 can be expressed at high levels.

[0149] Cytochrome b5 can be expressed in yeast cells by introducing a nucleic acid encoding cytochrome b5 or a homolog thereof. For example, the nucleic acid shown in SEQ ID NO:3, or having at least 60% homology or identity with it, or having at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or more homology or identity with SEQ ID NO:3, can be expressed by introducing a nucleic acid encoding cytochrome b5 or a homolog thereof into yeast cells. At least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its homologous products.

[0150] To test whether a functional variant of cytochrome b5 retains the desired activity, methods known in the art can be used; for example, spectrophotometry as described in Lamb et al., 1999.

[0151] Therefore, in some embodiments, yeast cells express the desaturase and fatty acyl-CoA reductase as described above, and further express the heterologous cytochrome b5 described herein. Specifically, yeast cells may express one or more desaturases selected from Cpo_CPRQ (SEQ ID NO:2), mutant Cpo_CPRQ (such as the S82 mutant or the S85 mutant, preferably the S85 mutant such as the S85A mutant), and functional variants thereof, and one or more reductases selected from Ase_FAR (SEQ ID NO:10), mutant Ase_FAR (such as the T198 mutant or the S413 mutant, preferably the T198A mutant or the S413A mutant), Aip_FAR (SEQ ID NO:61), Hs_FAR (SEQ ID NO:71), Has_FAR (SEQ ID NO:73), Hv_FAR (SEQ ID NO:75), Har_FAR (SEQ ID NO:12), and functional variants thereof, as well as cytochrome b5 as described above, such as cytochrome b5 from cotton bollworm (SEQ ID NO:4) or functional variants thereof. In addition to Cpo_CPRQ, its mutants or functional variants, yeast cells can also express another desaturase capable of introducing at least one double bond into a 12-carbon acyl-CoA fatty acid, as described above, such as Cpo_NPVE, Cpo_SPTQ, its mutants or functional variants.

[0152] Yeast cells can be further modified with any of the modifications described herein, particularly by: expression of heterologous cytochrome b5 reductase, expression of hemoglobin, mutation of one or more natural elongase genes resulting in complete or partial loss of activity, mutation of one or more natural thioesterase genes resulting in complete or partial loss of activity, mutation of one or more natural genes encoding one or more fatty aldehyde dehydrogenases, one or more fatty alcohol oxidases, peroxisome biogenetic factors and / or one or more fatty acyl synthases, expression of heterologous thioesterase genes and / or expression of fusion proteins of fatty acyl synthases and thioesterases.

[0153] Heterologous cytochrome b5 reductase (EC) Expression of 1.6.2.2)

[0154] Another modification that can lead to increased production of E8, E10-dodecadienyl coenzyme A and optionally methyl methacrylate and its derivatives is the expression of heterologous cytochrome b5 reductase (EC 1.6.2.2).

[0155] Cytochrome b5 reductase (also known as methemoglobin reductase) is an NADH-dependent enzyme that converts methemoglobin into hemoglobin.

[0156] NADH+H+ +2 Methemochrome B5 = NAD + +2 Ferrous Cytochrome B5

[0157] In some embodiments, the cytochrome b5 reductase is a naturally occurring cytochrome b5 reductase for lepidopteran species. In a particular embodiment, the cytochrome b5 reductase is a cytochrome b5 reductase from a species of the genus *Bollworm*, preferably a cytochrome b5 reductase from a species of the genus *Bollworm* such as the cotton bollworm, for example, the cytochrome b5 reductase shown in SEQ ID NO:24, or a functional variant thereof having at least 65% homology or identity, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0158] To test whether a functional variant of cytochrome b5 reductase retains the desired activity, methods known in the art can be used; for example, spectrophotometry as described in Lamb et al., 1999.

[0159] Cytochrome b5 reductase can be expressed at high levels.

[0160] Cytochrome b5 reductase can be expressed by introducing a nucleic acid encoding the cytochrome b5 reductase or a homolog thereof into yeast cells. For example, the nucleic acid shown in SEQ ID NO:23, or having at least 60% homology or identity with it, or having at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or more homology or identity with SEQ ID NO:23, can be introduced. At least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its homologous products.

[0161] Therefore, in some embodiments, yeast cells express the desaturase and fatty acyl-CoA reductase as described above, and further express the heterologous cytochrome b5 reductase described herein. Yeast cells may be further modified with any of the modifications described herein. Specifically, yeast cells may express one or more desaturases selected from Cpo_CPRQ (SEQ ID NO:2), Gmo_CPRQ (SEQ ID NO:77), mutant Cpo_CPRQ (such as S82 mutant or S85 mutant, preferably S85 mutant such as S85A mutant) and their functional variants, and one or more reductases selected from Ase_FAR (SEQ ID NO:10), mutant Ase_FAR (such as T198 mutant or S413 mutant, preferably T198A mutant or S413A mutant), Aip_FAR (SEQ ID NO:61), Hs_FAR (SEQ ID NO:71), Has_FAR (SEQ ID NO:73), Hv_FAR (SEQ ID NO:75), Har_FAR (SEQ ID NO:12) and their functional variants, as well as cytochrome b5 reductases as described above, such as cytochrome b5 reductase from cotton bollworm (SEQ ID NO:25) or their functional variants. In addition to Cpo_CPRQ or Gmo_CPRQ, their mutants or functional variants, yeast cells may also express another desaturase capable of introducing at least one double bond into a 12-carbon acyl-CoA fatty acid chain, as described above, such as Cpo_NPVE, Cpo_SPTQ, their mutants or functional variants. Yeast cells may also express cytochrome b5 as described above.

[0162] Yeast cells can be further modified with any of the modifications described herein, particularly by: expression of heterologous cytochrome b5, expression of hemoglobin, mutation of one or more natural elongase genes resulting in complete or partial loss of activity, mutation of one or more natural thioesterase genes resulting in complete or partial loss of activity, mutation of one or more natural genes encoding one or more fatty aldehyde dehydrogenases, one or more fatty alcohol oxidases, peroxisome biogenetic factors and / or one or more fatty acyl synthases, expression of heterologous thioesterase genes and / or expression of fusion proteins of fatty acyl synthases and thioesterases.

[0163] Hemoglobin expression

[0164] Another modification that facilitates the production of E8, E10-dodecadienyl coenzyme A and optionally methyl methacrylate and its derivatives is the expression of hemoglobin, particularly heterologous hemoglobin, in yeast cells.

[0165] As shown in the examples, particularly Example 6, the expression of hemoglobin in yeast cells expressing desaturases increased the production of E8, E10-C12:Me and E9 / Z9-C12:Me.

[0166] In some embodiments, the hemoglobin is the native hemoglobin of species of the genus Vitreoscilla (such as Vitreoscilla stercoraria). In specific embodiments, the hemoglobin is as shown in SEQ ID NO:6, or a functional variant thereof having at least 65% homology or identity with it, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0167] To test whether a functional variant of hemoglobin retains the desired activity, appropriate assays known in the art, such as colorimetric assays, can be performed.

[0168] Hemoglobin can be expressed at high levels.

[0169] Hemoglobin can be expressed by introducing a nucleic acid encoding the hemoglobin or a homolog thereof into yeast cells. For example, the nucleic acid shown in SEQ ID NO:5, or having at least 60% homology or identity with it, or having at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or more homology or identity with SEQ ID NO:5. At least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its homologous products.

[0170] Therefore, in some embodiments, yeast cells express the desaturase and fatty acyl-CoA reductase as described above, and further express the hemoglobin described herein. Specifically, yeast cells may express one or more desaturases selected from Cpo_CPRQ (SEQ ID NO:2), Gmo_CPRQ (SEQ ID NO:77), mutant Cpo_CPRQ (such as S82 mutant or S85 mutant, preferably S85 mutant such as S85A mutant) and their functional variants, and one or more reductases selected from Ase_FAR (SEQ ID NO:10), mutant Ase_FAR (such as T198 mutant or S413 mutant, preferably T198A mutant or S413A mutant), Aip_FAR (SEQ ID NO:61), Hs_FAR (SEQ ID NO:71), Has_FAR (SEQ ID NO:73), Hv_FAR (SEQ ID NO:75), Har_FAR (SEQ ID NO:12) and their functional variants, as well as hemoglobin as described above, such as hemoglobin from *Vibrio fecalis* (SEQ ID NO:6) or its functional variants. In addition to Cpo_CPRQ or Gmo_CPRQ, their mutants or functional variants, yeast cells may also express another desaturase capable of introducing at least one double bond into a 12-carbon acyl-CoA fatty acid chain, as described above, such as Cpo_NPVE, Cpo_SPTQ, their mutants or functional variants.

[0171] Yeast cells can be further modified with any of the modifications described herein, particularly by: expression of heterologous cytochrome b5, expression of cytochrome b5 reductase, mutation of one or more natural elongase genes resulting in complete or partial loss of activity, mutation of one or more natural thioesterase genes resulting in complete or partial loss of activity, mutation of one or more natural genes encoding one or more fatty aldehyde dehydrogenases, one or more fatty alcohol oxidases, peroxisome biogenetic factors and / or one or more fatty acyl synthases, expression of heterologous thioesterase genes and / or expression of fusion proteins of fatty acyl synthases and thioesterases.

[0172] Mutations in one or more elongase genes

[0173] Another modification that facilitates the production of E8, E10-dodecadienyl coenzyme A and optionally methyl methacrylate (MCMA) and its derivatives is a mutation in certain genes in yeast cells, particularly one or more elongase genes, wherein said mutation results in partial or complete loss of the corresponding elongase activity. Elongases catalyze the carbon chain elongation of several molecules, including fatty acids. In some embodiments, the elongase is a medium-chain acyl elongase. If yeast cells naturally containing several genes encoding elongases are used, the yeast cells can be further engineered to include mutations in one or more of said genes, resulting in partial or complete loss of activity of one or more elongases.

[0174] In some implementations, the yeast cells are Yersinia lipolytica cells, and the elongase is encoded by the ELO1 gene (SEQ ID NO:13).

[0175] In some embodiments, the mutation is a deletion that results in the complete loss of the corresponding elongase activity. In other embodiments, the elongase is inactivated, for example, by introducing one or more mutations (including complete or partial deletions, insertions, substitutions, or nonsense or missense mutations) into the gene (e.g., in the coding sequence, promoter, Kozak sequence, terminator, or other regulatory element). For example, a natural promoter or natural terminator may be replaced by another weaker promoter or another terminator, respectively. Other inactivation methods that result in partial or complete loss of activity include transcriptional repression and post-transcriptional inactivation (e.g., silencing), such as using RNAi or CRISPR / Cas systems to cause degradation of the relevant transcript, thereby preventing or at least reducing translation; and post-translational inactivation (e.g., protein repression). Examples of how to test whether a protein retains elongase activity are described in Example 4 or in Schneiter et al., 2000.

[0176] Therefore, in some embodiments, yeast cells express the desaturase and fatty acyl-CoA reductase as described above, and also contain one or more mutations in one or more genes encoding the elongase, wherein the mutations result in partial or complete loss of function, as described herein. Specifically, yeast cells may express one or more desaturases selected from Cpo_CPRQ (SEQ ID NO:2), mutant Cpo_CPRQ (such as the S82 mutant or the S85 mutant, preferably the S85 mutant such as the S85A mutant), and functional variants thereof, and one or more reductases selected from Ase_FAR (SEQ ID NO:10), mutant Ase_FAR (such as the T198 mutant or the S413 mutant, preferably the T198A mutant or the S413A mutant), Aip_FAR (SEQ ID NO:61), Hs_FAR (SEQ ID NO:71), Has_FAR (SEQ ID NO:73), Hv_FAR (SEQ ID NO:75), Har_FAR (SEQ ID NO:12), and functional variants thereof, and may also contain mutations as described above that result in partial or complete loss of elongase activity. In addition to Cpo_CPRQ or Gmo_CPRQ, their mutants or functional variants, yeast cells may also express another desaturase capable of introducing at least one double bond into a 12-carbon acyl-CoA fatty acid chain, as described above, such as Cpo_NPVE, Cpo_SPTQ, their mutants or functional variants.

[0177] Yeast cells can be further modified with any of the modifications described herein, particularly by: expression of heterologous cytochromes, expression of heterologous cytochrome b5 reductase, expression of hemoglobin, mutation of one or more native thioesterase genes resulting in complete or partial loss of activity, mutation of one or more native genes encoding one or more fatty aldehyde dehydrogenases, one or more fatty alcohol oxidases, peroxisome biogenetic factors and / or one or more fatty acyl synthases, expression of heterologous thioesterase genes and / or expression of fusion proteins of fatty acyl synthases and thioesterases.

[0178] Mutations in one or more thioesterase genes

[0179] Another modification that facilitates the production of E8, E10-dodecadienyl coenzyme A and optionally methyl thioesterase and its derivatives is the mutation of certain genes in yeast cells, particularly one or more thioesterase genes, wherein said mutations result in partial or complete loss of the activity of the corresponding thioesterase. If yeast cells naturally containing several genes encoding thioesterases are used, the yeast cells can be further engineered to include mutations in one or more of said genes, resulting in partial or complete loss of activity of one or more thioesterases.

[0180] In some embodiments, the yeast cells are *Yarrowia lipolytica* cells, and the thioesterases are encoded by the genes YAL10_F14729g (SEQ ID NO:19), YALI0_E18876g (SEQ ID NO:54), or YALI0_D03597g (SEQ ID NO:55). Therefore, in some embodiments, the *Yarrowia lipolytica* cells contain mutations, such as deletions, in the YAL10_F14729g gene (SEQ ID NO:19) that result in partial or complete loss of the corresponding thioesterase. In other embodiments, the *Yarrowia lipolytica* cells contain mutations, such as deletions, in the YALI0_E18876g gene (SEQ ID NO:54) that result in partial or complete loss of the corresponding thioesterase. In other embodiments, the *Yarrowia lipolytica* cells contain mutations, such as deletions, in the YALI0_D03597g (SEQ ID NO:55) that result in partial or complete loss of the corresponding thioesterase. In some embodiments, the *Yarrowia lipolytica* cells contain mutations in several thioesterase genes. For example, the cells may contain mutations, such as deletions, in YAL10_F14729g (SEQ ID NO:19) and YALI0_E18876g (SEQ ID NO:54); or mutations, such as deletions, in YAL10_F14729g (SEQ ID NO:19) and YALI0_D03597g (SEQ ID NO:55); or mutations, such as deletions, in YALI0_E18876g (SEQ ID NO:54) and YALI0_D03597g (SEQ ID NO:55). In some embodiments, the cells contain mutations, such as deletions, in YAL10_F14729g (SEQ ID NO:19), YALI0_E18876g (SEQ ID NO:54), and YALI0_D03597g (SEQ ID NO:55).

[0181] Therefore, in some embodiments, yeast cells express desaturases and fatty acyl-CoA reductases as described above, and also contain one or more mutations in one or more genes encoding thioesterases, wherein said mutations result in partial or complete loss of function, as described herein. Specifically, yeast cells may express one or more desaturases selected from Cpo_CPRQ (SEQ ID NO:2), Gmo_CPRQ (SEQ ID NO:77), mutant Cpo_CPRQ (such as the S82 mutant or S85 mutant, preferably the S85 mutant such as the S85A mutant), and their functional variants, and one or more desaturases selected from Ase_FAR (SEQ ID NO:10), mutant Ase_FAR (such as the T198 mutant or S413 mutant, preferably the T198A mutant or S413A mutant), Aip_FAR (SEQ ID NO:61), Hs_FAR (SEQ ID NO:71), Has_FAR (SEQ ID NO:73), Hv_FAR (SEQ ID NO:75), Har_FAR (SEQ ID NO:10), and other similar enzymes. Reductases of thioesterases (NO:12) and their functional variants, and one or more mutations in one or more genes encoding thioesterases, wherein said mutations result in partial or complete loss of function as described above, such as mutations in one or more of YAL10_F14729g (SEQ ID NO:19), YALI0_E18876g (SEQ ID NO:54), and YALI0_D03597g (SEQ ID NO:55). In addition to Cpo_CPRQ or Gmo_CPRQ, their mutants, or functional variants, yeast cells may also express another desaturase capable of introducing at least one double bond into a 12-carbon acyl-CoA fatty acid chain, as described above, such as Cpo_NPVE, Cpo_SPTQ, their mutants, or functional variants.

[0182] Yeast cells can be further modified with any of the modifications described herein, particularly by: expression of heterologous cytochrome b5, expression of heterologous cytochrome b5 reductase, expression of hemoglobin, mutation of one or more natural elongase genes resulting in complete or partial loss of activity, mutation of one or more natural genes encoding one or more fatty aldehyde dehydrogenases, one or more fatty alcohol oxidases, peroxisome biogenetic factors and / or one or more fatty acyl synthases, expression of heterologous thioesterase genes and / or expression of fusion proteins of fatty acyl synthases and thioesterases.

[0183] Other modifications

[0184] Yeast cells may also contain other modifications, such as at least one mutation that reduces the activity of enzymes involved in fatty acid metabolism. In some embodiments, the activity of one or more natural fatty aldehyde dehydrogenases, one or more fatty alcohol oxidases, peroxisome biogenesis factors, and / or one or more fatty acyl synthases is modified, preferably reducing or eliminating the activity. For example, yeast cells may also contain one or more mutations in the genes encoding fatty aldehyde dehydrogenases, fatty alcohol oxidases, and / or peroxisome biogenesis factors. Any of these enzymes can be inactivated, for example, by introducing one or more mutations (including complete or partial deletions, insertions, substitutions, or nonsense or missense mutations) in the gene (e.g., in the coding sequence, promoter, Kozak sequence, terminator, or other regulatory element). For example, a natural promoter or natural terminator may be replaced by another weaker promoter or another terminator, respectively. Other inactivation methods that result in partial or complete loss of activity include transcriptional repression and post-transcriptional inactivation (e.g., silencing), such as using RNAi systems or CRISPR / Cas systems to cause degradation of the relevant transcript, thereby preventing or at least reducing translation; and post-translational inactivation (e.g., protein repression). Enzyme activity can be modified in other ways using methods known in the art, such as modifying enzyme properties (e.g., intracellular localization) or increasing activity.

[0185] In some embodiments, the yeast cells are *Yarrowia lipolytica* cells as described above herein, further comprising modifications such as mutations in at least one of HFD1, HFD2, HFD3, HFD4, FAO1, GPAT, and PEX10, or modifications resulting in at least 60% homology or identity with them, such as at least 65% homology or identity, at least 70% homology or identity, at least 75% homology or identity, at least 80% homology or identity, at least 81% homology or identity, at least 82% homology or identity, at least 83% homology or identity, at least 84% homology or identity, at least 85% homology or identity, etc. A mutation modifying at least one protein with reduced activity, exhibiting at least 86% homology or identity, at least 87% homology or identity, at least 88% homology or identity, at least 89% homology or identity, at least 90% homology or identity, at least 91% homology or identity, at least 92% homology or identity, at least 93% homology or identity, at least 94% homology or identity, at least 95% homology or identity, at least 96% homology or identity, at least 97% homology or identity, at least 98% homology or identity, or at least 99% homology or identity.

[0186] In *Yarrowia lipolytica*, the fatty aldehyde dehydrogenase Hfd1 is encoded by HFD1 (YALIO_F23793g). It catalyzes the oxidation of fatty aldehydes to fatty acids. As detailed in application WO 2018 / 109163, a decrease in Hfd1 activity leads to an increase in the titer of desaturated fatty alcohols in yeast cells. Therefore, *Yarrowia lipolytica* cells according to this disclosure may also contain mutations in HFD1, such as deletions, resulting in partial or complete loss of Hfd1 activity. The reduction in Hfd1 activity can be achieved by other methods described herein.

[0187] The fatty aldehyde dehydrogenase Hfd2 is encoded by HFD2 (YALI_0E15400g). It catalyzes the oxidation of fatty aldehydes to fatty acids. Therefore, the Yersinia lipolytica cells according to this disclosure may also contain mutations in HFD2, such as deletions, resulting in partial or complete loss of Hfd2 activity. The reduction in Hfd2 activity can be achieved by other methods described herein.

[0188] The fatty aldehyde dehydrogenase Hfd3 is encoded by HFD3 (YALI0_A17875g). It catalyzes the oxidation of fatty aldehydes to fatty acids. Therefore, the Yersinia lipolytica cells according to this disclosure may also contain mutations in HFD3, such as deletions, resulting in partial or complete loss of Hfd3 activity. The reduction in Hfd3 activity can be achieved by other methods described herein.

[0189] In *Yarrowia lipolytica*, the fatty aldehyde dehydrogenase Hfd4 is encoded by HFD4 (YALI0_B01298g). It catalyzes the oxidation of fatty aldehydes to fatty acids. As described in detail in application WO 2018 / 109163, a decrease in Hfd4 activity leads to an increase in the titer of desaturated fatty alcohols in yeast cells. Therefore, *Yarrowia lipolytica* cells according to this disclosure may also contain mutations in HFD4, such as deletions, resulting in partial or complete loss of Hfd4 activity. The reduction in Hfd4 activity can be achieved by other methods described herein.

[0190] In some embodiments, the yeast cells also contain modifications, such as mutations, like deletions, resulting in at least 60% homology or identity with Hfd1, Hfd2, Hfd3, or Hfd4, or at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, or 85% homology or identity with Hfd1, Hfd2, Hfd3, or Hfd4. The activity of aliphatic aldehyde dehydrogenase is partially or completely lost, such as at least 86% homology or identity, at least 87% homology or identity, at least 88% homology or identity, at least 89% homology or identity, at least 90% homology or identity, at least 91% homology or identity, at least 92% homology or identity, at least 93% homology or identity, at least 94% homology or identity, at least 95% homology or identity, at least 96% homology or identity, at least 97% homology or identity, at least 98% homology or identity, or at least 99% homology or identity.

[0191] In *Yarrowia lipolytica*, the fatty alcohol oxidase Fao1 is encoded by FAO1 (YALI0B14014g). Its deletion leads to increased accumulation of ω-hydroxy fatty acids. As detailed in application WO 2018 / 109163, decreased Fao1 activity results in an increased titer of desaturated fatty alcohols in yeast cells. Therefore, *Yarrowia lipolytica* cells according to this disclosure may also contain mutations in FAO1, such as deletions, leading to partial or complete loss of Hfd1 activity. The reduction of Fao1 activity can be achieved by other methods described herein.

[0192] In some implementations, the yeast cells also contain mutations, such as deletions, resulting in at least 60% homology or identity with Fao1, or at least 65% homology or identity with Fao1, or at least 70% homology or identity, or at least 75% homology or identity, or at least 80% homology or identity, or at least 81% homology or identity, or at least 82% homology or identity, or at least 83% homology or identity, or at least 84% homology or identity, or at least 85% homology or identity, or at least 86% homology or identity, or at least The activity of fatty alcohol oxidase is partially or completely lost, with 87% or more homology or identity, such as at least 88% or more homology or identity, such as at least 89% or more homology or identity, such as at least 90% or more homology or identity, such as at least 91% or more homology or identity, such as at least 92% or more homology or identity, such as at least 93% or more homology or identity, such as at least 94% or more homology or identity, such as at least 95% or more homology or identity, such as at least 96% or more homology or identity, such as at least 97% or more homology or identity, such as at least 98% or more homology or identity, such as at least 99% or more homology or identity.

[0193] In *Yarrowia lipolytica*, peroxisome biogenesis factor 10 (Pex10) is encoded by PEX10 (YALI0C01023g). As described in detail in application WO 2018 / 109163, a decrease in Pex10 activity leads to an increase in the titer of desaturated fatty alcohols in yeast cells. Therefore, *Yarrowia lipolytica* cells according to this disclosure may also contain mutations in PEX10, such as deletions, resulting in partial or complete loss of Pex10 activity. The reduction in Pex10 activity can be achieved by other methods described herein.

[0194] In some implementations, the yeast cells also contain mutations, such as deletions, resulting in at least 60% homology or identity with Pex10, or at least 65% homology or identity with Pex10, or at least 70% homology or identity, or at least 75% homology or identity, or at least 80% homology or identity, or at least 81% homology or identity, or at least 82% homology or identity, or at least 83% homology or identity, or at least 84% homology or identity, or at least 85% homology or identity, or at least 86% homology or identity, or at least 87% homology or identity. The activity of peroxisome biogenic factors is partially or completely lost, exhibiting at least 88% or 89% homology or identity, at least 90% or 91% homology or identity, at least 92% or 93% homology or identity, at least 94% or 95% homology or identity, at least 96% or 97% homology or identity, at least 98% or 99% homology or identity.

[0195] In *Yarrowia lipolytica*, the glycerol-3-phosphoacyltransferase is encoded by GPAT (YALI0_C00209g). GPAT catalyzes the first reaction toward glycerol biosynthesis. This gene is essential in *Yarrowia lipolytica*. As detailed in application WO2018 / 109163, a decrease in GPAT activity leads to an increase in the titer of desaturated fatty alcohols in yeast cells. Therefore, *Yarrowia lipolytica* cells according to this disclosure may also contain mutations in GPAT, resulting in partial or complete loss of GPAT activity. The reduction in GPAT activity can be achieved by other methods described herein.

[0196] In some embodiments, the yeast cells also contain mutations that result in at least 60% homology or identity with GPAT, or at least 65% homology or identity with GPAT, or at least 70% homology or identity, or at least 75% homology or identity, or at least 80% homology or identity, or at least 81% homology or identity, or at least 82% homology or identity, or at least 83% homology or identity, or at least 84% homology or identity, or at least 85% homology or identity, or at least 86% homology or identity, or at least 87% homology. The activity of glycerol-3-phosphoacyltransferase is partially or completely lost, with at least 88% or 89% homology or identity, at least 90% or 91% homology or identity, at least 92% or 93% homology or identity, at least 94% or 95% homology or identity, at least 96% or 97% homology or identity, at least 98% or 99% homology or identity.

[0197] The partial or complete loss of any of the aforementioned enzyme activities can also be achieved, for example, by introducing one or more mutations (including complete or partial deletions, insertions, substitutions, or nonsense or missense mutations) into the gene (e.g., in the coding sequence, promoter, Kozak sequence, terminator, or other regulatory element). For example, a natural promoter or natural terminator can be replaced by another weaker promoter or another terminator, respectively. Other inactivation methods that result in partial or complete loss of activity include transcriptional repression and post-transcriptional inactivation (e.g., silencing), such as using RNAi systems or CRISPR / Cas systems to cause degradation of the relevant transcript, thereby preventing or at least reducing translation; and post-translational inactivation (e.g., protein repression). To determine whether a modification (such as a mutation or any of the modifications described above) results in complete or partial loss of activity, methods known in the art, such as those detailed above, can be used. For example, in the case of a deletion or modification that results in reduced transcription, amplification methods such as PCR can be used to confirm the absence of the relevant sequence. Protein expression can be studied using appropriate assays (e.g., Western blotting) or by measuring expression levels using markers such as fluorescent markers.

[0198] It can also be advantageous for yeast cells to express one or more modified fatty acid acyl synthases. This can help direct metabolic flux toward the production of desaturated products, such as E8, E10-dodecadienyl coenzyme A and desaturated fatty alcohols and their derivatives, such as ketamine and its derivatives. Therefore, in some embodiments, the yeast cells are further modified to express fatty acid acyl synthases having modified ketosynthase domains. In some embodiments, the yeast cells are *Yarrowia lipolytica* cells as described herein, wherein the cells also express the modified fatty acid synthase complex. In one embodiment, the fatty acid synthase complex is modified by mutating the gene encoding the α subunit of the complex. In some embodiments, the mutation is in the gene encoding FAS2 (SEQ ID NO: 18). In other embodiments, the mutation is in the gene encoding FAS1 (SEQ ID NO: 16). The mutation can result in modification of one or more residues 123 (L123) of SEQ ID NO: 16. Mutations can result in modifications to one or more of residues 1220 (I1220), 1217 (M1217), or 1226 (M1226) of SEQ ID NO:18, producing the variant FAS2. Those skilled in the art will know how to design such mutations.

[0199] Preferably, the mutation in FAS2 produces an I1220F variant, an I1220W variant, an I1220Y variant, or an I1220H variant of Fas2. In a specific embodiment, the mutation produces the I1220F variant. In some embodiments, the mutation produces an M1217F variant, an M1217W variant, an M1217Y variant, or an M1217H variant. In other embodiments, the mutation produces an M1226F variant, an M1226W variant, an M1226Y variant, or an M1226H variant.

[0200] Preferably, the mutation in FAS1 produces the L123V variant.

[0201] Yeast cells with more than one of the above mutations were also considered, such as two or three mutations at residues I1220, M1217 or M1226 of FAS2, and / or one mutation at residue 123 of FAS1.

[0202] Therefore, in some embodiments, yeast cells express desaturases and fatty acyl-CoA reductases as described above, and also contain one or more modifications as described in this section. Specifically, yeast cells may express one or more desaturases selected from Cpo_CPRQ (SEQ ID NO:2), Gmo_CPRQ (SEQ ID NO:77), mutant Cpo_CPRQ (such as the S82 mutant or S85 mutant, preferably the S85 mutant such as the S85A mutant), and their functional variants, and one or more desaturases selected from Ase_FAR (SEQ ID NO:10), mutant Ase_FAR (such as the T198 mutant or S413 mutant, preferably the T198A mutant or S413A mutant), Aip_FAR (SEQ ID NO:61), Hs_FAR (SEQ ID NO:71), Has_FAR (SEQ ID NO:73), Hv_FAR (SEQ ID NO:75), Har_FAR (SEQ ID NO:10), and other similar enzymes. Reductases of NO:12 and its functional variants, and one or more modifications, such as mutations resulting in partial or complete loss of function of one or more of Hfd1, Hfd2, Hfd3, Hfd4, Fao1, and Pex10 as described above, and / or expression of one or more modified fatty acyl synthases as described above. In addition to Cpo_CPRQ or Gmo_CPRQ, their mutants or functional variants, yeast cells may also express another desaturase capable of introducing at least one double bond into a 12-carbon fatty acyl-CoA, as described above, such as Cpo_NPVE, Cpo_SPTQ, their mutants or functional variants.

[0203] Yeast cells can be further modified with any of the modifications described herein, particularly by: expression of heterologous cytochrome b5, expression of heterologous cytochrome b5 reductase, expression of hemoglobin, inactivation of one or more natural elongases resulting in complete or partial loss of activity, inactivation of one or more natural thioesterases resulting in complete or partial loss of activity, expression of heterologous thioesterase genes and / or expression of fusion proteins of fatty acyl synthase and thioesterase.

[0204] Specifically, yeast cells may express one or more desaturases selected from Cpo_CPRQ (SEQ ID NO:2), Gmo_CPRQ (SEQ ID NO:77), mutant Cpo_CPRQ (such as S82 mutant or S85 mutant, preferably S85 mutant such as S85A mutant) and their functional variants, and one or more of Ase_FAR (SEQ ID NO:10), mutant Ase_FAR (such as T198 mutant or S413 mutant, preferably T198A mutant or S413A mutant), Aip_FAR (SEQ ID NO:61), Hs_FAR (SEQ ID NO:71), Has_FAR (SEQ ID NO:73), Hv_FAR (SEQ ID NO:75), Har_FAR (SEQ ID NO:76), and Gmo_CPRQ (SEQ ID NO:77), and their functional variants. NO:12) and its functional variants of reductases, and may also contain mutations in the following: HFD1 and HFD2; HFD1 and HFD3; HFD1 and HFD4; HFD1 and FAO1; HFD1 and PEX10; HFD2 and HFD3; HFD2 and HFD4; HFD2 and FAO1; HFD2 and PEX10; HFD3 and HFD4; HFD3 and FAO1; HFD3 and PEX10; HFD4 and FAO1; HFD4 and PEX10; F AO1 and PEX10; HFD1, HFD2 and HFD3; HFD1, HFD2 and HFD4; HFD1, HFD2 and FAO1; HFD1, HFD2 and PEX10; HFD1, HFD3 and HFD4; HFD1, HFD3 and FAO1; HFD1, HFD3 and PEX10; HFD1, HFD4 and FAO1; HFD1, HFD4 and PEX10; HFD1, FAO1 and PEX10; HFD2, HFD3 and H FD4; HFD2, HFD3 and FAO1; HFD2, HFD3 and PEX10; HFD2, HFD4 and FAO1; HFD2, HFD4 and PEX10; HFD2, FAO1 and PEX10; HFD3, HFD4 and FAO1; HFD3, HFD4 and PEX10; HFD3, FAO1 and PEX10; HFD4, FAO1 and PEX10; HFD1, HFD2, HFD3 and HFD4; HFD1, HFD2 HFD3 and FAO1; HFD1, HFD2, HFD3 and PEX10; HFD1, HFD2, HFD4 and FAO1; HFD1, HFD2, HFD4 and PEX10; HFD1, HFD2, FAO1 and PEX10; HFD1, HFD3, HFD4 and FAO1; HFD1, HFD3, HFD4 and PEX10; HFD1, HFD3, FAO1 and PEX10; HFD1, HFD4, FAO1 and PEX10;HFD2, HFD3, HFD4, and FAO1; HFD2, HFD3, HFD4, and PEX10; HFD2, HFD3, FAO1, and PEX10; HFD2, HFD4, FAO1, and PEX10; HFD3, HFD4, FAO1, and PEX10; HFD1, HFD2, HFD3, HFD4, and FAO1; HFD1, HFD2, HFD3, HFD4, and PEX10; HFD1, HFD3, HFD4, FAO1, and PEX10; HFD2, HFD3, HFD4, FAO1, and PEX10; HFD1, HFD2, HFD3, HFD4, FAO1, and PEX10; HFD1, HFD2, HFD3, HFD4, FAO1, and PEX10, or combinations thereof with corresponding variants having at least 60% homology or identity with them. Furthermore, yeast cells may also express modified fatty acyl synthases as described above, particularly mutants Fas1 and / or Fas2. In addition to Cpo_CPRQ or Gmo_CPRQ, their mutants or functional variants, yeast cells can also express another desaturase capable of introducing at least one double bond into a 12-carbon acyl-CoA fatty acid chain, as described above, such as Cpo_NPVE, Cpo_SPTQ, their mutants or functional variants.

[0205] Expression of heterothioesterase

[0206] Further engineering yeast cells by introducing thioesterases, particularly heterologous thioesterases, can be advantageous. Therefore, in some embodiments, nucleic acids encoding thioesterases are introduced into yeast cells, for example, on a vector or through genome integration. The thioesterase gene can be under the control of an inducible promoter or a constitutive promoter. Codon optimization of the nucleic acid encoding the thioesterase can be performed for yeast cells, as is known in the art. In particular, codon optimization of the nucleic acid can be performed for Yarrowia cells (such as Yarrowia lipolytica cells). As is known in the art, thioesterases can be expressed at high levels.

[0207] In some embodiments, the thioesterase is derived from an organism selected from *Cuphea palustris*, *Cuphea hookeriana*, *Cinnamomum camphora*, or *Escherichia coli*. In a preferred embodiment, the thioesterase is derived from *E. coli* or *Cinnamomum camphora*. In some embodiments, the thioesterase has at least 60% homology or identity with a thioesterase selected from: the thioesterase derived from *Cuphea palustris* as shown in SEQ ID NO:33, the thioesterase derived from *Cuphea hookeriana* as shown in SEQ ID NO:57, the thioesterase derived from *Cinnamomum camphora* as shown in SEQ ID NO:35, and the thioesterase derived from *E. coli* as shown in SEQ ID NO:26. Preferably, the thioesterase has at least 60% homology or identity with the thioesterase derived from *Cinnamomum camphora* as shown in SEQ ID NO:35 or the thioesterase derived from *E. coli* as shown in SEQ ID NO:26. In one embodiment, the thioesterase has at least 60% homology or identity with a thioesterase derived from camphor tree as shown in SEQ ID NO:35. In another embodiment, the thioesterase has at least 60% homology or identity with a thioesterase derived from Escherichia coli as shown in SEQ ID NO:26.

[0208] In another embodiment, the thioesterase has at least 60% homology or identity with the camphor-derived thioesterase as shown in SEQ ID NO:35, and has at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, or at least... 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% homology or identity.

[0209] In another embodiment, the thioesterase has at least 60% homology or identity with the thioesterase derived from *E. coli* as shown in SEQ ID NO:26, and has at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, or so on. At least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% homology or identity.

[0210] The nucleic acid encoding the thioesterase can be codon-optimized, as is known in the art. In one embodiment, the yeast cell is a *Yarrowia* cell, preferably a *Yarrowia lipophila* cell, and the nucleic acid is codon-optimized accordingly.

[0211] In one embodiment, at least one thioesterase is encoded by a nucleic acid having at least 60% homology or identity with the nucleic acid encoding a camphor-derived thioesterase as shown in SEQ ID NO:34, and having at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, or at least 73% homology or identity with the nucleic acid encoding a camphor-derived thioesterase as shown in SEQ ID NO:34. For example, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology or identity.

[0212] In one embodiment, at least one thioesterase is encoded by a nucleic acid having at least 60% homology or identity with the nucleic acid encoding a thioesterase derived from *E. coli* as shown in SEQ ID NO:25, and having at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, or at least 73% homology or identity with the nucleic acid encoding a thioesterase derived from *E. coli* as shown in SEQ ID NO:25. For example, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology or identity.

[0213] Therefore, in some embodiments, yeast cells express the desaturase and fatty acyl-CoA reductase as described above, and also express one or more thioesterases, such as one or more heterothioesterases, as described herein. Specifically, yeast cells may express one or more desaturases selected from Cpo_CPRQ (SEQ ID NO:2), Gmo_CPRQ (SEQ ID NO:77), mutant Cpo_CPRQ (such as the S82 mutant or S85 mutant, preferably the S85 mutant such as the S85A mutant) and their functional variants, and one or more reductases selected from Ase_FAR (SEQ ID NO:10), mutant Ase_FAR (such as the T198 mutant or S413 mutant, preferably the T198A mutant or S413A mutant), Aip_FAR (SEQ ID NO:61), Hs_FAR (SEQ ID NO:71), Has_FAR (SEQ ID NO:73), Hv_FAR (SEQ ID NO:75), Har_FAR (SEQ ID NO:4) and their functional variants, as well as one or more heterothioesterases such as SEQ ID NO:33, SEQ ID NO:57, SEQ ID NO:35 and / or SEQ ID NO:10. The thioesterases or their functional variants shown in NO:26. In addition to Cpo_CPRQ or Gmo_CPRQ, their mutants or functional variants, yeast cells may also express another desaturase capable of introducing at least one double bond into a 12-carbon acyl-CoA fatty acid chain, as described above, such as Cpo_NPVE, Cpo_SPTQ, their mutants or functional variants.

[0214] Yeast cells can be further modified with any of the modifications described herein, particularly by: expression of heterologous cytochrome b5, heterologous cytochrome b5 reductase, expression of hemoglobin, mutations in one or more natural elongase genes resulting in complete or partial loss of activity, mutations in one or more natural thioesterase genes resulting in complete or partial loss of activity, mutations in one or more natural genes encoding one or more fatty aldehyde dehydrogenases, one or more fatty alcohol oxidases, peroxisome biogenetic factors and / or one or more fatty acyl synthases, and / or expression of fusion proteins of fatty acyl synthases and thioesterases, as described above herein.

[0215] Expression of a fusion protein of fatty acyl synthase and thioesterase

[0216] In some embodiments, yeast cells also express a fusion protein of a truncated fatty acyl synthase and a truncated thioesterase, such as the fusion protein shown in SEQ ID NO:59, or a homolog thereof having at least 60% homology or identity with it. This fusion protein is a fusion of a truncated form of Fas1 from *Yarrowia lipolytica* and a truncated form of thioesterase TesA from *Escherichia coli*. It can be expressed by introducing a nucleic acid as shown in SEQ ID NO:58. The fusion protein can be expressed at high levels.

[0217] Therefore, in some embodiments, yeast cells also express the fusion protein as shown in SEQ ID NO:59 or having at least 60% homology or identity with it, such as at least 61% homology or identity, at least 62% homology or identity, at least 63% homology or identity, at least 64% homology or identity, at least 65% homology or identity, at least 66% homology or identity, at least 67% homology or identity, at least 68% homology or identity, at least 69% homology or identity, at least 70% homology or identity, at least 71% homology or identity, at least 72%, at least 73%, at least 74%, and so on. At least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its homologous products.

[0218] In some embodiments, the yeast cell contains a nucleic acid encoding the fusion protein, such as the nucleic acid shown in SEQ ID NO:58, or having at least 60% homology or identity with it, or having at least 61% homology or identity with SEQ ID NO:58, such as at least 62% homology or identity, such as at least 63% homology or identity, such as at least 64% homology or identity, such as at least 65% homology or identity, such as at least 66% homology or identity, such as at least 67% homology or identity, such as at least 68% homology or identity, such as at least 69% homology or identity, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, and so on. At least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its homologous products.

[0219] Therefore, in some embodiments, yeast cells express the desaturase and fatty acyl-CoA reductase as described above, and also express a fusion protein of truncated fatty acyl synthase and truncated thioesterase, such as the fusion protein shown in SEQ ID NO:59. Specifically, yeast cells may express one or more desaturases selected from Cpo_CPRQ (SEQ ID NO:2), mutant Cpo_CPRQ (such as the S82 mutant or the S85 mutant, preferably the S85 mutant such as the S85A mutant), and functional variants thereof, and one or more reductases selected from Ase_FAR (SEQ ID NO:10), mutant Ase_FAR (such as the T198 mutant or the S413 mutant, preferably the T198A mutant or the S413A mutant), Aip_FAR (SEQ ID NO:61), Hs_FAR (SEQ ID NO:71), Has_FAR (SEQ ID NO:73), Hv_FAR (SEQ ID NO:75), Har_FAR (SEQ ID NO:12), and functional variants thereof, as well as fusion proteins of truncated fatty acyl synthase and truncated thioesterase as shown in SEQ ID NO:59, or functional variants thereof. In addition to Cpo_CPRQ or Gmo_CPRQ, their mutants or functional variants, yeast cells may also express another desaturase capable of introducing at least one double bond into a 12-carbon acyl-CoA fatty acid chain, as described above, such as Cpo_NPVE, Cpo_SPTQ, their mutants or functional variants.

[0220] Yeast cells can be further modified with any of the modifications described herein, particularly by: expression of heterologous cytochrome b5, expression of heterologous cytochrome b5 reductase, expression of hemoglobin, mutation of one or more natural elongase genes resulting in complete or partial loss of activity, mutation of one or more natural thioesterase genes resulting in complete or partial loss of activity, mutation of one or more natural genes encoding one or more fatty aldehyde dehydrogenases, one or more fatty alcohol oxidases, peroxisome biogenetic factors and / or one or more fatty acyl synthases, and / or expression of heterologous thioesterase genes, as described above herein.

[0221] titer

[0222] The yeast cells disclosed herein can produce E8,E10-dodecadien-1-ol at a titer of at least 0.2 mg / L. In some embodiments, the titer of E8,E10-dodecadien-1-ol is at least 0.25 mg / L, such as at least 0.3 mg / L, at least 0.4 mg / L, at least 0.5 mg / L, at least 0.75 mg / L, at least 1 mg / L, at least 1.5 mg / L, at least 2.5 mg / L, at least 5.0 mg / L, at least 10 mg / L, at least 15 mg / L, at least 2 mg / L, etc. 0 mg / L, such as 25 mg / L, such as at least 50 mg / L, such as at least 100 mg / L, such as at least 250 mg / L, such as at least 500 mg / L, such as at least 750 mg / L, such as at least 1 g / L, such as at least 2 g / L, such as at least 3 g / L, such as at least 4 g / L, such as at least 5 g / L, such as at least 6 g / L, such as at least 7 g / L, such as at least 8 g / L, such as at least 9 g / L, such as at least 10 g / L or more.

[0223] The methods for determining the titer are known in the art.

[0224] Production of E8,E10-dodecadienyl acetate

[0225] Ketoconazole can be further converted into E8,E10-dodecadienyl acetate; as is known in the art, this can be carried out in vitro, for example by chemical conversion, or it can be carried out in vivo by the action of an acetyltransferase (EC 2.3.1.84) capable of converting at least a portion of the cellularly produced E8,E10-dodecadien-1-ol into E8,E10-dodecadienyl acetate.

[0226] In some embodiments, yeast cells are thus engineered to overexpress native acetyltransferases and / or to express heterologous acetyltransferases (optionally at high levels). In such embodiments, yeast cells are able to produce E8,E10-dodecadien-1-ol and E8,E10-dodecadienyl acetate.

[0227] In some embodiments, yeast cells express an acetyltransferase capable of converting at least a portion of cell-produced E8,E10-dodecadien-1-ol into E8,E10-dodecadienyl acetate, such as Sc_Atf1 acetyltransferase (SEQ ID NO:37) or having at least 60% homology or identity with it, or having at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, or as much as SEQ ID NO:37. At least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its homologous products.

[0228] The expression of acetyltransferases can be achieved by introducing nucleic acids, which can be codon-optimized for expression in yeast cells, such as the nucleic acid shown in SEQ ID NO:36, or having at least 60% homology or identity with it, such as at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, and so on. At least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its homologous products.

[0229] Therefore, in some embodiments, yeast cells express heterologous desaturases as described above, heterologous fatty acyl reductases as described above, and optionally any other modifications described above, and also express acetyltransferases capable of converting at least partially generated E8,E10-dodecadien-1-ol to E8,E10-dodecadienyl acetate.

[0230] Therefore, the yeast cells disclosed herein are capable of producing E8,E10-dodecadienyl acetate at a titer of at least 0.2 mg / L. In some embodiments, the titer of E8,E10-dodecadienyl acetate is at least 0.25 mg / L, such as at least 0.3 mg / L, at least 0.4 mg / L, at least 0.5 mg / L, at least 0.75 mg / L, at least 1 mg / L, at least 1.5 mg / L, at least 2.5 mg / L, at least 5.0 mg / L, at least 10 mg / L, at least 15 mg / L, or at least 2 mg / L. 0 mg / L, such as 25 mg / L, such as at least 50 mg / L, such as at least 100 mg / L, such as at least 250 mg / L, such as at least 500 mg / L, such as at least 750 mg / L, such as at least 1 g / L, such as at least 2 g / L, such as at least 3 g / L, such as at least 4 g / L, such as at least 5 g / L, such as at least 6 g / L, such as at least 7 g / L, such as at least 8 g / L, such as at least 9 g / L, such as at least 10 g / L or more.

[0231] The methods for determining the titer are known in the art.

[0232] Production of E8,E10-dodecadienal

[0233] It may also be of interest to further convert at least a portion of the E8,E10-dodecadien-1-ol produced by the cells into E8,E10-dodecadienal. This can be done by chemical transformation or by further engineering the yeast cells.

[0234] In some embodiments, yeast cells can be further engineered to enable them to convert at least a portion of E8,E10-dodecadien-1-ol to E8,E10-dodecadienal. This can be achieved by engineering yeast cells to further express aldehyde-forming fatty acid-CoA reductase (EC 1.2.1.50), alcohol dehydrogenase (EC 1.1.1.2), and / or fatty alcohol oxidase (EC 1.1.3.20) capable of converting at least a portion of E8,E10-dodecadien-1-ol to E8,E10-dodecadienal. In such embodiments, yeast cells are capable of producing E8,E10-dodecadien-1-ol and E8,E10-dodecadienal.

[0235] Therefore, nucleic acids encoding aldehyde-forming fatty acid-CoA reductase (EC 1.2.1.50), alcohol dehydrogenase (EC 1.1.1.2), and / or fatty alcohol oxidase (EC 1.1.3.20) capable of converting at least a portion of E8,E10-dodecadien-1-ol to E8,E10-dodecadienal can be introduced into yeast cells. The nucleic acids can be codon-optimized and expressed at high levels.

[0236] Therefore, in some embodiments, yeast cells express heterologous desaturases as described above, heterologous fatty acyl reductases as described above, and optionally any other modifications described above, and also express aldehyde-forming fatty acyl-CoA reductase (EC 1.2.1.50), alcohol dehydrogenase (EC 1.1.1.2), and / or fatty alcohol oxidase (EC 1.1.3.20) capable of converting at least a portion of E8,E10-dodecadien-1-ol to E8,E10-dodecadienal.

[0237] Therefore, the yeast cells disclosed herein are capable of producing E8,E10-dodecadienal at a titer of at least 0.2 mg / L. In some embodiments, the titer of E8,E10-dodecadienal is at least 0.25 mg / L, such as at least 0.3 mg / L, at least 0.4 mg / L, at least 0.5 mg / L, at least 0.75 mg / L, at least 1 mg / L, at least 1.5 mg / L, at least 2.5 mg / L, at least 5.0 mg / L, at least 10 mg / L, at least 15 mg / L, or at least 20 mg / L. g / L, such as 25 mg / L, such as at least 50 mg / L, such as at least 100 mg / L, such as at least 250 mg / L, such as at least 500 mg / L, such as at least 750 mg / L, such as at least 1 g / L, such as at least 2 g / L, such as at least 3 g / L, such as at least 4 g / L, such as at least 5 g / L, such as at least 6 g / L, such as at least 7 g / L, such as at least 8 g / L, such as at least 9 g / L, such as at least 10 g / L or more.

[0238] The methods for determining the titer are known in the art.

[0239] Chain shortening

[0240] In some embodiments, yeast cells are further modified to increase the availability of fatty acyl-CoA of a given chain length through chain shortening. Without being bound by theory, such modifications are expected to increase the availability of substrates with the desired carbon chain length, particularly those with a carbon chain length of 12, thereby increasing the production of E8,E10-dodecadienyl-CoA and optionally E8,E10-dodecadien-1-ol, and optionally E8,E10-dodecadienyl acetate and E8,E10-dodecadienal. This can be achieved by reducing the activity of native acyl-CoA oxidases in the microbial producing cells and by expressing specific acyl-CoA oxidases, desaturases, reductases, and acetyltransferases. Such modifications are described in detail in EP 19157910.1 (filed by the same applicant on February 19, 2019).

[0241] Therefore, in some implementations, the yeast cell is any yeast cell described above, and also:

[0242] i) Having one or more mutations that reduce the activity of one or more natural acyl-CoA oxidases; and

[0243] ii) expressing at least one set of enzymes comprising at least one acyl-CoA oxidase capable of oxidizing fatty acyl-CoA, wherein the set of enzymes is capable of shortening fatty acyl-CoA of a first carbon chain length X to a shortened fatty acyl-CoA having a second carbon chain length X', wherein X' ≤ X-2.

[0244] In such embodiments, the activity of acyl-CoA oxidases (i.e., one or more natural enzymes) normally present in yeast cells is reduced or eliminated by mutating the genes encoding said one or more enzymes in the cell. To guide carbon chain shortening to obtain fatty alcohols and their derivatives of a desired carbon chain length, one or more acyl-CoA oxidases are expressed in yeast cells. These acyl-CoA oxidases may be natural for yeast cells or they may be derived from another organism. If the cells do not already express them, or if increased activity or substrate specificity is required, genes encoding other enzymes required for the oxidation of fatty acyl-CoA of a given chain length may be introduced into the cells. The one or more acyl-CoA oxidases thus expressed allow fatty acyl-CoA to be oxidized and shortened to fatty acyl-CoA with a carbon chain length shorter than that of the substrate. Therefore, in some embodiments, the reduction in the activity of one or more natural acyl-CoA oxidases is a reduction in the activity of acyl-CoA with a carbon chain length less than X, such as less than X'.

[0245] In this disclosure, the term acyl-CoA oxidase refers to an enzyme capable of catalyzing the following reactions, such as those described in EC number 1.3.3.6:

[0246]

[0247] This enzyme belongs to the oxidoreductase family, specifically those that act on the CH-CH group of a donor using oxygen as an acceptor. The systematic name for this class of enzymes is acyl-CoA:O2-oxidoreductase. Other names used include fatty acyl-CoA oxidase, acyl-CoA oxidase, and fatty acyl-CoA oxidase.

[0248] The yeast cells disclosed herein can be engineered starting from yeast cells possessing one or more natural acyl-CoA oxidases. The modified yeast cells disclosed herein preferably have reduced activity of the one or more natural acyl-CoA oxidases; this can be achieved by using yeast cells with one or more mutations that result in reduced activity of at least one natural acyl-CoA oxidase. Natural acyl-CoA oxidases can be peroxisomeric, mitochondrial, or cytoplasmic. In some embodiments, the one or more mutations result in reduced activity of all natural acyl-CoA oxidases. With respect to reduced activity, it should be understood that the yeast cell, due to the mutation, has a reduced ability to catalyze the aforementioned reactions, particularly the conversion of acyl-CoA to the corresponding trans-2,3-dehydroacyl-CoA. In some embodiments, "reduced ability" refers to the complete or partial elimination of the ability to catalyze the reactions. In some embodiments, "reduced ability" refers to the ability to catalyze reactions limited to a subset of substrates that would be available for the reaction under normal conditions (i.e., by using enzymes with normal ability).

[0249] The yeast cells disclosed herein can express at least one set of enzymes, said enzymes comprising at least one acyl-CoA oxidase capable of oxidizing fatty acyl-CoA. In addition to at least one acyl-CoA oxidase, the set of enzymes also includes other enzymes required to convert fatty acyl-CoA of a given carbon chain length to fatty acyl-CoA of a shorter carbon chain length. These other enzymes may preferably be native to the yeast cells; in such embodiments, expressing the set of enzymes in the yeast cells only requires the introduction of a gene encoding an acyl-CoA oxidase.

[0250] In embodiments where acyl-CoA oxidase is native to yeast cells, the acyl-CoA oxidase can be modified as known in the art, for example by introducing a promoter such as a constitutive or inducible promoter, or by implementing a promoter that overexpresses acyl-CoA oxidase. The native acyl-CoA oxidase reintroduced into the first group of enzymes can be a mutant form with modified activities (such as modified substrate specificity) and / or modified activities (such as increased reaction efficiency).

[0251] In other embodiments, the acyl-CoA oxidase is derived from another organism. The acyl-CoA contained in the first group of enzymes can be an acyl-CoA oxidase derived from yeast, fungi, insects, mammals, birds, or plants, such as at least one acyl-CoA oxidase in the first group of enzymes being derived from yeast, fungi, insects, mammals, birds, or plants. For example, the acyl-CoA oxidase is derived from organisms selected from genera including *Yersinia*, *Saccharomyces*, *Noctua*, *Arabidopsis*, *Aspergillus*, *Cucurbita*, *Homo*, *Arthrobacter*, and *Raconia*, such as at least one acyl-CoA oxidase in the first group of enzymes being derived from organisms selected from genera including *Yersinia*, *Saccharomyces*, *Noctua*, *Arabidopsis*, *Aspergillus*, *Cucurbita*, *Homo*, *Arthrobacter*, and *Raconia*. In some embodiments, the at least one group of enzymes comprises an acyl-CoA oxidase derived from Yersinia lipophila, Saccharomyces cerevisiae, cutworm, Arabidopsis thaliana, Aspergillus nidulans, Cucurbita maxima, Homo sapiens, Paenarthrobacterureafaciens, or Rattus norvegicus.

[0252] The acyl-CoA oxidase introduced into yeast cells can therefore be a naturally occurring acyl-CoA oxidase from *Yarrowia lipolytica*, *Cephalotaxus fortunei*, *Arabidopsis thaliana*, *Aspergillus nidus*, *Cucumis melo*, *Homo sapiens*, *Arthrobacter ureagenesis*, or *Rhizopus purpurea*. Yeast cells can be as described above.

[0253] Therefore, the yeast cells of this disclosure can express at least one group of enzymes comprising at least one acyl-CoA oxidase capable of oxidizing fatty acyl-CoA, wherein the at least one acyl-CoA oxidase is selected from Yli_POX1 (XP_504703), Yli_POX2 (XP_505264), Yli_POX3 (XP_503244), Yli_POX4 (XP_504475), Yli_POX5 (XP_502199), Yli_POX6 (XP_503632), Ase_POX (SEQ ID NO:39), Ath_POX1 (SEQ ID NO:41), Ath_POX2 (SEQ ID NO:43), Ani_POX (SEQ ID NO:45), Cma_POX (SEQ ID NO:47), Hsa_POX1-2 (SEQ ID NO:49), Pur_POX (SEQ ID NO:45), Ase_POX1-2 (SEQ ID NO:49), Pur_POX1-2 (SEQ ID NO:49 ... NO:51), Sc_POX1 (SEQ ID NO:31) and Rno_POX2 (SEQ ID NO:53), or their functional variants having at least 60% homology or identity, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity.

[0254] In some embodiments, the expression of at least one acyl-CoA oxidase is achieved by introducing a nucleic acid encoding said at least one acyl-CoA oxidase. For example, yeast expressions encoding Yli_POX1 (YALI0_E32835g), Yli_POX2 (YALI0_F10857g), Yli_POX3 (YALI0_D24750g), Yli_POX4 (YALI0_E27654g), Yli_POX5 (YALI0_C23859g), Yli_POX6 (YALI0_E06567g), Ase_POX (SEQ ID NO:38), Ath_POX1 (SEQ ID NO:40), Ath_POX2 (SEQ ID NO:42), Ani_POX (SEQ ID NO:44), Cma_POX (SEQ ID NO:46), Hsa_POX (SEQ ID NO:48), Pur_POX (SEQ ID NO:50), and Sc_POX1 (SEQ ID NO:50) are expressed. SEQ ID NO:52 of NO:30 or encoding Rno_POX2, or a homolog thereof having at least 60% homology or identity, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0255] In some implementations, X' = 12.

[0256] Suitable acyl-CoA oxidases are described in detail in WO 2020 / 169389 (filed by the same applicant on 10 February 2020), particularly in the section on "acyl-CoA oxidases".

[0257] To obtain E8, E10-dodecadienyl coenzyme A and optionally ketamine, in embodiments utilizing chain shortening, yeast cells may therefore express, in addition to expressing at least one set of enzymes, an additional heterologous desaturase capable of introducing at least one double bond of E / Z conformation into fatty acyl-CoA having a carbon chain length X or X'. X or X' can be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms. In some embodiments, the desaturase is capable of introducing at least one double bond of E / Z conformation into fatty acyl-CoA having a chain length of X', wherein X' is as defined above. Suitable desaturases are described in detail in WO 2020 / 169389 (filed by the same applicant on February 10, 2020), particularly in the section on "Desaturases (FAD)". In particular, desaturases capable of converting C14:CoA to Z11-C14:CoA and / or E11-C14:CoA are of particular interest. For example, desaturase CroZ11 (SEQ ID NO:63) from the rose leafroller (Choristoneura rosaceana) or CpaE11 (SEQ ID NO:63) from the parallel color leafroller (Choristoneura parallela) could be used. NO:65) or having at least 60% homology or identity with it, such as at least 61% homology or identity, such as at least 62% homology or identity, such as at least 63% homology or identity, such as at least 64% homology or identity, such as at least 65% homology or identity, such as at least 66% homology or identity, such as at least 67% homology or identity, such as at least 68% homology or identity, such as at least 69% homology or identity, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as At least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity of its functional variants. The resulting Z11-C14:CoA and / or E11-C14:CoA can then undergo further chain shortening to obtain Z9-C12:CoA and / or E9-C12:CoA, which are subsequently desaturated by Cpo_CPRQ to obtain E8, E10-C12:CoA.

[0258] In such an implementation, the yeast cell thus expresses at least one desaturase as described in the “Desaturases” section above, such as Cpo_CPRQ or Gmo_CPRQ (preferably Cpo_CPRQ), its mutants or functional variants, and also expresses an additional heterologous desaturase capable of introducing at least one double bond of E / Z conformation into a fatty acyl-CoA having a carbon chain length of X or X', wherein X and X' are as described above. Specifically, the desaturase is capable of introducing at least one double bond of E / Z conformation into a fatty acyl-CoA with a carbon chain length of 14, which can then be shortened to a fatty acyl-CoA with a carbon chain length of 12 as described above—which can then be further desaturated to E8,E10-C12:CoA, and can then be converted to E8,E10-dodecadien-1-ol by the action of FAR as detailed above.

[0259] Methods for producing E8,E10-dodecadienyl coenzyme A, E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienyl acetate

[0260] The yeast cells described above can be used to produce E8,E10-dodecadienyl coenzyme A and optionally E8,E10-dodecadien-1-ol, which can be further converted into E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienyl acetate.

[0261] Methods for producing E8,E10-dodecadien-1-ol

[0262] This article provides a method for producing E8,E10-dodecadienyl-CoA and optionally E8,E10-dodecadien-1-ol in yeast cells, the method comprising the steps of providing yeast cells and incubating the yeast cells in a culture medium, wherein the yeast cells express:

[0263] i) at least one heterologous desaturase capable of introducing one or more double bonds into a fatty acyl-CoA with a carbon chain length of 12, thereby converting said fatty acyl-CoA into desaturated fatty acyl-CoA, wherein at least a portion of said desaturated fatty acyl-CoA is E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA); and

[0264] ii) Optionally, at least one heterologous fatty acyl-CoA reductase (EC 1.2.1.84) capable of converting at least a portion of the desaturated fatty acyl-CoA into a desaturated fatty alcohol, wherein the fatty acyl-CoA reductase is capable of converting at least a portion of the E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA) into E8,E10-dodecadien-1-ol.

[0265] This produces E8,E10-dodecadienyl coenzyme A and optionally E8,E10-dodecadien-1-ol.

[0266] The yeast cell can be any yeast cell described above in this article.

[0267] The method of the present invention preferably allows the production of E8,E10-dodecadienyl-1-ol at a titer of at least 0.2 mg / L. In some embodiments, the titer of E8,E10-dodecadien-1-ol is at least 0.25 mg / L, such as at least 0.3 mg / L, at least 0.4 mg / L, at least 0.5 mg / L, at least 0.75 mg / L, at least 1 mg / L, at least 1.5 mg / L, at least 2.5 mg / L, at least 5.0 mg / L, at least 10 mg / L, at least 15 mg / L, at least 2 mg / L, etc. 0 mg / L, such as 25 mg / L, such as at least 50 mg / L, such as at least 100 mg / L, such as at least 250 mg / L, such as at least 500 mg / L, such as at least 750 mg / L, such as at least 1 g / L, such as at least 2 g / L, such as at least 3 g / L, such as at least 4 g / L, such as at least 5 g / L, such as at least 6 g / L, such as at least 7 g / L, such as at least 8 g / L, such as at least 9 g / L, such as at least 10 g / L or more.

[0268] The method of the present invention allows for the production of E8,E10-dodecadien-1-ol at a titer of at least 0.2 mg / L. In some embodiments, the titer of E8,E10-dodecadien-1-ol is at least 0.25 mg / L, such as at least 0.3 mg / L, at least 0.4 mg / L, at least 0.5 mg / L, at least 0.75 mg / L, at least 1 mg / L, at least 1.5 mg / L, at least 2.5 mg / L, at least 5.0 mg / L, at least 10 mg / L, at least 15 mg / L, at least 2 mg / L, etc. 0 mg / L, such as 25 mg / L, such as at least 50 mg / L, such as at least 100 mg / L, such as at least 250 mg / L, such as at least 500 mg / L, such as at least 750 mg / L, such as at least 1 g / L, such as at least 2 g / L, such as at least 3 g / L, such as at least 4 g / L, such as at least 5 g / L, such as at least 6 g / L, such as at least 7 g / L, such as at least 8 g / L, such as at least 9 g / L, such as at least 10 g / L or more.

[0269] The methods for determining the titer are known in the art.

[0270] Methods for producing E8,E10-dodecadienyl acetate

[0271] In some embodiments, the method further includes the step of converting at least a portion of E8,E10-dodecadien-1-ol to E8,E10-dodecadienyl acetate via acetyltransferase expression or chemical conversion. Therefore, this document discloses a method for producing E8,E10-dodecadienyl acetate in yeast cells, the method comprising the following steps:

[0272] a) Providing yeast cells and incubating the yeast cells in a culture medium, wherein the yeast cells express:

[0273] i) at least one heterologous desaturase capable of introducing one or more double bonds into a fatty acyl-CoA with a carbon chain length of 12, thereby converting said fatty acyl-CoA into desaturated fatty acyl-CoA, wherein at least a portion of said desaturated fatty acyl-CoA is E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA); and

[0274] ii) at least one heterologous fatty acyl-CoA reductase (EC 1.2.1.84) capable of converting at least a portion of the desaturated fatty acyl-CoA into a desaturated fatty alcohol, wherein the fatty acyl-CoA reductase is capable of converting at least a portion of the E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA) into E8,E10-dodecadien-1-ol.

[0275] b) Convert at least a portion of E8,E10-dodecadien-1-ol to E8,E10-dodecadienyl acetate.

[0276] In some implementations, E8,E10-dodecadienyl acetate is obtained by engineering yeast cells as described above in “The Generation of E8,E10-dodecadienyl acetate”.

[0277] In other embodiments, the conversion of cell-produced E8,E10-dodecadien-1-ol to E8,E10-dodecadienyl acetate is carried out chemically, as is known in the art. For example, cell-produced E8,E10-dodecadien-1-ol can be recovered, and then acetyl chloride can be added to the E8,E10-dodecadien-1-ol, mixed, and incubated, for example, at room temperature, thereby converting at least a portion of cell-produced E8,E10-dodecadien-1-ol to E8,E10-dodecadienyl acetate.

[0278] In other embodiments, yeast cells produce E8,E10-dodecadienyl coenzyme A, which can be converted into lipids (such as triglycerides) or free fatty acids. The lipids or free fatty acids are then recovered and can be further converted into E8,E10-dodecadien-1-ol. As described above, E8,E10-dodecadien-1-ol can then be further converted into E8,E10-dodecadien-1-ol in vitro. In such embodiments, the conversion of cell-produced E8,E10-dodecadien-1-ol to E8,E10-dodecadienyl acetate is carried out chemically, as is known in the art.

[0279] Therefore, the method of the present invention can allow the production of E8,E10-dodecadienyl acetate with a titer of at least 0.2 mg / L. In some embodiments, the titer of E8,E10-dodecadienyl acetate is at least 0.25 mg / L, such as at least 0.3 mg / L, at least 0.4 mg / L, at least 0.5 mg / L, at least 0.75 mg / L, at least 1 mg / L, at least 1.5 mg / L, at least 2.5 mg / L, at least 5.0 mg / L, at least 10 mg / L, at least 15 mg / L, at least 2 mg / L, etc. 0 mg / L, such as 25 mg / L, such as at least 50 mg / L, such as at least 100 mg / L, such as at least 250 mg / L, such as at least 500 mg / L, such as at least 750 mg / L, such as at least 1 g / L, such as at least 2 g / L, such as at least 3 g / L, such as at least 4 g / L, such as at least 5 g / L, such as at least 6 g / L, such as at least 7 g / L, such as at least 8 g / L, such as at least 9 g / L, such as at least 10 g / L or more.

[0280] The methods for determining the titer are known in the art.

[0281] Methods for producing E8,E10-dodecadienal

[0282] In some embodiments, the method further includes the step of converting at least a portion of E8,E10-dodecadien-1-ol to E8,E10-dodecadienal by further engineering yeast cells or by chemical conversion. Therefore, this document discloses a method for producing E8,E10-dodecadienal in yeast cells, the method comprising the following steps:

[0283] a) Providing yeast cells and incubating the yeast cells in a culture medium, wherein the yeast cells express:

[0284] i) at least one heterologous desaturase capable of introducing one or more double bonds into a fatty acyl-CoA with a carbon chain length of 12, thereby converting said fatty acyl-CoA into desaturated fatty acyl-CoA, wherein at least a portion of said desaturated fatty acyl-CoA is E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA); and

[0285] ii) at least one heterologous fatty acyl-CoA reductase (EC 1.2.1.84) capable of converting at least a portion of the desaturated fatty acyl-CoA into a desaturated fatty alcohol, wherein the fatty acyl-CoA reductase is capable of converting at least a portion of the E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA) into E8,E10-dodecadien-1-ol.

[0286] b) Convert at least a portion of E8,E10-dodecadien-1-ol to E8,E10-dodecadienal.

[0287] In some implementations, E8,E10-dodecadienal is obtained by engineering yeast cells as described above in “The Generation of E8,E10-dodecadienal”.

[0288] In other embodiments, the method includes the step of converting at least a portion of E8,E10-dodecadien-1-ol to E8,E10-dodecadienal via chemical conversion. The chemical conversion is based on the oxidation of E8,E10-dodecadien-1-ol to E8,E10-dodecadienal. Methods for carrying out this conversion are known in the art. Preferred methods are environmentally friendly and minimize the amount of hazardous waste.

[0289] In other embodiments, yeast cells produce E8,E10-dodecadienyl coenzyme A, which can be converted into lipids (such as triglycerides) or free fatty acids, and then recovered and converted in vitro into E8,E10-dodecadien-1-ol, as described above. In such embodiments, the conversion of cell-produced E8,E10-dodecadien-1-ol to E8,E10-dodecadienal is carried out chemically, as is known in the art.

[0290] Therefore, in some embodiments, the chemical conversion can be metal-free, avoiding toxic heavy metal-based reagents such as manganese oxide, chromium oxide (Jones ox. PDC, PCC), or ruthenium compounds (TPAP, Ley-Griffith ox.). In some embodiments, the conversion does not involve reactions involving activated dimethyl sulfoxide, such as Swern oxidation or Pfitzner-Moffat type reactions. Such reactions may involve the stereochemical formation of trace amounts of strongly odorous organosulfur compounds, such as dimethyl sulfide, which may be difficult to remove from the target product. In some embodiments, the method includes the Dess-Martin reaction (Yadav et al., 2004; Meyer et al., 1994). In other embodiments, the chemical conversion includes oxidation with sodium hypochlorite under aqueous / organic two-phase conditions (Okada et al., 2014; Tamura et al., 2012; Li et al., 2009). In some implementations, chemical oxidation with 1-chlorobenzotriazole can be carried out in a dichloromethane medium containing 25% pyridine (Ferrell and Yao, 1972).

[0291] Alternatively, the oxidation of E8,E10-dodecadien-1-ol to E8,E10-dodecadienal can be facilitated by alcohol dehydrogenase. Those skilled in the art will know how to perform this enzymatic oxidation. For example, it can be performed by contacting purified enzymes, cell extracts, or whole cells with E8,E10-dodecadien-1-ol.

[0292] Therefore, in some embodiments, the method disclosed herein allows the production of E8,E10-dodecadienal at a titer of at least 0.2 mg / L. In some embodiments, the titer of E8,E10-dodecadienal is at least 0.25 mg / L, such as at least 0.3 mg / L, at least 0.4 mg / L, at least 0.5 mg / L, at least 0.75 mg / L, at least 1 mg / L, at least 1.5 mg / L, at least 2.5 mg / L, at least 5.0 mg / L, at least 10 mg / L, at least 15 mg / L, or at least 20 mg / L. g / L, such as 25 mg / L, such as at least 50 mg / L, such as at least 100 mg / L, such as at least 250 mg / L, such as at least 500 mg / L, such as at least 750 mg / L, such as at least 1 g / L, such as at least 2 g / L, such as at least 3 g / L, such as at least 4 g / L, such as at least 5 g / L, such as at least 6 g / L, such as at least 7 g / L, such as at least 8 g / L, such as at least 9 g / L, such as at least 10 g / L or more.

[0293] The methods for determining the titer are known in the art.

[0294] Recycle

[0295] In some implementations, the method further includes a step of recovering the obtained product.

[0296] In some embodiments, the method is used to produce E8,E10-dodecadien-1-ol, and therefore further includes a step of recovering the produced E8,E10-dodecadien-1-ol. In other embodiments, the method is used to produce E8,E10-dodecadienyl acetate, and therefore further includes a step of recovering the produced E8,E10-dodecadienyl acetate. In other embodiments, the method is used to produce E8,E10-dodecadienal, and therefore further includes a step of recovering the produced E8,E10-dodecadienal.

[0297] Methods for recovering products obtained by the method of the present invention are known in the art and may include extraction with a hydrophobic solvent (such as decane, hexane or vegetable oil).

[0298] Alternatively, the method described in application PCT / EP2020 / 076351 (filed by the same applicant on September 22, 2020) can also be used to recover desired products. For example, the method can be used to recover lipids (such as triglycerides) or fatty acids obtained from the conversion of E8,E10-dodecadienyl coenzyme A, or to recover the resulting E8,E10-dodecadien-1-ol, the resulting E8,E10-dodecadienyl acetate, and / or the resulting E8,E10-dodecadienal. The method utilizes the addition of an extractant to the culture medium in an amount equal to or greater than its turbidity concentration measured in aqueous solution (e.g., in a culture medium) at culture temperature, which greatly facilitates the recovery of hydrophobic compounds such as fatty alcohols, fatty alcohol acetates, and fatty aldehydes. Therefore, such a method can be advantageously used to facilitate the recovery of lipids (such as triglycerides) or fatty acids obtained through the conversion of E8,E10-dodecadienyl coenzyme A, E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate, and E8,E10-dodecadienal produced by the method of the present invention. Furthermore, it has been found that the addition of the extractant to the culture medium generally increases the titer of hydrophobic compounds produced by the cells and increases the secretion of the produced hydrophobic compounds from the cells.

[0299] Therefore, in some embodiments, the culture medium used in the method of the present invention contains an amount of extractant equal to or greater than its turbidity concentration measured in an aqueous solution (e.g., in the culture medium) at the culture temperature, wherein the extractant is a nonionic surfactant, preferably a nonionic ethoxylated surfactant such as an antifoaming agent, preferably selected from the following polyethoxylated surfactants: polyoxyethylene polyoxypropylene ether, mixtures of polyether dispersions, antifoaming agents containing polyethylene glycol monostearate such as dimethicone, fatty alcohol alkoxylates, polyethoxylated surfactants, and ethoxylated and propoxylated C16 -C 18 Alcohol-based defoamers and combinations thereof.

[0300] The turbidity concentration in the aqueous solution is determined at a given temperature, preferably at room temperature or at the temperature at which fermentation is to take place, such as 30°C, or at room temperature. As used herein, the term "extractant" refers to a nonionic surfactant, particularly an antifoaming agent, that promotes the recovery of hydrophobic compounds generated during fermentation. For example, a nonionic surfactant is a nonionic ethoxylated surfactant, such as a polyethoxylated surfactant selected from: polyoxyethylene polyoxypropylene ether, mixtures of polyether dispersions, antifoaming agents containing polyethylene glycol monostearate such as dimethicone, fatty alcohol alkoxylates, polyethoxylated surfactants, and ethoxylated and propoxylated C 16 -C 18 Alcohol-based defoamers, and combinations thereof. Example 7 of PCT / EP2020 / 076351 describes how to determine the turbidity concentration of surfactants.

[0301] In application PCT / EP2020 / 076351 (filed by the same applicant on 22 September 2020), the nonionic surfactant as a suitable extractant and the suitable amount of said nonionic surfactant are described in detail, particularly in the section entitled “Nonionic Ethoxylated Surfactants”.

[0302] In some embodiments, the culture medium used in the method of the present invention therefore comprises a nonionic surfactant, which is ethoxylated and propoxylated C 16 -C 18 Alcohol-based defoamers, such as C 16 -C 18 Alkyl alcohol ethoxylate propoxylate (CAS No. 68002-96-0), and the culture medium contains at least 1% vol / vol C 16 -C 18 Alkyl alcohol ethoxylates and propoxylates, such as at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 22.5%, at least 25%, at least 27.5%, or at least 30% vol / vol of C 16 -C 18 Alkyl alcohol ethoxylates, propoxylates, or more.

[0303] In other embodiments, the culture medium used in the method of the present invention comprises a nonionic surfactant, such as polyoxyethylene polyoxypropylene ether, for example... P407 (CAS No. 9003-11-6), and the culture medium contains at least 10% vol / vol of polyoxyethylene polyoxypropylene ether, such as P407, such as at least 11% vol / vol, such as at least 12% vol / vol, such as at least 13% vol / vol, such as at least 14% vol / vol, such as at least 15% vol / vol, such as at least 16% vol / vol, such as at least 17% vol / vol, such as at least 18% vol / vol, such as at least 19% vol / vol, such as at least 20% vol / vol, such as at least 25% vol / vol, such as at least 30% vol / vol, such as at least 35% vol / vol of polyoxyethylene polyoxypropylene ether. P407, or more.

[0304] In other embodiments, the culture medium used in the method of the present invention comprises a nonionic surfactant, which is a mixture of polyether dispersions such as defoamer 204, and the culture medium comprises at least 1% vol / vol of a mixture of polyether dispersions such as defoamer 204, such as at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 22.5%, at least 25%, at least 27.5%, at least 30% vol / vol of a mixture of polyether dispersions such as defoamer 204, or more.

[0305] In other embodiments, the culture medium used in the method of the present invention comprises a nonionic surfactant comprising polyethylene glycol monostearate such as dimethicone, and the culture medium comprises at least 1% vol / vol of polyethylene glycol monostearate or dimethicone, such as at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 22.5%, at least 25%, at least 27.5%, at least 30% vol / vol of polyethylene glycol monostearate or dimethicone, or more.

[0306] In other embodiments, the culture medium used in the method of the present invention comprises a nonionic surfactant, which is a fatty alcohol alkoxylate, and the culture medium contains at least 1% vol / vol of the fatty alcohol alkoxylate, such as at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 22.5%, at least 25%, at least 27.5%, or at least 30% vol / vol of the fatty alcohol alkoxylate, or more. Suitable fatty alcohol alkoxylates include... LF300 (CAS No. 196823-11-7) LF1300 (68002-96-0), SLF180 (CAS No. 196823-11-7) 2574 (CAS No. 68154-97-2) and Imbentin SG / 251 (CAS No. 68002-96-0) are preferred. LF300 or 2574.

[0307] In other embodiments, the culture medium used in the method of the present invention comprises a nonionic surfactant, Agnique BP420 (CAS No. 68002-96-0), and the culture medium contains at least 1% vol / vol of Agnique BP420, such as at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 22.5%, at least 25%, at least 27.5%, at least 30% vol / vol of Agnique BP420, or more.

[0308] In some embodiments, the culture medium contains an extractant in an amount greater than 50% of its turbidity concentration, such as at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 750%, at least 1000%, or more, and / or the culture medium contains an extractant in an amount at least twice its turbidity concentration, such as at least three times its turbidity concentration. If its turbidity concentration is at least 4 times, if its turbidity concentration is at least 5 times, if its turbidity concentration is at least 6 times, if its turbidity concentration is at least 7 times, if its turbidity concentration is at least 8 times, if its turbidity concentration is at least 9 times, if its turbidity concentration is at least 10 times, if its turbidity concentration is at least 12.5 times, if its turbidity concentration is at least 15 times, if its turbidity concentration is at least 17.5 times, if its turbidity concentration is at least 20 times, if its turbidity concentration is at least 25 times, if its turbidity concentration is at least 30 times.

[0309] The addition of an extractant, i.e., a nonionic surfactant such as a polyethoxylated surfactant, for example any nonionic surfactant, defoamer, or polyethoxylated surfactant described herein, results in the formation of an emulsion in the fermentation broth, wherein hydrophobic compounds produced by the microorganisms, namely E8,E10-dodecadienyl-CoA (or lipids or free fatty acids obtained by conversion of E8,E10-dodecadienyl-CoA), E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate, and / or E8,E10-dodecadienal, are present in the emulsion. In embodiments of the method of the present invention carried out with a culture medium containing the extractant, the method may therefore further include the step of disrupting the emulsion to recover the product phase containing the extractant and the hydrophobic compounds. Once the emulsion is disrupted, the fermentation broth separates into three phases: an aqueous phase primarily containing water and hydrated compounds; a phase containing cells and cell debris; and a product phase primarily containing extractants and E8,E10-dodecadienyl-CoA (or lipids or free fatty acids obtained through the conversion of E8,E10-dodecadienyl-CoA), E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate, and / or E8,E10-dodecadienal. Thus, a composition consisting of these three phases is obtained. This is described in detail in application PCT / EP2020 / 076351 (filed by the same applicant on September 22, 2020), particularly in the section entitled "Product Phase Containing Hydrophobic Compounds".

[0310] In some embodiments, a majority of E8,E10-dodecadienyl coenzyme A (or lipids or free fatty acids obtained by converting E8,E10-dodecadienyl coenzyme A), and optionally a majority of E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate, and / or E8,E10-dodecadienal are present in the product phase. For example, at least 50% of E8,E10-dodecadienyl coenzyme A (or lipids or free fatty acids obtained by converting E8,E10-dodecadienyl coenzyme A) and optionally E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate, and / or E8,E10-dodecadienal are present in the product phase, such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or so on. At least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% of E8,E10-dodecadienyl coenzyme A (or lipids or free fatty acids obtained by conversion of E8,E10-dodecadienyl coenzyme A) and optionally E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienal are present in the product phase. In some embodiments, the product phase comprises at least 50% of E8,E10-dodecadienyl coenzyme A (or lipids or free fatty acids obtained by conversion of E8,E10-dodecadienyl coenzyme A) initially present in the fermentation broth and optionally E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate, and / or E8,E10-dodecadienal, such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% of E8,E10-dodecadienyl coenzyme A (or lipids or free fatty acids obtained by conversion of E8,E10-dodecadienyl coenzyme A) and optionally E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienal.

[0311] The step of breaking down the emulsion can be carried out as is known in the art, for example by subjecting the emulsion to a phase separation step, such as by centrifugation.

[0312] Following the step of disrupting the emulsion, a product phase comprising an extractant and lipids or free fatty acids obtained by converting E8,E10-dodecadienyl-CoA, and optionally E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate, and / or E8,E10-dodecadienal. In such embodiments, the method may further include the step of separating the lipids or free fatty acids obtained by converting E8,E10-dodecadienyl-CoA, and optionally E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate, and / or E8,E10-dodecadienal from the extractant. This can be carried out by methods known in the art, such as by distillation (e.g., distillation under reduced pressure) or by column purification, or any other suitable method. The extractant can be recycled to a fermenter or bioreactor.

[0313] Nucleic acid constructs

[0314] Nucleic acid constructs for modifying yeast cells are also provided, the constructs comprising:

[0315] i) at least one first polynucleotide encoding at least one heterodesaturase capable of introducing one or more double bonds into a 12-carbon acyl-CoA fatty acid chain, thereby converting the fatty acid-CoA into desaturated fatty acid-CoA, wherein at least a portion of the desaturated fatty acid-CoA is E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA); and

[0316] ii) Optionally encoding a second polynucleotide of at least one heterologous fatty acyl-CoA reductase (EC 1.2.1.84), said at least one heterologous fatty acyl-CoA reductase being capable of converting at least a portion of said desaturated fatty acyl-CoA into desaturated fatty alcohol, said fatty acyl-CoA reductase being capable of converting at least a portion of said E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA) into E8,E10-dodecadien-1-ol.

[0317] Nucleic acid constructs can be used to obtain the yeast cells described herein, namely yeast cells capable of producing E8,E10-dodecadienyl-CoA and optionally E8,E10-dodecadien-1-ol. The term "nucleic acid construct" herein may refer to a single physical entity, i.e., a single molecule, such as a vector or plasmid containing a first polynucleotide and optionally a second polynucleotide, or it may refer to multiple nucleic acid molecules, such as a first polynucleotide contained in one plasmid or vector and a second polynucleotide contained in another plasmid or vector.

[0318] Nucleic acid constructs may also include one or more of the following:

[0319] iii) Polynucleotides encoding heterologous cytochrome b5, such as the polynucleotide shown in SEQ ID NO:3 or its homologs having at least 60% homology or identity with it;

[0320] iv) Polynucleotides encoding heterologous cytochrome b5 reductase, such as the polynucleotide shown in SEQ ID NO:23 or its homologs having at least 60% homology or identity with it;

[0321] v) a polynucleotide encoding hemoglobin, such as the polynucleotide shown in SEQ ID NO:5 or a homolog thereof having at least 60% homology or identity with it; and / or

[0322] vi) A polynucleotide encoding a thioesterase, such as the polynucleotide shown in SEQ ID NO:25 or SEQ ID NO:34, or a homolog thereof having at least 60% homology or identity with it.

[0323] Polynucleotides can contain several copies of any of the aforementioned genes and can be codon-optimized to ensure proper expression in the yeast cells to which they will be introduced.

[0324] In some embodiments, at least one heterologous desaturase capable of introducing one or more double bonds into a 12-carbon acyl-CoA to convert the acyl-CoA into a desaturated acyl-CoA (wherein at least a portion of the desaturated acyl-CoA is E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA)) is Cpo_CPRQ (SEQ ID NO:2) as described above or a functional variant thereof having at least 60% homology or identity with it. In such embodiments, the first polynucleotide comprises SEQ ID NO:1 or a homolog thereof having at least 60% homology or identity with it, as described above herein.

[0325] In some embodiments, at least one heterologous desaturase is a mutant Cpo_CPRQ, such as a Cpo_CPRQ mutant with a mutation at position 85, or a functional variant thereof having at least 60% homology or identity with it. In some embodiments, the mutation is the S85A mutation. In some embodiments, the desaturase is a mutant Cpo_CPRQ, such as a Cpo_CPRQ mutant with a mutation at position 82. In some embodiments, the mutation is the S82A mutation, or a functional variant thereof having at least 60% homology or identity with it.

[0326] In other embodiments, at least one heterologous desaturase capable of introducing one or more double bonds into a 12-carbon-chain acyl-CoA to convert the acyl-CoA into a desaturated acyl-CoA (wherein at least a portion of the desaturated acyl-CoA is E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA)) is Gmo_CPRQ (SEQ ID NO:77) or a functional variant thereof having at least 60% homology or identity with it, as described above. In such embodiments, the first polynucleotide comprises SEQ ID NO:78 or a homolog thereof having at least 60% homology or identity with it, as described herein.

[0327] In some embodiments, yeast cells express several desaturases capable of introducing one or more double bonds into a 12-carbon acyl-CoA fatty acid. In such embodiments, preferably, at least one of the desaturases is Cpo_CPRQ, Gmo_CPRQ, mutants thereof, or functional variants thereof, as detailed herein, and the first polynucleotide comprises or consists of SEQ ID NO:1 or a homolog thereof having at least 60% homology or identity with it. In such embodiments, the nucleic acid construct may contain additional first polynucleotides, each encoding a desaturase as described above herein. For example, the nucleic acid construct contains a first polynucleotide encoding Cpo_CPRQ, Gmo_CPRQ, or a homolog thereof, and also contains additional first polynucleotides encoding another desaturase, preferably Cpo_NPVE (SEQ ID NO:67) or Cpo_SPTQ (SEQ ID NO:69) or a functional variant thereof having at least 60% homology or identity with it. Therefore, in some embodiments, the additional first polynucleotide comprises SEQ ID NO:66 or SEQ ID NO:68, or a homolog thereof having at least 60% homology or identity with it.

[0328] The nucleic acid construct may also contain a second polynucleotide encoding a FAR. The FAR is preferably an insect FAR, such as a natural FAR for insects of the genera *Helicoverpa*, *Helicoverpa*, *Helicoverpa virescens*, or *Helicoverpa spp.*. For example, the FAR is natural for *Agrotissegetum*, *Agrotis ipsilon*, *Heliothis subflexa*, *Helicoverpa assulta*, *Helicoverpa virescens*, or *Helicoverpa spp.*

[0329] In some embodiments, the FAR is Ase_FAR (SEQ ID NO:10), i.e., the FAR naturally occurring in *Gnaphalium affine*. In some embodiments, the heterologous FAR is a functional variant of Ase_FAR that retains the ability to convert E8,E10-C12:CoA to E8,E10-dodecadien-1-ol. For example, the functional variant has at least 65% homology or identity with it. In some embodiments, the FAR is a mutant Ase_FAR, such as a mutant with a mutation at position 198 or 413. In some embodiments, the Ase_FAR mutant is the T198A mutant. In other embodiments, the Ase_FAR mutant is the S413A mutant. In such embodiments, the second polynucleotide comprises or consists of SEQ ID NO:9 or its homolog having at least 60% homology or identity with it.

[0330] In other embodiments, the FAR is Aip_FAR (SEQ ID NO:61), which is the FAR naturally found in the cutworm. In some embodiments, the heterologous FAR is a functional variant of Aip_FAR that retains the ability to convert E8,E10-C12:CoA to E8,E10-dodecadien-1-ol. In such embodiments, the second polynucleotide comprises or consists of SEQ ID NO:60 or its homologs having at least 60% homology or identity with it.

[0331] In other embodiments, the FAR is Hs_FAR (SEQ ID NO:71), which is the FAR naturally found in Heliothissubflexa. In some embodiments, the heterologous FAR is a functional variant of Hs_FAR that retains the ability to convert E8,E10-C12:CoA to E8,E10-dodecadien-1-ol. In such embodiments, the second polynucleotide comprises or consists of SEQ ID NO:70 or its homologs having at least 60% homology or identity with it.

[0332] In other embodiments, FAR is Has_FAR (SEQ ID NO:73), which is the FAR naturally found in the tobacco burrowing moth. In some embodiments, the heterologous FAR is a functional variant of Has_FAR that retains the ability to convert E8,E10-C12:CoA to E8,E10-dodecadien-1-ol. In such embodiments, the second polynucleotide comprises or consists of SEQ ID NO:72 or its homologs having at least 60% homology or identity with it.

[0333] In other embodiments, the FAR is Hv_FAR (SEQ ID NO:75), which is the FAR naturally found in the tobacco shoot borer. In some embodiments, the heterologous FAR is a functional variant of Hv_FAR that retains the ability to convert E8,E10-C12:CoA to E8,E10-dodecadien-1-ol. In such embodiments, the second polynucleotide comprises or consists of SEQ ID NO:74 or its homologs having at least 60% homology or identity with it.

[0334] In other embodiments, the FAR is Har_FAR (SEQ ID NO:12), which is the FAR naturally found in the cotton bollworm (Helicoverpa armigera). In some embodiments, the heterologous FAR is a functional variant of Har_FAR that retains the ability to convert E8,E10-C12:CoA to E8,E10-dodecadien-1-ol. In such embodiments, the second polynucleotide comprises or consists of SEQ ID NO:13 or its homologs having at least 60% homology or identity with it.

[0335] In other embodiments, the FAR is a codling moth FAR, such as Cpo_FAR or a functional variant thereof, which retains the ability to convert E8,E10-C12:CoA to E8,E10-dodecadien-1-ol. In such embodiments, the second polynucleotide comprises or consists of SEQ ID NO:75 or its homologs having at least 60% homology or identity with it.

[0336] In implementations that require the expression of several FARs, the second polynucleotide can be multiple second polynucleotides, each encoding one FAR.

[0337] In some embodiments, the nucleic acid construct contains at least one additional polynucleotide, which may be a nucleic acid molecule different from the first and / or second polynucleotide, or it may be part of a nucleic acid molecule identical to the first and / or second polynucleotide.

[0338] In some embodiments, the additional polynucleotide encodes heterologous cytochrome b5, such as cytochrome b5 shown in SEQ ID NO:3, or a homolog thereof having at least 60% homology or identity with it. In some embodiments, cytochrome b5 is a naturally occurring cytochrome b5 for lepidopteran species. In a particular embodiment, cytochrome b5 is cytochrome b5 from a species of the genus *Botrytis*, preferably cytochrome b5 from *Botrytis cinerea* shown in SEQ ID NO:4, or a functional variant thereof having at least 60% homology or identity with it. In such embodiments, the additional polynucleotide comprises or consists of SEQ ID NO:3 or a homolog thereof having at least 60% homology with it.

[0339] In some embodiments, the additional polynucleotide encodes a heterologous cytochrome b5 reductase, such as the cytochrome b5 reductase shown in SEQ ID NO:24, or a homolog thereof having at least 60% homology or identity with it. In some embodiments, the cytochrome b5 reductase is a natural cytochrome b5 reductase for species of the genus *Boltria*. In a particular embodiment, the cytochrome b5 reductase is a cytochrome b5 reductase from a species of the genus *Boltria*, preferably the cytochrome b5 reductase from *Boltria* shown in SEQ ID NO:24, or a functional variant thereof having at least 60% homology or identity with it. In such embodiments, the additional polynucleotide comprises or consists of the following: SEQ ID NO:23 or a homolog thereof having at least 60% homology with it.

[0340] In some embodiments, the additional polynucleotide encodes a heterologous hemoglobin, such as the hemoglobin shown in SEQ ID NO:6, or a homolog thereof having at least 60% homology or identity with it. In some embodiments, the hemoglobin is a hemoglobin native to species of the genus *Vibrio fusiforme*. In a particular embodiment, the hemoglobin is the hemoglobin from *Vibrio fusiforme* as shown in SEQ ID NO:6, or a functional variant thereof having at least 60% homology or identity with it. In such embodiments, the additional polynucleotide comprises or consists of the following: SEQ ID NO:5 or a homolog thereof having at least 60% homology with it.

[0341] In some embodiments, the additional polynucleotide encodes a thioesterase, such as the thioesterase shown in SEQ ID NO:6, or a homolog thereof having at least 60% homology or identity with it. In some embodiments, the thioesterase is natural for species of the genera *Cuphea*, *Cinnamomum*, or *Escherichia*. In specific embodiments, the thioesterase is a hemoglobin from *Cuphea hygrophila*, *Cuphea hygrophila*, *Cinnamomum*, or *Escherichia coli*, as shown in SEQ ID NO:33, SEQ ID NO:57, SEQ ID NO:35, or SEQ ID NO:26, or a functional variant thereof having at least 60% homology or identity with it. In such embodiments, the additional polynucleotide comprises or consists of the following: SEQ ID NO:34, SEQ ID NO:56, SEQ ID NO:34, or SEQ ID NO:25, or a homolog thereof having at least 60% homology with it.

[0342] In some embodiments, the nucleic acid construct comprises a first polynucleotide as described above and optionally a second polynucleotide as described above, and also expresses one or more other polynucleotides as described above. In some embodiments, the nucleic acid construct therefore comprises a first polynucleotide and optionally a second polynucleotide, and also comprises one of the following:

[0343] • At least one additional polynucleotide encoding heterologous cytochrome b5; or

[0344] • At least one additional polynucleotide encoding heterologous cytochrome b5 reductase; or

[0345] • At least one additional polynucleotide encoding hemoglobin; or

[0346] • At least one additional polynucleotide encoding a thioesterase.

[0347] In other embodiments, the nucleic acid construct comprises a first polynucleotide and optionally a second polynucleotide, and further comprises:

[0348] • At least one additional polynucleotide encoding heterologous cytochrome b5; and

[0349] • At least one additional polynucleotide encoding heterologous cytochrome b5 reductase;

[0350] or

[0351] • At least one additional polynucleotide encoding heterologous cytochrome b5; and

[0352] • At least one additional polynucleotide encoding hemoglobin;

[0353] or

[0354] • At least one additional polynucleotide encoding heterologous cytochrome b5; and

[0355] • At least one additional polynucleotide encoding a thioesterase;

[0356] or:

[0357] • At least one additional polynucleotide encoding heterologous cytochrome b5 reductase; and

[0358] • At least one additional polynucleotide encoding hemoglobin;

[0359] or:

[0360] • At least one additional polynucleotide encoding heterologous cytochrome b5 reductase; and

[0361] • At least one additional polynucleotide encoding a thioesterase;

[0362] or:

[0363] • At least one additional polynucleotide encoding hemoglobin; and

[0364] • At least one additional polynucleotide encoding a thioesterase.

[0365] In other embodiments, the nucleic acid construct comprises a first polynucleotide and optionally a second polynucleotide, and further comprises:

[0366] • At least one additional polynucleotide encoding heterologous cytochrome b5; and

[0367] • At least one additional polynucleotide encoding heterologous cytochrome b5 reductase; and

[0368] • At least one additional polynucleotide encoding hemoglobin;

[0369] or

[0370] • At least one additional polynucleotide encoding heterologous cytochrome b5; and

[0371] • At least one additional polynucleotide encoding heterologous cytochrome b5 reductase; and

[0372] • At least one additional polynucleotide encoding a thioesterase;

[0373] or

[0374] • At least one additional polynucleotide encoding heterologous cytochrome b5 reductase; and

[0375] • At least one additional polynucleotide encoding hemoglobin; and

[0376] • At least one additional polynucleotide encoding a thioesterase;

[0377] or

[0378] • At least one additional polynucleotide encoding heterologous cytochrome b5; and

[0379] • At least one additional polynucleotide encoding hemoglobin; and

[0380] • At least one additional polynucleotide encoding a thioesterase.

[0381] In some implementations, the nucleic acid construct comprises a first polynucleotide and optionally a second polynucleotide, and also comprises all of the following:

[0382] • At least one additional polynucleotide encoding heterologous cytochrome b5; and

[0383] • At least one additional polynucleotide encoding heterologous cytochrome b5 reductase; and

[0384] • At least one additional polynucleotide encoding hemoglobin; and

[0385] • At least one additional polynucleotide encoding a thioesterase.

[0386] The nucleic acid construct may also contain additional polynucleotides for introducing any of the other modifications described above into yeast cells, particularly polynucleotides that, upon introduction into yeast cells, result in the activity of one or more natural fatty aldehyde dehydrogenases, one or more fatty alcohol oxidases, peroxisome biogenic factors, and / or one or more fatty acyl synthases; preferably, the activity is reduced or eliminated.

[0387] As is known in the art, nucleic acid constructs may contain additional elements required for or promoting the expression of the polynucleotide contained therein, such as promoters located upstream of the coding sequence contained in the polynucleotide, for example, inducible, repressive or constitutive promoters.

[0388] As a formulation of pheromone composition

[0389] In some embodiments, the method of the present invention further includes the step of formulating E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienal produced by yeast cells into a pheromone composition, as known in the art.

[0390] E8,E10-dodecadienyl coenzyme A, E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienal can be obtained by the method of the present invention.

[0391] This disclosure also provides E8,E10-dodecadienyl coenzyme A (or lipids or free fatty acids obtained by converting E8,E10-dodecadienyl coenzyme A), E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate, and / or E8,E10-dodecadienal that can be obtained by the methods of the present invention. When insect desaturases and / or reductases are expressed in yeast cells, the resulting mixture of products (e.g., E8,E10-dodecadienyl coenzyme A and / or fatty alcohols containing E8,E10-dodecadien-1-ol) typically has a composition similar to that produced in the pheromone glands of insects. This allows for the production of pheromone mixtures suitable for a variety of insects, rather than generating separate pheromone components in a separate process, which then need to be mixed in appropriate proportions. However, the resulting mixture of products (e.g., E8,E10-dodecadienyl coenzyme A and / or fatty alcohols) may contain characteristic byproducts produced by the organism.

[0392] Therefore, in some embodiments of the production of E8,E10-dodecadienyl coenzyme A, the produced fatty acyl coenzyme A comprises at least 1% such as at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20% of desaturated fatty acyl coenzyme A having desaturation at a position different from the desired fatty acyl coenzyme A, and / or at least 1% such as at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20% of the corresponding saturated fatty acyl coenzyme A.

[0393] In embodiments for the production of E8,E10-dodecadien-1-ol, the resulting fatty alcohol comprises at least 1% (e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%) of a desaturated fatty alcohol having desaturation at a position different from the desired fatty alcohol, and / or at least 1% (e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%) of the corresponding saturated fatty alcohol. If the mixture of fatty alcohols recovered from the fermentation broth is chemically oxidized to an aldehyde or acetylated to an acetate, a corresponding mixture of aldehydes and acetates is produced.

[0394] In some embodiments, the method of the present invention is used to produce E8,E10-dodecadienal. In some embodiments, the yeast cells and method of the present invention result in the production of a mixture of aliphatic aldehydes, said mixture comprising E8,E10-dodecadienal, but also comprising odd-chain aliphatic aldehydes. The term "odd-chain" aliphatic aldehyde refers to an aliphatic aldehyde having a carbon chain length of an odd number of carbon atoms (e.g., 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23 carbon atoms). The term "even-chain" aliphatic aldehyde refers to an aliphatic aldehyde having a carbon chain length of an even number of carbon atoms (e.g., 8, 10, 12, 14, 16, 18, 20, or 22 carbon atoms).

[0395] In some embodiments, the method of the present invention is used to produce E8,E10-dodecadienyl acetate. In some embodiments, the yeast cells and method of the present invention result in the production of a mixture of fatty alcohol acetates, said mixture comprising E8,E10-dodecadienyl acetate, but also comprising odd-chain fatty alcohol acetates. The term "odd-chain" fatty alcohol acetate refers to a fatty alcohol acetate having a carbon chain length of an odd number of carbon atoms (e.g., 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23 carbon atoms). The term "even-chain" fatty alcohol acetate refers to a fatty alcohol acetate having a carbon chain length of an even number of carbon atoms (e.g., 8, 10, 12, 14, 16, 18, 20, or 22 carbon atoms).

[0396] Pheromones

[0397] E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate, and / or E8,E10-dodecadienal produced by yeast cells can be formulated into pheromone compositions, as known in the art. Such pheromone compositions can be used as integrated pest management products, in methods for monitoring the presence of pests, or in methods for interfering with pest mating.

[0398] The pheromone compositions disclosed herein can be used as biological pest control agents. Such compositions can be sprayed or dispensed onto cultures, in fields, or in orchards. They can also be soaked, for example, on rubber septa, or mixed with other components, as is known in the art. This can cause mating disturbance, thereby preventing pest reproduction, or it can be used in combination with trapping devices to capture pests. Non-limiting examples of pests that can be combated using the pheromone compositions of the present invention include: cotton bollworm (Helicoverpa armigera), striped stemborer (Chilo suppressalis), diamondback moth (Plutellaxylostella), cabbage moth (Mamestra brassicae), large cabbage-heart caterpillar (Crocidolomia binotalis), European corn stalk borer (Sesamia nonagrioides), currant clearwing moth (Synanthedon tipuliformis), and artichoke plume moth (Platyptilia carduidactylal). Therefore, using the compositions of the present invention in cultures can increase crop yields with virtually no environmental impact.

[0399] The relative amounts of different compounds in the pheromone composition of this invention can vary depending on the nature of the crop and / or the pests to be controlled; geographical differences may also exist. Therefore, determining the optimal relative amounts may require conventional optimization.

[0400] In some embodiments of this disclosure, the pheromone composition may also comprise one or more other compounds, such as liquid or solid carriers or matrices. Suitable carriers or matrices include, for example, vegetable oils, refined mineral oils or fractions thereof, rubber, plastics, silica, diatomaceous earth, wax molds, and cellulose powder.

[0401] The pheromone composition can be formulated as is known in the art. For example, it can be in the form of a solution, gel, or powder. The pheromone composition can be formulated to facilitate dispensing, as is known in the art.

[0402] Reagent test kit

[0403] This document provides a component kit for carrying out the methods of the present invention. The component kit may contain “ready-to-use” yeast cells as described herein. In one embodiment, the yeast cells are *Yarrowia* cells (e.g., *Yarrowia lipolytica* cells) or *Saccharomyces* cells (e.g., *Saccharomyces cerevisiae* cells).

[0404] Alternatively, the component kit may also contain a nucleic acid construct encoding a desired activity to be introduced into yeast cells. The nucleic acid construct can be provided as a variety of nucleic acid constructs, such as a variety of vectors, each encoding one or more desired activities. Useful nucleic acid constructs have been described above.

[0405] The component kit may also contain nucleic acid constructs for introducing mutations that result in partial or complete loss of function, such as any of the mutations described above.

[0406] The component kit may optionally contain yeast cells to be modified.

[0407] In some implementations, the component kit contains all of the above items.

[0408] Methods for monitoring the presence of pests or interfering with their mating

[0409] E8,E10-dodecadien-1-ol and optionally E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienal produced by the yeast cells and methods disclosed herein can be used in methods for monitoring the presence of pests or interfering with the mating of pests.

[0410] Therefore, this paper also provides a method for monitoring the presence of harmful organisms or interfering with their mating, the method comprising the following steps:

[0411] i) E8,E10-dodecadien-1-ol and optionally E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienal are produced by the methods described herein.

[0412] ii) Formulating the E8,E10-dodecadien-1-ol and optionally the E8,E10-dodecadienyl acetate and / or the E8,E10-dodecadienal into a pheromone composition; and

[0413] iii) Use the pheromone composition as an integrated pest management composition.

[0414] Any yeast cells and methods described above can be used in such methods.

[0415] Example

[0416] Example 1: Construction of biological blocks

[0417] All heterologous genes were synthesized by GeneArt (Life Technologies) in codon-optimized forms for *Yarrowia lipolytica*. All genes were amplified by PCR using Phusion U hot-start DNA polymerase (ThermoFisher) to obtain fragments for cloning into yeast expression vectors. Primers are listed in Table 1, and the resulting DNA fragments (bioblocks) are listed in Table 2. PCR products were isolated on 1% agarose gels containing Midori Green Advance (Nippon Genetics Europe GmbH). Correctly sized PCR products were excised from the gels and purified using Nucleospin gels and a PCR cleanup kit (Macherey-Nagel).

[0418] Table 1 - Primers

[0419]

[0420]

[0421]

[0422]

[0423]

[0424] Table 2. DNA fragments (bioclasts) obtained by PCR using the indicated templates and primers

[0425]

[0426]

[0427]

[0428]

[0429] 1 Holkenbrink et al. 2020

[0430] 2 Holkenbrink et al. 2017

[0431] Example 2: Construction of plasmids

[0432] The integrative yeast vector with the USER cassette was linearized at 37°C for 2 hours using FastDigest SfaAI (ThermoFisher), followed by digestion at 65°C for 1 hour using Nb.Bsml (New England Biolabs). The vector containing sticky ends was separated by gel electrophoresis, excised from the gel, and purified using a Nucleospin gel and PCR cleanup kit (Macherey-Nagel). DNA fragments were cloned into the prepared vector via USER cloning, as described by Holkenbrink et al., 2017. The reaction was transformed into chemically competent *E. coli* DHα cells, and the cells were plated on Lysogeny Broth (LB) agar plates containing 100 mg / L ampicillin. The plates were incubated overnight at 37°C, and colonies were screened by colony PCR. Plasmids were purified from the overnight *E. coli* liquid culture, and the correct clones were confirmed by sequencing. The constructed vectors are listed in Table 3.

[0433] Table 3. Integrated Expression Vectors

[0434]

[0435]

[0436] Example 3: Construction of the strain

[0437] As described by Holkenbrink et al., 2017, yeast strains were constructed by transforming DNA vectors. The integrative vector was linearized with FastDigest NotI prior to transformation. When needed, helper vectors that promote integration into specific genomic regions were co-transformed with integrative plasmids or DNA repair fragments listed in Table 4. Strains were selected on yeast peptone dextran (YPD) agar using appropriate antibiotics. Correct genotypes were confirmed by colony PCR and, if necessary, sequencing. The wild-type strain of *Yarrowia lipolytica* was transformed with plasmid pCfB6364 (EP19204554) to produce strain ST6029. Then, the genes HFD1 (YALI0_F23793g), HFD2 (YALI0_E15400g), HFD3 (YALI0_A17875g), HFD4 (YALI0_B01298g), FAO1 (YALI0_B14014g), and PEX10 (YALI1_C01416g) were deleted to produce strain ST6629 (Borodina et al., 2018). Strains ST6029 and ST6629 were used as parental strains to construct all other strains. The resulting strains are listed in Table 5.

[0438] Table 4. Auxiliary carriers

[0439]

[0440]

[0441]

[0442] Table 5. Yeast strains

[0443]

[0444]

[0445]

[0446]

[0447]

[0448] Example 4: Cultivation of the strain, extraction and analysis of fatty acid methyl esters and fatty alcohols

[0449] The strain was inoculated from a YPD agar plate (10 g / L yeast extract, 10 g / L peptone, 20 g / L glucose, 15 g / L agar) into 2.5 mL of YPG medium (10 g / L yeast extract, 10 g / L peptone, 40 g / L glycerol) in 24-well EnzyScreen plates. Initial OD 600 The concentration was 0.1–0.2. The plates were incubated at 28°C with shaking at 300 rpm. After 22 h, the plates were centrifuged at 4°C and 3,000 x g for 5 min. The supernatant was discarded, and the cells were resuspended in 1.25 mL of production medium per well (Borodina et al., 2018). The medium was replenished with 2.5 μL of methyl dodecanoate. The plates were incubated at 28°C for 28 h with shaking at 300 rpm.

[0450] To analyze fatty alcohols, 200 μL of culture medium was extracted with 990 μL ethyl acetate:ethanol (84:15) and 10 μL Z10-17:Me (2 mg / mL) as an internal standard. The samples were vortexed for 20 seconds and incubated at room temperature for 1 hour, followed by vortexing for 5 minutes. 300 μL H2O was added to each sample. The samples were vortexed and centrifuged at 21 °C and 3,000 x g for 5 min. The upper organic phase was analyzed by gas chromatography-mass spectrometry (GC-MS). GC-MS analysis was performed on a Hewlett Packard 6890GC connected to a mass-selective detector HP 5973. The GC was equipped with an INNOWAX column (30 m × 0.25 mm × 0.25 μm) and helium was used as the carrier gas (average velocity: 33 cm / s). MS was operated in electron collision mode (70 eV), scanning between m / z 30 and 400, with the injector configured in splitless mode at 220 °C. The column oven temperature was set to 80 °C and held for 1 min, then increased to 210 °C at a rate of 10 °C / min, held at 210 °C for 15 min, then increased to 230 °C at a rate of 10 °C / min, and held at 230 °C for 20 min. Compounds were identified by comparing their retention times and mass spectra with those of reference compounds available in laboratory collections. Compounds were quantified by recorded total ion current (TIC). Data were analyzed using Agilent ChemStation software and iWork Numbers.

[0451] To analyze fatty acids, 1 mL of each vial was collected by centrifugation at 4 °C and 3,000 x g for 5 min. Each precipitate was extracted with 1000 μL of 1M HCl in anhydrous methanol. The sample was vortexed for 20 seconds and placed in an 80 °C water bath for 2 hours. The sample was vortexed for 10 seconds every 30 minutes. After cooling the sample to room temperature, 1000 μL of 1M NaOH in anhydrous methanol, 500 μL of saturated NaCl aqueous solution, 990 μL of hexane, and 10 μL of Z10-17:Me (2 mg / mL) as an internal standard were added. The sample was vortexed and centrifuged at 21 °C and 3,000 x g for 5 min. The upper organic phase was analyzed by GC-MS as described above.

[0452] Example 5: Production of E8,E10-C12:OH in Yersinia lipolytica

[0453] The strain ST8494, derived from strain ST6629, expresses the cotton bollworm fatty acyl reductase Har_FAR (two copies) and the codling moth desaturase Cpo_CPRQ. Strain ST6629 is a lipophilic Yersinia strain engineered to reduce fatty alcohol degradation and the accumulation of stored lipids (Holkenbrink et al., 2020).

[0454] The strain was cultured, extracted, and analyzed as described in Example 4, except that, for the analysis of the fatty alcohols formed, six vials (1.25 mL each) were combined and harvested by centrifugation at 4°C and 3,000 g for 5 min. The concentration of fatty alcohols was calculated based on an internal standard.

[0455] Among the strains engineered to reduce fatty alcohol degradation, strain ST8494, which combines the expression of the desaturase CpoCPRQ and the fatty alcohol reductase HarFAR, showed a production of 4.4 mg / L E8,E10-C12:OH (Table 6).

[0456] Table 6. Concentration of fatty alcohols in strain ST8494.

[0457] strain E9 / Z9-C12:OH (mg / L) E8,E10-C12:OH (mg / L) ST8494 20.1 4.4

[0458] Example 6: Increased production of E8,E10-C12:Me and E8,E10-C12:OH in Yersinia lipolytica

[0459] Strain ST8406 was derived from strain ST6629 and additionally expressed the CpoCPRQ desaturase. Strain ST9066, derived from ST8406, expressed two copies of Cpo_CPRQ. The strains were cultured, extracted, and analyzed as described in Example 4. The concentrations of fatty acid methyl esters and fatty alcohols were calculated based on internal standards (Tables 7-10).

[0460] Expression of an additional copy of the desaturase Cpo_CPRQ from the codling moth (ST9066) resulted in a 2.8-fold and a 1.5-fold increase in the yields of E8, E10-C12:Me, and E9 / Z9-C12:Me, respectively (Table 7). This indicates that overexpression of the desaturase can lead to increased yields of E8, E10-C12:Me, and E9 / Z9-C12:Me.

[0461] Table 7. Concentrations of fatty acid methyl esters in strains ST8406 and ST9066

[0462] strain E9 / Z9-C12:Me (mg / L) E8,E10-C12:Me (mg / L) ST8406 3.81±0.52 0.43±0.00 ST9066 5.75±1.12 1.22±0.52

[0463] Strains ST8411 and ST8416, which combined the expression of the desaturase Cpo_CPRQ from the codling moth with the expression of cytochrome b5 (HarCyb5, SEQ ID NO:4) from the cotton bollworm or with the expression of hemoglobin (VHb, SEQ ID NO:6) from *Vibrio fecalith*, produced 18% and 22% more E8,E10-C12:Me, respectively, than the reference strain ST8406, which only expressed the desaturase from the codling moth. These strains also showed increased E9 / Z9-C12:Me production (Table 8). These data suggest that the expression of desaturases combined with cytochrome b5 or with hemoglobin can produce more E8,E10-C12:Me compared to strains expressing only the desaturase.

[0464] Table 8. Concentrations of fatty acid methyl esters in strains ST8406, ST8411, and ST8416

[0465] strain E9 / Z9-C12:Me (mg / L) E8,E10-C12:Me (mg / L) ST8406 1.29±0.20 0.22±0.09 ST8411 1.60±0.16 0.26±0.03 ST8416 1.68±0.10 0.27±0.02

[0466] In addition to expressing cytochrome b5 (HarCyb5) from *Helicobacter pylori* (strain ST8411), expression of hemoglobin (VHb) from *Vibrio fecalis* (ST9115) resulted in additional increases of 21% and 41% in E8, E10-C12:Me, and E9 / Z9-C12:Me titers, respectively (Table 9). These data indicate that co-expression of desaturases with cytochrome b5 and hemoglobin can produce more E8, E10-C12:Me, and E9 / Z9-C12:Me compared to strains expressing only one of the three.

[0467] Table 9. Concentrations of fatty acid methyl esters in strains ST8406, ST8411, and ST9115

[0468] strain E9 / Z9-C12:Me (mg / L) E8,E10-C12:Me (mg / L) ST8406 2.99±0.64 0.34±0.04 ST8411 3.40±0.57 0.42±0.00 ST9115 4.83±0.62 0.51±0.05

[0469] The strain ST9250, expressing fatty acyl reductase (Ase_FAR) from the yellow cutworm, showed the production of C12:OH, E9 / Z9-C12:OH and E8,E10-C12:OH, while the strain ST9249, expressing fatty acyl reductase (Ta_FAR, SEQ ID NO:8) from the barn owl, showed only the production of C12:OH (Table 10).

[0470] Table 10. Concentrations of fatty alcohols in strains ST9066, ST9249, and ST9250. ND: Not detected.

[0471]

[0472]

[0473] Example 7: Increased E8,E10-C12:Me yield in the Δelo1 lipophilic yeast strain

[0474] The intrinsic elongase gene ELO1 (YALI0_F06754g, SEQ ID NO:13) was deleted in strain ST8406 to generate strain ST9060. The strain was cultured, extracted, and analyzed as described in Example 4. The concentration of fatty acid methyl esters was calculated based on internal standards (Table 11). Compared with strain ST8406, strain ST9060 showed a 2.2-fold and 1.6-fold increase in the yields of E8, E10-C12:Me, and E9 / Z9-C12:Me, respectively. These data indicate that the deletion of the elongase gene can increase the yields of E8, E10-C12:Me, and E9 / Z9-C12:Me.

[0475] Table 11. Concentrations of fatty acid methyl esters in strains ST8406 and ST9060

[0476] strain E9 / Z9-C12:Me (mg / L) E8,E10-C12:Me (mg / L) ST8406 4.77±0.65 0.63±0.10 ST9060 7.40±1.93 1.39±0.33

[0477] Example 8: Increased E8,E10-C12:Me yield in *Yarrowia lipolyticis* strains containing deletions of genes YALI0_F14729g, YALI0_E18876g, or YALI0_D03597g.

[0478] The intrinsic lipase-coding genes YALI0_F14729g (SEQ ID NO:19), YALI0_E18876g (SEQ ID NO:54), and YALI0_D03597g (SEQ ID NO:55) of *Yalexinia lipolytica*, all encoding putative thioesterases, were deleted in strain ST8406, resulting in strains ST9061, ST9062, and ST9063, respectively. The strains were cultured, extracted, and analyzed as described in Example 4. The concentrations of fatty acid methyl esters were calculated based on internal standards (Table 12). Compared to strain ST8406, strain ST9061 showed a 1.6-fold and 1.7-fold increase in the yields of E8, E10-C12:Me, and E9 / Z9-C12:Me, respectively. Compared to strain ST8406, strain ST9062 showed a 1.2-fold and 1.3-fold increase in the yields of E8, E10-C12:Me, and E9 / Z9-C12:Me, respectively. Compared with strain ST8406, strain ST9063 showed a 1.1-fold increase in the production of E8, E10-C12:Me, and E9 / Z9-C12:Me. These data suggest that the absence of endogenous putative thioesterases can increase the production of E8, E10-C12:Me, and E9 / Z9-C12:Me.

[0479] Table 12. Concentrations of fatty acid methyl esters in strains ST8406, ST9061, ST9062, and ST9063

[0480] strain E9 / Z9-C12:Me (mg / L) E8,E10-C12:Me (mg / L) ST8406 1.97±0.12 0.29±0.01 ST9061 3.35±0.44 0.46±0.06 ST9062 2.49±0.34 0.35±0.03 ST9063 2.24±0.47 0.31±0.04

[0481] Example 9: Production of E8,E10-C12:Me in a *Yarrowia lipolytica* strain containing amino acid modifications in the desaturase Cpo_CPRQ.

[0482] In strain ST8406, the amino acid at position 85 of protein Cpo_CPRQ was modified from serine (S) to alanine (A), resulting in strain ST9072. The strain was cultured, extracted, and analyzed as described in Example 4. The concentration of fatty acid methyl esters was calculated based on internal standards (Table 13).

[0483] Compared to strain ST8406, strain ST9072 expressing Cpo_CPRQ_S85A showed a 213% increase in the yield of E8, E10-C12:Me. These data suggest that Cpo_CPRQ can be engineered to increase the yield of E8, E10-C12:Me and E9 / Z9-C12:Me.

[0484] Table 13. Concentrations of fatty acid methyl esters in strains ST8406 and ST9072

[0485]

[0486]

[0487] Example 10: Production of E8,E10-C12:Me and E8,E10-C12:OH in a *Yarrowia lipolytica* strain containing a combination of amino acid modifications (S85A) in the desaturase Cpo_CPRQ and other beneficial modifications.

[0488] Strain ST9278, derived from ST9060, contains two copies of Cpo_CPRQ and a deletion of ELO1. Strain ST9279, derived from ST9060, contains one copy of Cpo_CPRQ, one copy of Cpo_CPRQ_S85A, and a deletion of ELO1.

[0489] The strains were cultured, extracted, and analyzed as described in Example 4. The concentrations of fatty acid methyl esters were calculated based on the internal standard (Table 14).

[0490] Compared with strain ST9279, which expresses one copy of Cpo_CPRQ, one copy of Cpo_CPRQ_S85A and lacks ELO1, strain ST9278, which expresses two copies of Cpo_CPRQ and lacks the ELO1 gene, showed lower yields of E9 / Z9-C12:Me and E8,E10-C12:Me.

[0491] Table 14. Concentrations of fatty acid methyl esters in strains ST9060, ST9278, and ST9279

[0492] strain E9 / Z9-C12:Me (mg / L) E8,E10-C12:Me (mg / L) ST9060 12.63±2.07 2.07±0.10 ST9278 20.70±2.46 5.47±0.74 ST9279 21.91±4.24 6.15±1.62

[0493] Among other modifications, strain ST9355, derived from ST9279, also expresses VHb and HarCyb5. Among other modifications, strain ST9356, derived from ST9355, also expresses HarCyb5 and HarCyb5 reductase (SEQ ID NO:24). Among other modifications, strain ST9357, derived from ST9356, also contains the deletion of the intrinsic Yersinia lipolyticis gene YALI0_F14729g. Among other modifications, strain ST9358, derived from ST9357, also expresses Ase_FAR. Among other modifications, strain ST9387, derived from ST9279, also expresses Ase_FAR. The strains were cultured, extracted, and analyzed as described in Example 4. The concentrations of fatty acid methyl esters and fatty alcohols were calculated based on internal standards (Tables 15 and 16).

[0494] Table 15. Concentrations of fatty acid methyl esters in strains ST9279, ST9355, and ST9356

[0495] strain E9 / Z9-C12:Me (mg / L) E8,E10-C12:Me (mg / L) ST9279 12.6±1.9 5.4±0.7 ST9355 11.9±0.7 6.4±0.5 ST9356 12.8±1.5 7.3±0.1 ST9357 11.3±0.1 7.2±0.2 ST9358 11.1±1.5 6.0±1.2 ST9387 11.0±3.2 4.0±0.7

[0496] Table 16. Concentrations of fatty alcohols in strains ST9279, ST9355, ST9356, ST9357, ST9358 and ST9387

[0497]

[0498]

[0499] These data suggest that beneficial modifications can be combined to achieve higher titers of E8,E10-C12:Me and E9 / Z9-C12:Me, as well as E8,E10-C12:OH and E9 / Z9-C12:OH.

[0500] Example 11: Production of E8,E10-C12:OH in strains containing amino acid modifications in the reductase Ase_FAR

[0501] In strain ST9250, amino acid position 198 of the Ase_FAR protein was modified from threonine (T) to alanine (A), resulting in strain ST9335. In strain ST9250, amino acid position 423 of the Ase_FAR protein was modified from serine (S) to alanine (A), resulting in strain ST9336. The strains were cultured, extracted, and analyzed as described in Example 4. The concentration of fatty alcohols was calculated based on the internal standard.

[0502] Example 12: Production of E8,E10-C12:OH in a *Yersinia lipolytica* strain containing amino acid modifications of fatty acid synthase 1 (FAS1) and fatty acid synthase 2 (FAS2).

[0503] In strain ST9387, amino acid at position 1220 of FAS2 (SEQ ID NO:18) of *Yersinia lipolytica* was modified from isoleucine (I) to phenylalanine (F), resulting in strain ST9388. In strain ST9387, amino acid at position 1220 of FAS2 of *Yersinia lipolytica* was modified from isoleucine (I) to tryptophan (W), resulting in strain ST9420. In strain ST9420, amino acid at position 123 of FAS1 (SEQ ID NO:16) of *Yersinia lipolytica* was modified from leucine (L) to valine (V), resulting in strain ST9421. The strains were cultured, extracted, and analyzed as described in Example 4, except that methyl dodecanoate was not added to the production medium. The concentration of fatty alcohols was calculated based on an internal standard.

[0504] Example 13: Amino acid modification in FAS2 containing *Yersinia lipolytica* (FAS2(I1220F)) and production of E8,E10-C12:OH in *Yersinia lipolytica* strains containing thioesterases from *E. coli* for the formation of C12 fatty acids.

[0505] Strain ST9397 expressed a fusion of a truncated form of FAS1 from *Yersinia lipolytica* and a truncated form of the thioesterase TesA from *Escherichia coli* (Xu et al., 2016) (SEQ ID NO: 59). Strain ST9397 was transformed with a plasmid containing fatty acyl-CoA synthase from *Yersinia lipolytica* to produce strain ST9398. The strain was cultured, extracted, and analyzed as described in Example 4, except that glass tubes were used and fatty alcohols were extracted from the total culture medium. The concentration of fatty alcohols was calculated based on internal standards (Table 17).

[0506] The expression of fatty acyl-CoA synthase from Yersinia lipolyticis did not significantly affect the production of E8 and E10-12:OH.

[0507] Table 17: Concentrations of E9 / Z9-12:OH and E8, E10-12:OH in strains ST9397 and ST9398

[0508] strain E9 / Z9-12:OH (mg / L) E8,E10-C12:OH (mg / L) ST9397 0.2±0 0.1±0 ST9398 0.2±0 0.1±0

[0509] Example 14: Production of E8,E10-C12:OH by chain shortening of peroxisomes in Yersinia lipolyticis

[0510] To increase the amount of C12:CoA precursor in strain ST9395, the following five endogenous peroxisome oxidases of Yersinia lipolyticis were deleted: POX1, POX2, POX3, POX4 and POX5 (corresponding to YALI0_E32835g, YALI0_F10857g, YALI0_E32835g, YALI0_E27654g, YALI0_E27654g), and heterologous peroxisome oxidases, such as Cma_POX (SEQ ID NO:47) from Cucurbita moschata, were expressed instead.

[0511] To increase the amount of Δ9-12:CoA precursor, the above strains additionally expressed Δ11-14 desaturases, such as CroZ11 (SEQ ID NO:63) from the rose leafroller or CpaE11 (SEQ ID NO:65) from the parallel color leafroller. This resulted in the production of Z / E11-14:CoA, which was shortened to Z / E9-12:CoA and then further converted to E8,E10-C12:Me via the desaturase Cpo_CPRQ (SEQ ID NO:1).

[0512] The strains were cultured, extracted, and analyzed as described in Example 4. Cultures of strains ST9600, ST9607, and ST9616 were supplemented with methyl myristate. The concentrations of fatty alcohols were calculated based on internal standards.

[0513] Example 15: Generation of E8,E10-C12:Me and E8,E10-C12:OH in Saccharomyces cerevisiae

[0514] The desaturase gene Cpo_CPRQ was amplified from cDNA in the pheromone gland tissue of the codling moth using primers attB1_Cpo_CPRQ_F and attB1_Cpo_CPRQ_R.

[0515] PCR products were separated by agarose gel electrophoresis and purified using a Wizard SV Gel and PCR cleanup system (Promega Biotech AB, Sweden). The purified DNA was cloned into the pDONR221 vector using Gateway cloning technology (Life Technologies). The resulting vector was confirmed by Sanger sequencing, and the gene was subcloned into the pYEX-CHT vector (Patel et al., 2003), which was then transformed into a Saccharomyces cerevisiae strain lacking OLE1 and ELO1 (MATaelo1::HIS3 ole1::LEU2 ade2 his3 leu2 ura3) (Schneiter et al., 2000). To select positive transformants, cells were cultured on synthetic complete medium containing 0.7% YNB (containing ammonium sulfate), uracil- and leucine-deficient auxotropic medium (Formedium LTD, England), 2% glucose, 1% tergitol (Nonidet NP-40, Sigma-Aldrich, Sweden), 0.01% adenine (Sigma-Aldrich, Sweden), and 0.5 mM oleic acid (Sigma-Aldrich, Sweden). After incubating the plates at 30°C for four days, individual colonies were inoculated into 10 ml of selective medium. The cultures were incubated at 30°C for 48 h and then inoculated into 10 ml of selective medium supplemented with 0.5 mM fatty acid methyl ester precursor containing 2 mM CuSO4, with an OD600 of 0.4. After 48 hours of incubation, cells were harvested by centrifugation at 3000 rpm. The supernatant was discarded, and total lipids were extracted in glass tubes using 3.75 ml of methanol / chloroform (2:1, v / v). Add 1 ml of HAc (0.15 M) and 1.25 ml of water and vortex the tube. Centrifuge the tube at 2000 rpm for 2 min and transfer the bottom chloroform phase to a new glass tube. To convert the lipids to fatty acid methyl esters (FAME), evaporate the solvent under a nitrogen stream. Add 1 ml of 2% sulfuric acid in methanol, vortex the suspension, and incubate at 90 °C for 1 h. Then add 1 ml of water, mix, and extract the FAME with 1 ml of hexane. Analyze the sample by GC-MS on a Hewlett Packard 6890GC coupled with a mass-selective detector HP5973. The GC was equipped with an HP-88 column (30 m × 0.25 mm × 0.25 μm) and helium was used as the carrier gas (average velocity: 33 ms). MS was operated in electron collision mode (70 eV) and the injector was configured in splitless mode at 220 °C. Set the column oven temperature to 80℃ and hold for 1 minute. Then increase the temperature to 210℃ at a rate of 10℃ / min and hold at 210℃ for 15 minutes. Then increase the temperature to 230℃ at a rate of 10℃ / min and hold at 230℃ for 20 minutes.E8, E10-12:OAc, used as reference standards, were purchased from Bedoukian, USA, and converted to the corresponding alcohols by hydrolysis using a solution of 0.5M KOH in methanol. As described in (Bjostad and Roelofs, 1984), pyridinium dichromate in dimethylformamide was used to oxidize fatty alcohols to the corresponding acids.

[0516] The chromatograms in Figure 2 show that in the Saccharomyces cerevisiae strain expressing Cpo_CPRQ, E9-12:Me and E8, E10-12:Me can be produced by 12:Me and E9-12:Me, respectively.

[0517] Example 16: Production of E8,E10-C12:Me in Yersinia lipolytica via Cpo_SPTQ, Cpo_NPVE, and Cpo_CPRQ

[0518] Strain ST10136, derived from ST6629, expresses one copy of Cpo_SPTQ. Strain ST10137, also derived from ST6629, expresses one copy of Cpo_NPVE. Strain ST9064, derived from ST8406, expresses one copy of Cpo_CPRQ and one copy of Cpo_SPTQ. Strain ST9065, derived from ST8406, expresses one copy of Cpo_CPRQ and one copy of Cpo_NPVE. Strain ST9066, derived from ST8406, expresses two copies of Cpo_CPRQ. Strain ST10138, derived from ST9065, expresses one copy of Cpo_CPRQ, one copy of Cpo_NPVE, and one copy of Cpo_SPTQ.

[0519] The strains were cultured, extracted, and analyzed as described in Example 4. The concentrations of fatty acid methyl esters were calculated based on the internal standard (Table 18).

[0520] Expression of Cpo_SPTQ (ST10136) does not result in the production of E9-C12:Me, Z9-C12:Me, or E8, E10-C12:Me. Expression of Cpo_NPVE (ST10137) results in the production of E9-C12:Me and Z9-C12:Me, but not E8, E10-C12:Me. Additional expression of Cpo_SPTQ or Cpo_NPVE in ST8406 (ST9064 and ST9065, respectively) does not result in an increase in E8, E10-C12:Me. Expression of an additional copy of Cpo_CPRQ (ST9066) in ST8406 results in a 2.8-fold and 2.1-fold increase in the production of E8, E10-C12:Me, and E9 / Z9-C12:Me, respectively. Compared to ST8406, combined expression of Cpo_CPRQ, Cpo_SPTQ, and Cpo_NPVE (ST10138) did not lead to an increase in E8,E10-C12:Me. This indicates that only the expression of Cpo_CPRQ results in the generation of E8,E10-C12:Me.

[0521] Table 18. Concentrations of fatty acid methyl esters in strains ST10136, ST10137, ST8406, ST9064, ST9065, ST9066 and ST10138

[0522] strain E9-C12:Me (mg / L) Z9-C12:Me (mg / L) E8,E10-C12:Me (mg / L) ST10136 0.00±0.00 0.00±0.00 0.00±0.00 ST10137 2.06±0.89 3.82±1.80 0.00±0.00 ST8406 5.02±0.88 0.33±0.03 0.66±0.15 ST9064 4.42±0.00 0.29±0.00 0.63±0.00 ST9065 6.04±0.22 2.29±0.10 0.86±0.10 ST9066 10.55±0.07 0.58±0.04 1.87±0.06 ST10138 6.38±0.99 2.66±0.41 0.76±0.06

[0523] Example 17: Production of E8,E10-C12:OH by expressing multiple copies of a biosynthetic enzyme

[0524] Strain ST9358 was explained in Example 10. Strain ST9495 was derived from strain ST9357 (described in Example 10) and expressed additional copies of the desaturase Cpo_CPRQ and fatty acyl reductase Ase_FAR. The strain was cultured, extracted, and analyzed as described in Example 4, except that glass tubes were used and fatty alcohols were extracted from the total culture medium. The concentration of fatty alcohols was calculated based on an internal standard.

[0525] Table 19 shows that additional gene copies of Cpo_CPRQ and Ase_FAR can increase the yield of E8,E10-12:OH to 7.1 mg / L.

[0526] Table 19. Concentrations of fatty alcohols in strains ST9358 and ST9495

[0527] strain E9-C12:OH (mg / L) E8,E10-C12:OH (mg / L) ST9358 0.3±0.0 0.1±0.0 ST9495 22.6±4.5 7.1±1.5

[0528] Example 18: Production of montmorillonite by expressing various fatty acyl reductases

[0529] Strains ST9358 and ST9623 were derived from strain ST9357. They also expressed fatty acyl reductases from *Cephalotaxus fortunei* and *Cephalotaxus sinensis*, respectively. The strains were cultured, extracted, and analyzed as described in Example 4.

[0530] The results in Table 20 show that the two fatty acyl reductases can produce E9-C12:OH and E8,E10-C12:OH.

[0531] Table 20. Concentrations of fatty alcohols in strains ST9357, ST9358, and ST9623

[0532] strain E9-C12:OH (mg / L) E8,E10-C12:OH (mg / L) ST9357 0±0 0±0 ST9358 1.1±1.2 0.5±0.5 ST9623 1.9±0.3 0.6±0.1

[0533] sequence

[0534]

[0535]

[0536]

[0537]

[0538]

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[0558] Terms and Conditions

[0559] 1. A yeast cell capable of producing E8,E10-dodecadienyl-CoA and optionally E8,E10-dodecadien-1-ol, said yeast cell expressing at least one heterologous desaturase capable of introducing one or more double bonds into a 12-carbon acyl-CoA fatty acid chain, thereby converting said fatty acyl-CoA into desaturated fatty acyl-CoA, wherein at least a portion of said desaturated fatty acyl-CoA is E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA), optionally said yeast cell belonging to a genera selected from: *Blavus*, *Candida*, *Cryptococcus*, *Clostridium*, *Ophiocoria*, *Morchella*, *Mucor*, *Pythium*, *Rhodotorula*, *Rhodotorula*. The genera *Mother*, *Mycosaccharomyces*, *Yeast*, and *Yeastia*, optionally wherein the yeast cells belong to species selected from the following: *Blancium trispora*, *C. revkaufi*, *C. tropicalis*, *Cryptococcus*, *Syngonium spp. ...

[0560] 2. A yeast cell capable of producing E8,E10-dodecadienyl-CoA and optionally E8,E10-dodecadien-1-ol, said yeast cell expressing at least one heterodesaturase capable of introducing one or more double bonds into a 12-carbon acyl-CoA fatty acid chain to convert said fatty acyl-CoA into desaturated fatty acyl-CoA, wherein at least a portion of said desaturated fatty acyl-CoA is E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA).

[0561] 3. The yeast cell according to any one of the preceding clauses, wherein the yeast cell is capable of producing E8,E10-dodecadien-1-ol, and the yeast cell further expresses at least one heterologous fatty acyl-CoA reductase (EC 1.2.1.84) capable of converting at least a portion of the desaturated fatty acyl-CoA into desaturated fatty alcohol, wherein the fatty acyl-CoA reductase is capable of converting at least a portion of the E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA) into E8,E10-dodecadien-1-ol.

[0562] 4. A yeast cell capable of producing E8,E10-dodecadien-1-ol, said yeast cell expressing:

[0563] i) at least one heterologous desaturase capable of introducing one or more double bonds into a fatty acyl-CoA with a carbon chain length of 12, thereby converting said fatty acyl-CoA into desaturated fatty acyl-CoA, wherein at least a portion of said desaturated fatty acyl-CoA is E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA); and

[0564] ii) At least one heterologous fatty acyl-CoA reductase (EC 1.2.1.84) capable of converting at least a portion of the desaturated fatty acyl-CoA into desaturated fatty alcohol, wherein the fatty acyl-CoA reductase is capable of converting at least a portion of the E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA) into E8,E10-dodecadien-1-ol.

[0565] 5. The yeast cell according to any one of the preceding clauses, wherein the yeast cell belongs to a genera selected from the following: *Blavus*, *Candida*, *Cryptococcus*, *Clostridium*, *Oleoma*, *Morchella*, *Mucor*, *Pythium*, *Rhodotorula*, *Rhodotorula*, *Hydrocotyle*, *Yeast*, and *Yersinia*.

[0566] 6. The yeast cell according to any one of the preceding clauses, wherein the yeast cell belongs to a species selected from:

[0567] *Blancium trispora*, *C. revkaufi*, *C. tropicalis*, *Cryptococcus*, *Syngonium spp.*, *Syngonium spp.*, *Syngonium spp.*, *Syngonium spp.*, *Syngonium spp.*, *Syngonium spp.*, *Syngonium spp.*, *Syngonium alpinum*, *Syngonium spp. ...

[0568] 7. The yeast cell according to any one of the preceding clauses, wherein the yeast cell belongs to the genus *Yersinia* or *Yersinia*, preferably the yeast cell is *Yersinia lipolytica* cell or *Saccharomyces cerevisiae* cell.

[0569] 8. The yeast cell according to any one of the preceding clauses, wherein the at least one desaturase is Gmo_CPRQ (SEQ ID NO:77) or Cpo_CPRQ (SEQ ID NO:2), or is combined with SEQ ID NO:77 or SEQ ID NO:2. NO:2 has a functional variant of at least 65% homology or identity, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, preferably the at least one desaturase is Cpo_CPRQ or a functional variant thereof;Or, the at least one desaturase is at least two desaturases, wherein at least one of the two desaturases is Gmo_CPRQ (SEQ ID NO:77) or Cpo_CPRQ (SEQ ID NO:2), or has at least 65% homology or identity with SEQ ID NO:77 or SEQ ID NO:2, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its functional variants, preferably at least one desaturase is Cpo_CPRQ or a functional variant thereof, and the other desaturase is a desaturase capable of introducing at least one double bond into a fatty acyl-CoA with a carbon chain length of 12, such as Z9-12 desaturase, preferably Cpo_NPVE (SEQ ID NO). SEQ ID NO:67) or Cpo_SPTQ (SEQ ID NO:69) or its functional variants having at least 65% homology or identity with SEQ ID NO:67 or SEQ ID NO:69, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0570] 9. The yeast cell according to any one of the preceding clauses, wherein the desaturase is a mutant of Cpo_CPRQ with a mutation such as the S85A mutation at position 85.

[0571] 10. The yeast cell according to any one of the preceding clauses, wherein the at least one heterologous desaturase is at least two different heterologous desaturases, such as Cpo_CPRQ shown in SEQ ID NO:2 and a mutant of Cpo_CPRQ having a mutation at position 85, such as the S85A mutation.

[0572] 11. The yeast cell according to any one of the preceding clauses, wherein the fatty acyl-CoA reductase is selected from the following: Ase_FAR (SEQ ID NO:10), Aip_FAR (SEQ ID NO:61), Hs_FAR (SEQ ID NO:71), Has_FAR (SEQ ID NO:73), Hv_FAR (SEQ ID NO:75), Har_FAR (SEQ ID NO:12), Cpo_FAR (SEQ ID NO:13), ... NO:76), and its functional variants having at least 65% homology or identity, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0573] 12. The yeast cell according to any one of the preceding clauses, wherein the fatty acyl-CoA reductase is a mutant of Ase_FAR, such as having a mutation at position 198 or 413 (preferably the T198A mutation or the S413A mutation).

[0574] 13. The yeast cell according to any one of the preceding clauses, wherein the heterologous desaturase is expressed at a high level.

[0575] 14. The yeast cell according to any one of the preceding clauses, wherein the heterologous fatty acyl-CoA reductase is expressed at a high level.

[0576] 15. The yeast cell according to any one of the preceding clauses, wherein the yeast cell is further modified to increase the availability of E8,E10-C12:CoA.

[0577] 16. The yeast cell according to any one of the preceding clauses, further expressing heterologous cytochrome b5, such as cytochrome b5 from lepidopteran species, such as cytochrome b5 from cotton bollworm, preferably cytochrome b5 HarCyb5 shown in SEQ ID NO:4 or a functional variant thereof having at least 65% homology or identity with it, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity.

[0578] 17. The yeast cell according to any one of the preceding clauses, further expressing a heterologous cytochrome b5 reductase (EC1.6.2.2), such as cytochrome b5 reductase from lepidopteran species such as the cotton bollworm, preferably the cytochrome b5 reductase from the cotton bollworm as shown in SEQ ID NO:24 or a functional variant thereof having at least 65% homology or identity, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity.

[0579] 18. The yeast cell according to any one of the preceding clauses, which also expresses hemoglobin, such as hemoglobin from *Vibrio faecalis*, preferably the hemoglobin from *Vibrio faecalis* shown in SEQ ID NO:6, or a functional variant thereof having at least 65% homology or identity with it, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0580] 19. The yeast cell according to any one of the preceding clauses further comprises a mutation in one or more genes encoding an elongase that results in partial or complete loss of elongase activity, such as a mutation in the ELO1 gene (SEQ ID NO: 13) that results in partial or complete loss of Elo1 activity, preferably said mutation being a deletion.

[0581] 20. The yeast cell according to any one of the preceding clauses further comprises a mutation in one or more genes encoding a thioesterase that results in partial or complete loss of thioesterase activity, such as a mutation in the YAL10_F14729g gene (SEQ ID NO:19), a mutation in the YALI0_E18876g gene (SEQ ID NO:54), or a mutation in the YALI0_D03597g gene (SEQ ID NO:55), preferably said mutation being a deletion.

[0582] 21. The yeast cell according to any one of the preceding clauses, further comprising at least one modification, such as at least one mutation causing a reduction in the activity of at least one of Hfd1, Hfd2, Hfd3, Hfd4, Fao1, GPAT, and Pex10; or having at least one modification causing at least 60% homology or identity with, such as at least 65% homology or identity, such as at least 70% homology or identity, such as at least 75% homology or identity, such as at least 80% homology or identity, such as at least 81% homology or identity, such as at least 82% homology or identity, such as at least 83% homology or identity, such as at least 84% homology or identity. The following are examples of mutations that reduce the activity of at least one protein: at least 85% homology or identity, at least 86% homology or identity, at least 87% homology or identity, at least 88% homology or identity, at least 89% homology or identity, at least 90% homology or identity, at least 91% homology or identity, at least 92% homology or identity, at least 93% homology or identity, at least 94% homology or identity, at least 95% homology or identity, at least 96% homology or identity, at least 97% homology or identity, at least 98% homology or identity, or at least 99% homology or identity.

[0583] 22. The yeast cell according to any one of the preceding clauses, wherein the yeast cell further expresses a fatty acyl synthase variant having a modified ketone synthase domain, wherein the fatty acyl synthase variant is a variant of Fas1 (SEQ ID NO:16) or Fas2 (SEQ ID NO:18), such as a mutant Fas1 having a mutation at position 123, preferably an L123V mutation, or a mutant Fas2 having a mutation at position 1220, preferably an I1220F or I1220W mutation.

[0584] 23. A yeast cell according to any one of the preceding clauses, wherein the yeast cell also expresses a thioesterase such as a heterothioesterase, optionally wherein the thioesterase is expressed at a high level.

[0585] 24. The yeast cell according to Clause 23, wherein the thioesterase has at least 60% homology or identity with the thioesterase from *Cinnamomum camphora* as shown in SEQ ID NO:33, the thioesterase from *Cinnamomum camphora* as shown in SEQ ID NO:57, the thioesterase from *Cinnamomum camphora* as shown in SEQ ID NO:35, or the thioesterase from *Escherichia coli* as shown in SEQ ID NO:26, preferably the thioesterase has at least 60% homology or identity with the thioesterase from *Cinnamomum camphora* as shown in SEQ ID NO:35 or the thioesterase from *Escherichia coli* as shown in SEQ ID NO:26.

[0586] 25. The yeast cell according to any one of the preceding clauses, wherein the yeast cell further expresses a fusion protein of a truncated fatty acyl synthase and a truncated thioesterase, such as the fusion protein shown in SEQ ID NO:59 or a homolog thereof having at least 60% homology or identity with it.

[0587] 26. The yeast cell according to any one of the preceding clauses, wherein the yeast cell comprises nucleic acid encoding the heterologous desaturase and nucleic acid encoding the heterologous fatty acyl-CoA reductase.

[0588] 27. The yeast cell according to Clause 26, wherein the nucleic acid encoding the heterologous desaturase and / or the nucleic acid encoding the heterologous fatty acyl-CoA reductase are present in high copy numbers.

[0589] 28. A yeast cell according to any one of clauses 26 to 27, wherein the nucleic acid encoding the heterodesaturase is as shown in SEQ ID NO:1 or its homolog having at least 60% homology or identity with it, or as shown in SEQ ID NO:78 or its homolog having at least 60% homology or identity with it.

[0590] 29. A yeast cell according to any one of clauses 26 to 28, wherein the nucleic acid encoding the heterologous fatty acyl-CoA reductase is as shown in SEQ ID NO:9 or a homolog thereof having at least 60% homology or identity with it.

[0591] 30. The yeast cell according to any one of the preceding clauses, wherein the yeast cell comprises nucleic acid encoding the heterologous cytochrome b5, nucleic acid encoding the heterologous cytochrome b5 reductase, nucleic acid encoding the hemoglobin, nucleic acid encoding the fatty acid synthase variant, nucleic acid encoding the thioesterase, and / or nucleic acid encoding the fusion protein.

[0592] 31. The yeast cell according to claim 30, wherein the nucleic acid encoding the heterologous cytochrome b5, the nucleic acid encoding the heterologous cytochrome b5 reductase, the nucleic acid encoding the hemoglobin, the nucleic acid encoding the fatty acid synthase variant, and / or the nucleic acid encoding the thioesterase are present in high copy numbers.

[0593] 32. The yeast cell according to any one of the preceding clauses, wherein the nucleic acid encoding the heterologous desaturase, the nucleic acid encoding the heterologous fatty acyl-CoA reductase, the nucleic acid encoding the heterologous cytochrome b5, the nucleic acid encoding the heterologous cytochrome b5 reductase, the nucleic acid encoding the hemoglobin, the nucleic acid encoding the fatty acid synthase variant, and / or the nucleic acid encoding the thioesterase are codon-optimized for expression in the yeast cell.

[0594] 33. A yeast cell according to any one of claims 30 to 32, wherein the nucleic acid encoding the heterologous cytochrome b5 is as shown in SEQ ID NO:3 or a homolog thereof having at least 60% homology or identity with it, the nucleic acid encoding the heterologous cytochrome b5 reductase is as shown in SEQ ID NO:23 or a homolog thereof having at least 60% homology or identity with it, the nucleic acid encoding the hemoglobin is as shown in SEQ ID NO:5 or a homolog thereof having at least 60% homology or identity with it, and / or the nucleic acid encoding the thioesterase is as shown in SEQ ID NO:25 or SEQ ID NO:34 or a homolog thereof having at least 60% homology or identity with it.

[0595] 34. The yeast cell according to any one of the preceding clauses, wherein the yeast cell is capable of producing E8,E10-dodecadien-1-ol in titers of at least 0.5 mg / L, such as at least 0.6 mg / L, such as at least 0.7 mg / L, such as at least 0.8 mg / L, such as at least 0.9 mg / L, such as at least 1 mg / L, such as at least 1.5 mg / L, such as at least 2.5 mg / L, such as at least 5.0 mg / L, such as at least 1 ... Less than 15 mg / L, such as at least 20 mg / L, such as 25 mg / L, such as at least 50 mg / L, such as at least 100 mg / L, such as at least 250 mg / L, such as at least 500 mg / L, such as at least 750 mg / L, such as at least 1 g / L, such as at least 2 g / L, such as at least 3 g / L, such as at least 4 g / L, such as at least 5 g / L, such as at least 6 g / L, such as at least 7 g / L, such as at least 8 g / L, such as at least 9 g / L, such as at least 10 g / L or more.

[0596] 35. The yeast cell according to any one of the preceding clauses, wherein the yeast cell further expresses an acetyltransferase (EC 2.3.1.84) capable of converting at least a portion of the E8,E10-dodecadien-1-ol into E8,E10-dodecadienyl acetate, thereby enabling the yeast cell to produce E8,E10-dodecadienyl acetate.

[0597] 36. The yeast cell according to Clause 35, wherein the acetyltransferase is a heterologous acetyltransferase (AcT) expressed from the yeast cell or a native acetyltransferase overexpressed from the yeast cell.

[0598] 37. A yeast cell according to any one of clauses 35 or 36, wherein the acetyltransferase is Sc_Atf1 (SEQ ID NO:37) or has at least 60% homology or identity with it, and has at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, or at least 72%. Variations of at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity.

[0599] 38. The yeast cell according to any one of the preceding clauses, wherein the yeast cell further expresses an aldehyde-forming fatty acyl-CoA reductase (EC 1.2.1.50), an alcohol dehydrogenase (EC 1.1.1.2), and / or a fatty alcohol oxidase (EC 1.1.3.20) capable of converting at least a portion of the E8,E10-dodecadien-1-ol to E8,E10-dodecadienal.

[0600] 39. The yeast cell according to any one of the preceding clauses, wherein the yeast cell further:

[0601] i) Having one or more mutations that reduce the activity of one or more natural acyl-CoA oxidases; and

[0602] ii) expressing at least one set of enzymes comprising at least one acyl-CoA oxidase capable of oxidizing fatty acyl-CoA, wherein the set of enzymes is capable of shortening fatty acyl-CoA of a first carbon chain length X to a shortened fatty acyl-CoA having a second carbon chain length X', wherein X' ≤ X-2.

[0603] 40. The yeast cell as described in Clause 39, wherein X' = 12.

[0604] 41. A yeast cell according to any one of claims 39 to 40, wherein the yeast cell further expresses a desaturase capable of introducing at least one double bond into a fatty acyl-CoA of carbon chain length X, such as CroZ11 desaturase (SEQ ID NO: 63) or CpaE11 desaturase (SEQ ID NO: 65), or in combination with SEQ ID NO: 63, SEQ ID NO: 65. NO:65 has a functional variant of at least 65% homology or identity, such as at least 70% homology or identity, such as at least 71% homology or identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0605] 42. The yeast cell according to any one of clauses 39 to 42, wherein the natural acyl-CoA oxidase of i) and / or the acyl-CoA oxidase of ii) are peroxisomal acyl-CoA oxidases.

[0606] 43. The yeast cell according to any one of clauses 39 to 41, wherein the at least one acyl-CoA oxidase of ii) is a natural acyl-CoA oxidase or a heterologous acyl-CoA oxidase, which is optionally overexpressed compared with a reference yeast strain that does not express the at least one group of enzymes, preferably at least one acyl-CoA oxidase in the enzyme group of ii) is a heterologous acyl-CoA oxidase.

[0607] 44. The yeast cell according to any one of clauses 39 to 43, wherein the enzyme group of ii) comprises acyl-CoA oxidase derived from organisms selected from the genera *Yersinia*, *Noctua*, *Arabidopsis*, *Aspergillus*, *Cucurbita*, *Homo*, *Arthrobacter*, and *Rats*, preferably, at least one of the first group enzymes comprises acyl-CoA oxidase derived from *Yersinia lipolytica*, *Cephalotaxus sinensis*, *Arabidopsis*, *Aspergillus nidus*, *Cucurbita*, *Homo sapiens*, *Arthrobacter ureagenesis*, or *Rat*. Selectedly, at least one acyl-CoA oxidase of the first group of enzymes is selected from Yli_POX1 (XP_504703), Yli_POX2 (XP_505264), Yli_POX3 (XP_503244), Yli_POX4 (XP_504475), Yli_POX5 (XP_502199), Yli_POX6 (XP_503632), Ase_POX (SEQ ID NO:39), Ath_POX1 (SEQ ID NO:41), Ath_POX2 (SEQ ID NO:43), Ani_POX (SEQ ID NO:45), Cma_POX (SEQ ID NO:47), Hsa_POX1-2 (SEQ ID NO:49), Pur_POX (SEQ ID NO:51), Sc_POX1 (SEQ ID NO:31), and Rno_POX2 (SEQ ID NO:51). Acyl-CoA oxidase (NO:53), or its functional variants having at least 60% homology or identity, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0608] 45. A method for producing E8,E10-dodecadienyl coenzyme A and optionally E8,E10-dodecadien-1-ol in yeast cells, the method comprising the steps of providing yeast cells and incubating the yeast cells in a culture medium, wherein the yeast cells express:

[0609] i) at least one heterologous desaturase capable of introducing one or more double bonds into a fatty acyl-CoA with a carbon chain length of 12, thereby converting said fatty acyl-CoA into desaturated fatty acyl-CoA, wherein at least a portion of said desaturated fatty acyl-CoA is E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA); and

[0610] ii) Optionally, at least one heterologous fatty acyl-CoA reductase (EC 1.2.1.84) capable of converting at least a portion of the desaturated fatty acyl-CoA into a desaturated fatty alcohol, wherein the fatty acyl-CoA reductase is capable of converting at least a portion of the E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA) into E8,E10-dodecadien-1-ol.

[0611] This produces E8,E10-dodecadienyl coenzyme A and optionally E8,E10-dodecadien-1-ol.

[0612] 46. ​​The method according to Clause 45, wherein the yeast cell is as defined in any one of Clauses 1 to 44.

[0613] 47. The method according to any one of clauses 45 to 46, further comprising the steps of converting E8,E10-dodecadienyl-CoA into lipids (such as triglycerides) or free fatty acids, recovering said lipids or free fatty acids, and converting said lipids or free fatty acids into E8,E10-dodecadien-1-ol.

[0614] 48. The method according to any one of clauses 45 to 47, further comprising the step of recovering the E8,E10-dodecadien-1-ol.

[0615] 49. The method according to any one of clauses 45 to 48, further comprising the step of converting at least a portion of E8,E10-dodecadien-1-ol to E8,E10-dodecadienyl acetate by expression of acetyltransferase or by chemical conversion.

[0616] 50. The method according to Clause 49, wherein the acetyltransferase is a heterologous acetyltransferase (EC 2.3.1.84) expressed from the yeast cells or a native acetyltransferase overexpressed from the yeast cells, wherein the acetyltransferase is capable of converting at least a portion of the E8,E10-dodecadien-1-ol to E8,E10-dodecadienyl acetate, thereby further producing E8,E10-dodecadienyl acetate.

[0617] 51. The method according to claim 50, wherein the acetyltransferase is Sc_Atf1 (SEQ ID NO:37) or a functional variant thereof having at least 75% homology or identity with Sc_Atf1 (SEQ ID NO:37), such as at least 80% homology or identity, such as at least 85% homology or identity, such as at least 90% homology or identity, such as at least 91% homology or identity, such as at least 92% homology or identity, such as at least 93% homology or identity, such as at least 94% homology or identity, such as at least 95% homology or identity, such as at least 96% homology or identity, such as at least 97% homology or identity, such as at least 98% homology or identity, such as at least 99% homology or identity, such as at least 100% homology or identity.

[0618] 52. The method according to any one of clauses 45 to 51, further comprising the step of recovering the E8,E10-dodecadienyl acetate.

[0619] 53. The method according to any one of claims 45 to 52, further comprising the step of converting at least a portion of the E8,E10-dodecadien-1-ol to E8,E10-dodecadienal by expressing an aldehyde-forming fatty acyl-CoA reductase (EC 1.2.1.50), an alcohol dehydrogenase (EC 1.1.1.2), and / or a fatty alcohol oxidase (EC 1.1.3.20) capable of converting at least a portion of the E8,E10-dodecadien-1-ol to E8,E10-dodecadienal or by chemical conversion, thereby further producing E8,E10-dodecadienal.

[0620] 54. The method according to Clause 53 further includes the step of recovering the E8,E10-dodecadienal.

[0621] 55. The method according to any one of clauses 45 to 54, wherein the culture medium comprises an extractant in an amount equal to or greater than its turbidity concentration in an aqueous solution, wherein the extractant is a nonionic ethoxylated surfactant such as an antifoaming agent, preferably selected from the group consisting of: polyoxyethylene polyoxypropylene ether, mixtures of polyether dispersions, antifoaming agents containing polyethylene glycol monostearate such as dimethicone, fatty alcohol alkoxylates, polyethoxylated surfactants, and ethoxylated and propoxylated C 16 -C 18 Alcohol-based defoamers and combinations thereof.

[0622] 56. The method described according to Clause 55, wherein:

[0623] -The nonionic ethoxylated surfactant is ethoxylated and propoxylated C 16 -C18 Alcohol-based defoamers, such as C 16 -C 18 Alkyl alcohol ethoxylate propoxylate (CAS No. 68002-96-0), and wherein the culture medium contains at least 1% vol / vol of C 16 -C 18 Alkyl alcohol ethoxylates and propoxylates, such as at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 22.5%, at least 25%, at least 27.5%, or at least 30% vol / vol of C 16 -C 18 Alkyl alcohol ethoxylates, propoxylates, or more,

[0624] - The nonionic ethoxylated surfactant is a polyoxyethylene polyoxypropylene ether, for example P407 (CAS No. 9003-11-6), and wherein the culture medium contains at least 10% vol / vol of polyoxyethylene polyoxypropylene ether, such as P407, such as at least 11% vol / vol, such as at least 12% vol / vol, such as at least 13% vol / vol, such as at least 14% vol / vol, such as at least 15% vol / vol, such as at least 16% vol / vol, such as at least 17% vol / vol, such as at least 18% vol / vol, such as at least 19% vol / vol, such as at least 20% vol / vol, such as at least 25% vol / vol, such as at least 30% vol / vol, such as at least 35% vol / vol of polyoxyethylene polyoxypropylene ether, such as P407, or more,

[0625] - The nonionic ethoxylated surfactant is a mixture of polyether dispersions (such as defoamer 204), and the culture medium contains at least 1% vol / vol of the mixture of polyether dispersions (such as defoamer 204), such as at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 22.5%, at least 25%, at least 27.5%, or at least 30% vol / vol of the mixture of polyether dispersions (such as defoamer 204), or more; and / or

[0626] - The nonionic ethoxylated surfactant is a nonionic ethoxylated surfactant comprising polyethylene glycol monostearate such as dimethicone, and the culture medium comprises at least 1% vol / vol of polyethylene glycol monostearate or dimethicone, such as at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 22.5%, at least 25%, at least 27.5%, at least 30% vol / vol of polyethylene glycol monostearate or dimethicone, or more;

[0627] - The nonionic ethoxylated surfactant is a fatty alcohol alkoxylate, preferably selected from... LF300 (CAS No. 196823-11-7) LF1300 (68002-96-0), SLF180 (CAS No. 196823-11-7) 2574 (CAS No. 68154-97-2) and Imbentin SG / 251 (CAS No. 68002-96-0) are preferred. LF300 or 2574, and wherein the culture medium contains at least 1% vol / vol of fatty alcohol alkoxylates, such as at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 22.5%, at least 25%, at least 27.5%, at least 30% vol / vol of fatty alcohol alkoxylates or more;

[0628] - The nonionic ethoxylated surfactant is Agnique BP420 (CAS No. 68002-96-0), and the culture medium contains at least 1% vol / vol of Agnique BP420, such as at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 22.5%, at least 25%, at least 27.5%, at least 30% vol / vol of Agnique BP420, or more.

[0629] 57. The method according to any one of clauses 45 to 56, wherein the culture medium contains an extractant in an amount greater than 50% of its turbidity concentration, such as at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 750%, at least 1000%, or more, and / or wherein the culture medium contains an extractant in an amount at least twice its turbidity concentration, such as at least 100% of its turbidity concentration, or ... At least 3 times its turbidity concentration, at least 4 times its turbidity concentration, at least 5 times its turbidity concentration, at least 6 times its turbidity concentration, at least 7 times its turbidity concentration, at least 8 times its turbidity concentration, at least 9 times its turbidity concentration, at least 10 times its turbidity concentration, at least 12.5 times its turbidity concentration, at least 15 times its turbidity concentration, at least 17.5 times its turbidity concentration, at least 20 times its turbidity concentration, at least 25 times its turbidity concentration, at least 30 times its turbidity concentration.

[0630] 58. The method according to any one of clauses 45 to 57, wherein the E8,E10-dodecadienyl coenzyme A is converted into lipids or free fatty acids, and wherein the lipids or free fatty acids produced by the yeast cells, the E8,E10-dodecadien-1-ol, and optionally the E8,E10-dodecadienyl acetate and / or the E8,E10-dodecadienal are present in an emulsion in the fermentation broth, the method further comprising the step of disrupting the emulsion to obtain a composition comprising a product phase comprising the extractant and the lipids or free fatty acids, the E8,E10-dodecadien-1-ol, and optionally the E8,E10-dodecadienyl acetate and / or the E8,E10-dodecadienal, optionally wherein:

[0631] - The step of disrupting the emulsion includes or consists of the following: a phase separation step of the fermentation broth, such as a centrifugation step, to obtain a composition consisting of three phases: an aqueous phase, a phase containing cells and cell debris, and a product phase comprising the extractant and the lipids or free fatty acids, E8,E10-dodecadien-1-ol and optionally the E8,E10-dodecadienyl acetate and / or the E8,E10-dodecadienal, and / or

[0632] -The product phase comprises at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or more of the lipids or free fatty acids, E8,E10-dodecadien-1-ol and optionally the E8,E10-dodecadienyl acetate and / or the E8,E10-dodecadienal that were initially present in the fermentation broth.

[0633] 59. The method according to any one of clauses 45 to 58, further comprising the following steps:

[0634] - The lipids or free fatty acids, the E8,E10-dodecadien-1-ol, and optionally the E8,E10-dodecadienyl acetate and / or the E8,E10-dodecadienal are recovered, preferably by a distillation step such as vacuum distillation or by column purification.

[0635] - Chemically convert at least a portion of the E8,E10-dodecadien-1-ol to E8,E10-dodecadienal and / or E8,E10-dodecadienyl acetate.

[0636] - Optionally, the E8,E10-dodecadienal and / or E8,E10-dodecadienyl acetate are recovered.

[0637] 60. The method according to any one of clauses 45 to 59, further comprising the step of formulating the recovered E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienal into a pheromone composition.

[0638] 61. The method according to any one of clauses 45 to 60, wherein the pheromone composition further comprises one or more additional compounds, such as a liquid or solid carrier or matrix.

[0639] 62. A nucleic acid construct for modifying yeast cells, the construct comprising:

[0640] i) at least one first polynucleotide encoding at least one heterodesaturase capable of introducing one or more double bonds into a 12-carbon acyl-CoA fatty acid chain, thereby converting the fatty acid-CoA into desaturated fatty acid-CoA, wherein at least a portion of the desaturated fatty acid-CoA is E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA); and

[0641] ii) Optionally encoding a second polynucleotide of at least one heterologous fatty acyl-CoA reductase (EC 1.2.1.84), said at least one heterologous fatty acyl-CoA reductase being capable of converting at least a portion of said desaturated fatty acyl-CoA into desaturated fatty alcohol, said fatty acyl-CoA reductase being capable of converting at least a portion of said E8,E10-dodecadienyl-CoA (E8,E10-C12:CoA) into E8,E10-dodecadien-1-ol.

[0642] 63. The nucleic acid construct according to Clause 62, wherein:

[0643] a) The at least one desaturase is Gmo_CPRQ (SEQ ID NO:77), Cpo_CPRQ (SEQ ID NO:2), or a functional variant thereof having at least 80% identity with SEQ ID NO:77 or SEQ ID NO:2, and having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:77 or SEQ ID NO:2, preferably the at least one desaturase is Cpo_CPRQ or a functional variant thereof; or

[0644] (b) The at least one desaturase is at least two desaturases, wherein at least one of the two desaturases is Gmo_CPRQ (SEQ ID NO:77), Cpo_CPRQ (SEQ ID NO:2) or a functional variant thereof having at least 80% identity with SEQ ID NO:2 having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:2. Preferably, the at least one desaturase is Cpo_CPRQ or a functional variant thereof, and the other desaturase is a desaturase capable of introducing at least one double bond into a fatty acyl-CoA with a carbon chain length of 12, such as Z9-12 desaturase.

[0645] 64. The nucleic acid construct according to any one of clauses 62 to 63, wherein the at least one heterologous desaturase is at least two desaturases, and wherein the other desaturase is selected from Cpo_NPVE (SEQ ID NO:67), Cpo_SPTQ (SEQ ID NO:69) or a functional variant thereof having at least 60% homology or identity with SEQ ID NO:67 or SEQ ID NO:69 and having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:67 or SEQ ID NO:69.

[0646] 65. A nucleic acid construct according to any one of clauses 62 to 64, wherein the first polynucleotide comprises SEQ ID NO:1 or SEQ ID NO:78 (preferably SEQ ID NO:1), or has at least 60% homology or identity with it, and has a content of, for example, 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, or at least 80% with SEQ ID NO:1 or SEQ ID NO:78 (preferably SEQ ID NO:1). For example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology or identity with its homologs.

[0647] 66. A nucleic acid construct according to any one of claims 62 to 65, wherein the at least one heterodesaturase is at least two heterodesaturases, and wherein the first polynucleotide further comprises the nucleic acid shown in SEQ ID NO:66 or SEQ ID NO:68, or has at least 60% homology or identity with it, such as 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, or as shown in the first polynucleotide. At least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology or identity with its homologs.

[0648] 67. The nucleic acid construct according to any one of Clauses 62 to 66, wherein the heterodesaturase is as defined in any one of Clauses 1 to 44.

[0649] 68. The nucleic acid construct according to any one of clauses 62 to 67, wherein the at least one desaturase is a mutant of Cpo_CPRQ with a mutation such as the S85A mutation at position 85.

[0650] 69. The nucleic acid construct according to any one of clauses 62 to 68, wherein the heterologous fatty acyl-CoA reductase is as defined in any one of clauses 1 to 44.

[0651] 70. The nucleic acid construct according to any one of claims 62 to 69, wherein the second polynucleotide comprises or is composed of: SEQ ID NO:9, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:11, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:11, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79 ... NO:76 and its homologs that have at least 60% homology or identity, such as at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0652] 71. The nucleic acid construct according to any one of clauses 62 to 70, further comprising one or more of the following:

[0653] iii) Polynucleotides encoding heterologous cytochrome b5, such as the polynucleotide shown in SEQ ID NO:3 or its homologs having at least 60% homology or identity with it;

[0654] iv) Polynucleotides encoding heterologous cytochrome b5 reductase, such as the polynucleotide shown in SEQ ID NO:23 or its homologs having at least 60% homology or identity with it;

[0655] v) Polynucleotides encoding hemoglobin, such as the polynucleotide shown in SEQ ID NO:5 or its homologs having at least 60% homology or identity with it;

[0656] vi) Polynucleotides encoding fatty acyl synthase variants with modified ketone synthase domains; and / or

[0657] vii) A polynucleotide encoding a thioesterase, such as the polynucleotide shown in SEQ ID NO:25 or SEQ ID NO:34, or a homolog thereof having at least 60% homology or identity with it.

[0658] 72. The nucleic acid construct according to any one of clauses 62 to 71, wherein the heterologous cytochrome b5, the heterologous cytochrome b5 reductase, the hemoglobin, the fatty acyl synthase variant, and / or the thioesterase are as defined in any one of clauses 1 to 44.

[0659] 73. A method for monitoring the presence of harmful organisms or interfering with the mating of harmful organisms, the method comprising the following steps:

[0660] i) Producing E8,E10-dodecadien-1-ol and optionally E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienal by the method according to any one of clauses 45 to 61.

[0661] ii) Formulating the E8,E10-dodecadien-1-ol and optionally the E8,E10-dodecadienyl acetate and / or the E8,E10-dodecadienal into a pheromone composition; and

[0662] iii) Use the pheromone composition as an integrated pest management composition.

[0663] 74. E8,E10-dodecadienyl coenzyme A, E8,E10-dodecadien-1-ol, E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienal can be obtained by the method according to any one of clauses 45 to 61.

[0664] 75. A component kit comprising an instruction manual and:

[0665] a) Yeast cells according to any one of clauses 1 to 44; and / or

[0666] b) A nucleic acid construct for modifying yeast cells according to any one of clauses 62 to 72 and optionally the yeast cells to be modified, wherein, after expression of the polynucleotide contained in the nucleic acid construct, the modified yeast cells are able to produce E8,E10-dodecadienyl-CoA and optionally E8,E10-dodecadien-1-ol. sequence list <110> Ferrobio <120> Yeast cells and methods for producing E8,E10-dodecadienyl coenzyme A, methyl methacrylate (MCMA) and its derivatives <130> P5484PC00 <160> 80 <170> PatentIn version 3.5 <210> 1 <211> 1047 <212> DNA <213> Artificial sequence <220> <223> CPO_CPRQ desaturase (AHW98354) for codon optimization against Yersinia lipophila; mRNA coding sequence <220> <221> Unclassified features <222> (1)..(1047) <223> CPO_CPRQ desaturase (AHW98354) for codon optimization against Yersinia lipophila; mRNA coding sequence <400> 1 atgcctcccc gagagtctaa gaaggtggcc ctgcgatctt acgagacccc tgtcgcttct 60 ctccctcctc gaaagtacga gattatctac ctcaacctct tcctgcacat cgctggacat 120 atctccgccg tctacggcct gtatctgtgc ttcaccgccg cccagtggaa gaccatcttc 180 tttgcctacc tgtggctgtt gatgggcgag ctcggcgtgg tgtgtggcgc tcacagattg 240 tggtctcacc gttctttcaa ggtgaagcct cctctcgaga tcatgctgat gctgttcaac 300 tgtattggat tccagaacac cgccactgac tgggtccgaa accaccggct ccatcacaag 360 cactctgaca ctgacgccga cccccataac tctaaccgag gaatgctgtt ctcccacatt 420 ggctggctgt gtgtgcgaaa gcacccagat gttaaagaac gaggcaagac caccgacatg 480 tctgacatct actctaaccc cgtgctccga ttccagaaga agcacaaggt accccttttc 540 ggcgccatgt gtttcggcct gcccaccctt attcccaccc tgtggggaga ggacatcgtc 600 accgcttggc acgtcaacct gctgcgattc gttcttaatc tgaactctat cctgctggtc 660 aactccattg ctcataagta cggcacccga ccctacgatc gaaccatctg ccctcgacaa 720 aacaccacct gtaacatgat gactcttgga gagggcttcc acaactacca ccacaccttt 780 ccttgggact accgatctgc cgagctggga aagaactacc tgaacttcac caagtggttc 840 atcgacttct tcgccctgat tggatgggcc tacgacctga agaccgttcc tgacgatatg 900 atccagcgac gaatgaaaag aaccggagac ggatccaact cgtggggatg gggagacaag 960 gacatgacta aggaggagcg agactctgct actatcattt atcccgagaa gaaggatgat 1020 attaagatga tctccaaaaa gaactaa 1047 <210> 2 <211> 348 <212> PRT <213> Codling moth (Cydia pomonella) <220> <221> Unclassified features <222> (1)..(348) <223> CPO_CPRQ desaturase for codling moth (AHW98354) <400> 2 Met Pro Pro Arg Glu Ser Lys Lys Val Ala Leu Arg Ser Tyr Glu Thr 1 5 10 15 Pro Val Ala Ser Leu Pro Pro Arg Lys Tyr Glu Ile Ile Tyr Leu Asn 20 25 30 Leu Phe Leu His Ile Ala Gly His Ile Ser Ala Val Tyr Gly Leu Tyr 35 40 45 Leu Cys Phe Thr Ala Ala Gln Trp Lys Thr Ile Phe Phe Ala Tyr Leu 50 55 60 Trp Leu Leu Met Gly Glu Leu Gly Val Val Cys Gly Ala His Arg Leu 65 70 75 80 Trp Ser His Arg Ser Phe Lys Val Lys Pro Pro Leu Glu Ile Met Leu 85 90 95 Met Leu Phe Asn Cys Ile Gly Phe Gln Asn Thr Ala Thr Asp Trp Val 100 105 110 Arg Asn His Arg Leu His His Lys His Ser Asp Thr Asp Ala Asp Pro 115 120 125 His Asn Ser Asn Arg Gly Met Leu Phe Ser His Ile Gly Trp Leu Cys 130 135 140 Val Arg Lys His Pro Asp Val Lys Glu Arg Gly Lys Thr Thr Asp Met 145 150 155 160 Ser Asp Ile Tyr Ser Asn Pro Val Leu Arg Phe Gln Lys Lys His Lys 165 170 175 Val Pro Leu Phe Gly Ala Met Cys Phe Gly Leu Pro Thr Leu Ile Pro 180 185 190 Thr Leu Trp Gly Glu Asp Ile Val Thr Ala Trp His Val Asn Leu Leu 195 200 205 Arg Phe Val Leu Asn Leu Asn Ser Ile Leu Leu Val Asn Ser Ile Ala 210 215 220 His Lys Tyr Gly Thr Arg Pro Tyr Asp Arg Thr Ile Cys Pro Arg Gln 225 230 235 240 Asn Thr Thr Cys Asn Met Met Thr Leu Gly Glu Gly Phe His Asn Tyr 245 250 255 His His Thr Phe Pro Trp Asp Tyr Arg Ser Ala Glu Leu Gly Lys Asn 260 265 270 Tyr Leu Asn Phe Thr Lys Trp Phe Ile Asp Phe Phe Ala Leu Ile Gly 275 280 285 Trp Ala Tyr Asp Leu Lys Thr Val Pro Asp Asp Met Ile Gln Arg Arg 290 295 300 Met Lys Arg Thr Gly Asp Gly Ser Asn Ser Trp Gly Trp Gly Asp Lys 305 310 315 320 Asp Met Thr Lys Glu Glu Arg Asp Ser Ala Thr Ile Ile Tyr Pro Glu 325 330 335 Lys Lys Asp Asp Ile Lys Met Ile Ser Lys Lys Asn 340 345 <210> 3 <211> 384 <212> DNA <213> Artificial sequence <220> <223> Codon-optimized cytochrome b5 (AAC33731) from *Yarrowia lipophila*; mRNA coding sequence <220> <221> Unclassified features <222> (1)..(384) <223> Codon-optimized cytochrome b5 (AAC33731) from *Yarrowia lipophila*; mRNA coding sequence <400> 3 atgaccgtgc gacagttcac ccgagtcgag gtgtctaagt ggaccactcg agaggaagcc 60 gtgttcatca tcgacaacgt ggtgtacaac gtgaccaagt tcctggacga gcaccccggt 120 ggacacgagg tgctggtgaa cgtggccggc aaggacgcct ctgaggactt cgacgacgtg 180 ggccactctc tggacgccaa ggaactgatg aagaagtacg tcgtcggcga ggtggtcgag 240 gccgagcgac gacacatcca gaagcgacag atctcttggg aagattctaa ggtggactct 300 gactcttctt tcacctcttc gtggaagttc cccgtgctgc tgggcatcgt ggtgaccctg 360 ctgtacacct acctgttcgg ctaa 384 <210> 4 <211> 127 <212> PRT <213> Helicoverpa armigera <220> <221> Unclassified features <222> (1)..(127) <223> Cotton bollworm cytochrome b5 (AAC33731) <400> 4 Met Thr Val Arg Gln Phe Thr Arg Val Glu Val Ser Lys Trp Thr Thr 1 5 10 15 Arg Glu Glu Ala Val Phe Ile Ile Asp Asn Val Val Tyr Asn Val Thr 20 25 30 Lys Phe Leu Asp Glu His Pro Gly Gly His Glu Val Leu Val Asn Val 35 40 45 Ala Gly Lys Asp Ala Ser Glu Asp Phe Asp Asp Val Gly His Ser Leu 50 55 60 Asp Ala Lys Glu Leu Met Lys Lys Tyr Val Val Gly Glu Val Val Glu 65 70 75 80 Ala Glu Arg Arg His Ile Gln Lys Arg Gln Ile Ser Trp Glu Asp Ser 85 90 95 Lys Val Asp Ser Asp Ser Ser Ser Phe Thr Ser Ser Trp Lys Phe Pro Val 100 105 110 Leu Leu Gly Ile Val Val Thr Leu Leu Tyr Thr Tyr Leu Phe Gly 115 120 125 <210> 5 <211> 441 <212> DNA <213> Artificial sequence <220> <223> Codon-optimized hemoglobin from *Yarrowia lipolyticis* (AAT01097); mRNA coding sequence <220> <221> Unclassified features <222> (1)..(441) <223> Codon-optimized hemoglobin from *Yarrowia lipolyticis* (AAT01097); mRNA coding sequence <400> 5 atgctggacc agcagaccgt ggacacctct aaggccaccg tgcctgtgct gaaggaacac 60 ggcgtgacca tcaccacccac cttctaccag aacctgttcg ctaagcaccc cgaggtgcga 120 cccctgttcg atatgggccg acaggcctct ctcgagcagc ccaaggctct ggccatgacc 180 gtggggagccg ccgctcagaa catcgagaac ctgcctgcca ttctgcccgc cgtgcagaag 240 atcgccgtca agcactgcca ggccggcgtg gccgctcgac actaccccat cgtgggccaa 300 gagctgctgg gcgccatcaa ggaactgctg ggtgacgccg ccaccgacga catcctggac 360 gcctggggca aggcctacgg cgtgatcgcc gacgtgttca tccaggtcga ggccgacctg 420 tacgcccagg acgccgagta a 441 <210> 6 <211> 146 <212> PRT <213> Vitreoscilla stercoraria <220> <221> Unclassified features <222> (1)..(146) <223> fecal hygroscopic oscillation bacillus hemoglobin (AAT01097) <400> 6 Met Leu Asp Gln Gln Thr Val Asp Thr Ser Lys Ala Thr Val Pro Val 1 5 10 15 Leu Lys Glu His Gly Val Thr Ile Thr Thr Thr Phe Tyr Gln Asn Leu 20 25 30 Phe Ala Lys His Pro Glu Val Arg Pro Leu Phe Asp Met Gly Arg Gln 35 40 45 Ala Ser Leu Glu Gln Pro Lys Ala Leu Ala Met Thr Val Gly Ala Ala 50 55 60 Ala Gln Asn Ile Glu Asn Leu Pro Ala Ile Leu Pro Ala Val Gln Lys 65 70 75 80 Ile Ala Val Lys His Cys Gln Ala Gly Val Ala Ala Arg His Tyr Pro 85 90 95 Ile Val Gly Gln Glu Leu Leu Gly Ala Ile Lys Glu Leu Leu Gly Asp 100 105 110 Ala Ala Thr Asp Asp Ile Leu Asp Ala Trp Gly Lys Ala Tyr Gly Val 115 120 125 Ile Ala Asp Val Phe Ile Gln Val Glu Ala Asp Leu Tyr Ala Gln Asp 130 135 140 Ala Glu 145 <210> 7 <211> 1557 <212> DNA <213> Artificial sequence <220> <223> A codon-optimized oxalis fatty acyl reductase (NP_001289627) targeting *Yarrowia lipolyticis*; mRNA coding sequence. <220> <221> Unclassified features <222> (1)..(1557) <223> A codon-optimized oxalis fatty acyl reductase (NP_001289627) targeting *Yarrowia lipolyticis*; mRNA coding sequence. <400> 7 atggtgtcta tccccgagta ctacgagggc aagaacatcc tgctgaccgg cgccaccggc 60 ttcatgggca aggtgctgct cgagaagctg ctgcgatctt gccccaaggt gaaggccgtg 120 tacgtgctgg tgcgacacaa ggccggacag acccctgagg ctcgaatcga ggaaatcacc 180 aactgcaagc tgttcgaccg actgcgagat gagcagcccg acttcaaggc caagatcatc 240 gtgatcacct ctgagctgac ccagcctgag ctggacctgt ctgagcccat caaggaaaag 300 ctgatcgagc gaatcaacat catcttccac tgcgccgcca ccgtgcgatt caacgagact 360 ctccgagatg ccgtgcagct gaacgtgacc gctactcagc agctcctgtt cctggctcag 420 cgaatgaaga acctggaagt gttcatgcac gtgtctaccg cctacgccta ctgcaaccga 480 aagcagatcg aagagatcgt gtaccctcct ccagtggacc ccaagaagct gattgactct 540 ctcgagtgga tggacgacgg cctggtgaac gacatcaccc ctaagctcat cggcgaccga 600 cctaacacct acacttacac caaggctctg gccgagtacg tggtgcagca agagggcgcc 660 aagctgaaca ccgccatcat tcgaccctct atcgtgggcg cctcttggaa ggaacccttt 720 cctggctgga tcgacaactt caacggcccc tctggcctgt tcattgccgc cggaaagggc 780 atcctgcgaa ccatgcgagc ctctaactct gccgtggccg acctggtgcc tgtggacgtg 840 gtggtgaaca ccactctggc cgctgcctgg tactctggcg tgaaccgacc tcgaaacgtg 900 atgatctaca actgcaccac cggcggcact aaccccttcc actggggcga agtgggctac 960 cacatcaacc tgaacttcaa gatcaaccct ctcgagaacg ccgtgcgaca ccccaactgt 1020 tctctgcagt ctaaccctct gctccatcag tactggaccg ccgtgtctca caccatgcct 1080 gcctttctgc tggacctcct gctgcgactg accggacaca agccctggat gatgaagacc 1140 atcactcgac tgcacaaggc catgatgctc ctcgagtact tcacctccaa ctcttggatc 1200 tggaacaccg agaacatgac catgctgatg aaccagctga accccgagga caagaagacc 1260 ttcaacttcg acgtgcgaca gctgcactgg gctgagtaca tggaaaacta ctgcatgggc 1320 accaagaagt acgtcctgaa cgaggaaatg tctggactgc ccgctgccag aaagcacctg 1380 aacaagctgc gaaacatccg atacggcttc aacaccgtgc tggtcatcct gatctggcga 1440 atcttcattg cccgatctca gatggcccga aacatctggt acttcgtggt gtctctgtgc 1500 tacaagttcc tgtcttactt ccgagcctct tctaccatgc gatactctaa gctgtag 1557 <210> 8 <211> 515 <212> PRT <213> Barn owl (Tyto alba) <220> <221> Features not yet classified <222> (1)..(515) <223> Barn owl fatty acyl reductase (NP_001289627) <400> 8 Met Val Ser Ile Pro Glu Tyr Tyr Glu Gly Lys Asn Ile Leu Leu Thr 1 5 10 15 Gly Ala Thr Gly Phe Met Gly Lys Val Leu Leu Glu Lys Leu Leu Arg 20 25 30 Ser Cys Pro Lys Val Lys Ala Val Tyr Val Leu Val Arg His Lys Ala 35 40 45 Gly Gln Thr Pro Glu Ala Arg Ile Glu Glu Ile Thr Asn Cys Lys Leu 50 55 60 Phe Asp Arg Leu Arg Asp Glu Gln Pro Asp Phe Lys Ala Lys Ile Ile 65 70 75 80 Val Ile Thr Ser Glu Leu Thr Gln Pro Glu Leu Asp Leu Ser Glu Pro 85 90 95 Ile Lys Glu Lys Leu Ile Glu Arg Ile Asn Ile Ile Phe His Cys Ala 100 105 110 Ala Thr Val Arg Phe Asn Glu Thr Leu Arg Asp Ala Val Gln Leu Asn 115 120 125 Val Thr Ala Thr Gln Gln Leu Leu Phe Leu Ala Gln Arg Met Lys Asn 130 135 140 Leu Glu Val Phe Met His Val Ser Thr Ala Tyr Ala Tyr Cys Asn Arg 145 150 155 160 Lys Gln Ile Glu Glu Ile Val Tyr Pro Pro Pro Val Asp Pro Lys Lys 165 170 175 Leu Ile Asp Ser Leu Glu Trp Met Asp Asp Gly Leu Val Asn Asp Ile 180 185 190 Thr Pro Lys Leu Ile Gly Asp Arg Pro Asn Thr Tyr Thr Tyr Thr Lys 195 200 205 Ala Leu Ala Glu Tyr Val Val Gln Gln Glu Gly Ala Lys Leu Asn Thr 210 215 220 Ala Ile Ile Arg Pro Ser Ile Val Gly Ala Ser Trp Lys Glu Pro Phe 225 230 235 240 Pro Gly Trp Ile Asp Asn Phe Asn Gly Pro Ser Gly Leu Phe Ile Ala 245 250 255 Ala Gly Lys Gly Ile Leu Arg Thr Met Arg Ala Ser Asn Ser Ala Val 260 265 270 Ala Asp Leu Val Pro Val Asp Val Val Val Asn Thr Thr Leu Ala Ala 275 280 285 Ala Trp Tyr Ser Gly Val Asn Arg Pro Arg Asn Val Met Ile Tyr Asn 290 295 300 Cys Thr Thr Gly Gly Thr Asn Pro Phe His Trp Gly Glu Val Gly Tyr 305 310 315 320 His Ile Asn Leu Asn Phe Lys Ile Asn Pro Leu Glu Asn Ala Val Arg 325 330 335 His Pro Asn Cys Ser Leu Gln Ser Asn Pro Leu Leu His Gln Tyr Trp 340 345 350 Thr Ala Val Ser His Thr Met Pro Ala Phe Leu Leu Asp Leu Leu Leu 355 360 365 Arg Leu Thr Gly His Lys Pro Trp Met Met Lys Thr Ile Thr Arg Leu 370 375 380 His Lys Ala Met Met Leu Leu Glu Tyr Phe Thr Ser Asn Ser Trp Ile 385 390 395 400 Trp Asn Thr Glu Asn Met Thr Met Leu Met Asn Gln Leu Asn Pro Glu 405 410 415 Asp Lys Lys Thr Phe Asn Phe Asp Val Arg Gln Leu His Trp Ala Glu 420 425 430 Tyr Met Glu Asn Tyr Cys Met Gly Thr Lys Lys Tyr Val Leu Asn Glu 435 440 445 Glu Met Ser Gly Leu Pro Ala Ala Arg Lys His Leu Asn Lys Leu Arg 450 455 460 Asn Ile Arg Tyr Gly Phe Asn Thr Val Leu Val Ile Leu Ile Trp Arg 465 470 475 480 Ile Phe Ile Ala Arg Ser Gln Met Ala Arg Asn Ile Trp Tyr Phe Val 485 490 495 Val Ser Leu Cys Tyr Lys Phe Leu Ser Tyr Phe Arg Ala Ser Ser Thr 500 505 510 Met Arg Tyr 515 <210> 9 <211> 1377 <212> DNA <213> Artificial sequence <220> <223> A codon-optimized fatty acyl reductase (AGP26039) targeting *Yarrowia lipolyticis*; mRNA coding sequence. <220> <221> Unclassified features <222> (1)..(1377) <223> A codon-optimized fatty acyl reductase (AGP26039) targeting *Yarrowia lipolyticis*; mRNA coding sequence. <400> 9 atgcccgtgc tgacctcgcg agaggacgag aagctgtctg tgcccgagtt ctacgccggc 60 aagtctatct tcgtgaccgg cggcaccgga ttcctcggca aggtgttcat tgagaagctg 120 ctctactgct gccccgacat cgacaagatc tacatgctga tccgagagaa gaagaacctg 180 tctatcgacg agcgaatgtc taagttcctg gacgaccctc tgttctctcg actgaaggaa 240 gaacgacccg gcgacctcga gaagatcgtg ctgatccccg gcgacatcac cgctcctaac 300 ctgggcctgt ctgccgagaa cgaacgaatc ctgctcgaga aggtgtccgt gatcatcaac 360 tctgccgcca ccgtgaagtt caacgagccc ctgcctatcg cctggaagat caacgtcgag 420 ggcacccgaa tgctgctggc cctgtctcga cgaatgaagc gaatcgaggt gtttatccac 480 atctctaccg cctactctaa cgcctcttct gaccgaatcg tggtggacga gattctgtac 540 cccgctcctg ccgacatgga ccaggtgtac cagctcgtga aggacggcgt gaccgaggaa 600 gagactgagc gactgctgaa cggactgccc aacacctaca ccttcaccaa ggctctgacc 660 gagcacctgg tggccgagca ccagacctac gtgcccacca tcatcattcg accctccgtg 720 gtggcctcta tcaaggacga gcccatccga ggctggctgt gcaactggtt cggcgccacc 780 ggcatctctg tgttcaccgc caagggcctg aaccgagtgc tgctcggaaa ggcctctaac 840 atcgtggacg tgatccccgt ggactacgtg gccaacctgg tgatcgtggc tggcgccaag 900 tctggcggcc agaagtctga cgagctgaag atctataact gctgttcttc tgactgcaac 960 cccgtgactc tgaagaagat catcaaggaa ttcaccgagg acaccatcaa gaacaagtct 1020 cacatcatgc ctctgcctgg ctggttcgtg ttcaccaagt acaagtggct gctgaccctc 1080 ctgaccatca tcttccagat gctgcccatg tacctggccg acgtgtaccg agtcctgacc 1140 ggcaagattc cccggtacat gaagctgcac cacctggtca ttcagacccg actgggaatc 1200 gacttcttca cctctcactc ttgggtgatg aagaccgacc gagtgcgaga gctgttcggc 1260 tctctgtctc tggccgagaa gcacatgttc ccttgcgacc cctcttccat cgactggacc 1320 gactacctgc agtcttactg ctacggcgtg cgacgattcc tggaaaagaa gaagtag 1377 <210> 10 <211> 458 <212> PRT <213> Yellow ground tiger (Agrotis segetum) <220> <221> Unclassified features <222> (1)..(458) <223> Yellow cutworm fatty acyl reductase (AGP26039) <400> 10 Met Pro Val Leu Thr Ser Arg Glu Asp Glu Lys Leu Ser Val Pro Glu 1 5 10 15 Phe Tyr Ala Gly Lys Ser Ile Phe Val Thr Gly Gly Thr Gly Phe Leu 20 25 30 Gly Lys Val Phe Ile Glu Lys Leu Leu Tyr Cys Cys Pro Asp Ile Asp 35 40 45 Lys Ile Tyr Met Leu Ile Arg Glu Lys Lys Asn Leu Ser Ile Asp Glu 50 55 60 Arg Met Ser Lys Phe Leu Asp Asp Pro Leu Phe Ser Arg Leu Lys Glu 65 70 75 80 Glu Arg Pro Gly Asp Leu Glu Lys Ile Val Leu Ile Pro Gly Asp Ile 85 90 95 Thr Ala Pro Asn Leu Gly Leu Ser Ala Glu Asn Glu Arg Ile Leu Leu 100 105 110 Glu Lys Val Ser Val Ile Ile Asn Ser Ala Ala Thr Val Lys Phe Asn 115 120 125 Glu Pro Leu Pro Ile Ala Trp Lys Ile Asn Val Glu Gly Thr Arg Met 130 135 140 Leu Leu Ala Leu Ser Arg Arg Met Lys Arg Ile Glu Val Phe Ile His 145 150 155 160 Ile Ser Thr Ala Tyr Ser Asn Ala Ser Ser Asp Arg Ile Val Val Asp 165 170 175 Glu Ile Leu Tyr Pro Ala Pro Ala Asp Met Asp Gln Val Tyr Gln Leu 180 185 190 Val Lys Asp Gly Val Thr Glu Glu Glu Thr Glu Arg Leu Leu Asn Gly 195 200 205 Leu Pro Asn Thr Tyr Thr Phe Thr Lys Ala Leu Thr Glu His Leu Val 210 215 220 Ala Glu His Gln Thr Tyr Val Pro Thr Ile Ile Ile Arg Pro Ser Val 225 230 235 240 Val Ala Ser Ile Lys Asp Glu Pro Ile Arg Gly Trp Leu Cys Asn Trp 245 250 255 Phe Gly Ala Thr Gly Ile Ser Val Phe Thr Ala Lys Gly Leu Asn Arg 260 265 270 Val Leu Leu Gly Lys Ala Ser Asn Ile Val Asp Val Ile Pro Val Asp 275 280 285 Tyr Val Ala Asn Leu Val Ile Val Ala Gly Ala Lys Ser Gly Gly Gln 290 295 300 Lys Ser Asp Glu Leu Lys Ile Tyr Asn Cys Cys Ser Ser Asp Cys Asn 305 310 315 320 Pro Val Thr Leu Lys Lys Ile Ile Lys Glu Phe Thr Glu Asp Thr Ile 325 330 335 Lys Asn Lys Ser His Ile Met Pro Leu Pro Gly Trp Phe Val Phe Thr 340 345 350 Lys Tyr Lys Trp Leu Leu Thr Leu Leu Thr Ile Ile Phe Gln Met Leu 355 360 365 Pro Met Tyr Leu Ala Asp Val Tyr Arg Val Leu Thr Gly Lys Ile Pro 370 375 380 Arg Tyr Met Lys Leu His His Leu Val Ile Gln Thr Arg Leu Gly Ile 385 390 395 400 Asp Phe Phe Thr Ser His Ser Trp Val Met Lys Thr Asp Arg Val Arg 405 410 415 Glu Leu Phe Gly Ser Leu Ser Leu Ala Glu Lys His Met Phe Pro Cys 420 425 430 Asp Pro Ser Ser Ile Asp Trp Thr Asp Tyr Leu Gln Ser Tyr Cys Tyr 435 440 445 Gly Val Arg Arg Phe Leu Glu Lys Lys Lys 450 455 <210> 11 <211> 1368 <212> DNA <213> Artificial sequence <220> <223> Codon-optimized fatty acyl reductase from *Yarrowia lipolyticis* (ATJ44471); mRNA coding sequence <220> <221> Unclassified features <222> (1)..(1368) <223> Codon-optimized fatty acyl reductase from *Yarrowia lipolyticis* (ATJ44471); mRNA coding sequence <400> 11 atggtggtcc tgacctctaa ggagactaag ccctccgtgg ccgagttcta cgctggcaag 60 tctgtcttca tcaccggcgg aaccggtttc ctgggcaagg tcttcattga gaagctgctg 120 tactcctgtc ccgacatcgg caacatctac atgctgatcc gagagaagaa gggactgtct 180 gtgtccgagc gaattaagca cttcctggac gaccccctgt tcacccgact gaaggagaag 240 cgacccgccg acctggagaa gatcgtgctg attcccggag acatcaccgc tcccgacctg 300 ggtattacct ctgagaacga gaagatgctg atcgagaagg tgtctgtcat cattcactcc 360 gccgctaccg tcaagttcaa cgagcccctg cccaccgcct ggaagatcaa cgtggaggga 420 acccgaatga tgctggctct gtctcgacga atgaagcgaa ttgaggtctt catccacatt 480 tccaccgcct acaccaacac caaccgagag gtggtggacg agatcctgta ccctgctcct 540 gctgacattg accaggtgca ccgatacgtc aaggacggta tctctgagga agagactgag 600 aagattctga acggccgacc caacacctac accttcacca aggccctgac cgagcacctg 660 gtggctgaga accaggctta cgtgcccacc atcattgtcc gaccctccgt ggtcgccgct 720 atcaaggacg agcccattaa gggatggctg ggtaactggt acggagctac cggactgacc 780 gtgttcaccg ctaagggtct gaaccgagtc atctacggcc actcttccaa catcgtggac 840 ctgattcccg tggactacgt cgccaacctg gtcattgccg ctggcgctaa gtcttccaag 900 tccaccgagc tgaaggtgta caactgttgc tcttccgcct gcaaccccat caccattgga 960 aagctgatgt ctatgttcgc cgaggacgct atcaagcaga agtcctacgc tatgcccctg 1020 cccggttggt acatcttcac caagtacaag tggctggtcc tgctgctgac cattctgttc 1080 caggtcatcc ccgcctacat taccgacctg taccgacacc tgatcggcaa gaacccccga 1140 tacattaagc tgcagtctct ggtcaaccag acccgatctt ccattgactt cttcacctct 1200 cactcctggg tcatgaaggc tgaccgagtc cgagagctgt tcgcctctct gtcccccgct 1260 gacaagtacc tgttcccctg tgaccccacc gacatcaact ggacccacta cattcaggac 1320 tactgctggg gagtgcgaca cttcctggag aagaagtcct acgagtag 1368 <210> 12 <211> 456 <212> PRT <213> Helicoverpa armigera <220> <221> Unclassified features <222> (1)..(456) <223> Cotton bollworm fatty acyl reductase (ATJ44471) <400> 12 Met Val Val Leu Thr Ser Lys Glu Thr Lys Pro Ser Val Ala Glu Phe 1 5 10 15 Tyr Ala Gly Lys Ser Val Phe Ile Thr Gly Gly Thr Gly Phe Leu Gly 20 25 30 Lys Val Phe Ile Glu Lys Leu Leu Tyr Ser Cys Pro Asp Ile Gly Asn 35 40 45 Ile Tyr Met Leu Ile Arg Glu Lys Lys Gly Leu Ser Val Ser Glu Arg 50 55 60 Ile Lys Gln Phe Leu Asp Asp Pro Leu Phe Thr Arg Leu Lys Glu Lys 65 70 75 80 Arg Pro Ala Asp Leu Glu Lys Ile Val Leu Ile Pro Gly Asp Ile Thr 85 90 95 Ala Pro Asp Leu Gly Ile Thr Ser Glu Asn Glu Lys Ile Leu Ile Glu 100 105 110 Lys Val Ser Val Ile Ile His Ser Ala Ala Thr Val Lys Phe Asn Glu 115 120 125 Pro Leu Pro Thr Ala Trp Lys Ile Asn Val Glu Gly Thr Arg Met Met 130 135 140 Leu Ala Leu Ser Arg Arg Met Lys Arg Ile Glu Val Phe Ile His Ile 145 150 155 160 Ser Thr Ala Tyr Thr Asn Thr Asn Arg Glu Val Val Asp Glu Ile Leu 165 170 175 Tyr Pro Ala Pro Ala Asp Ile Asp Gln Val His Gln Tyr Val Lys Asp 180 185 190 Gly Ile Ser Glu Glu Glu Thr Glu Lys Ile Leu Asn Gly Arg Pro Asn 195 200 205 Thr Tyr Thr Phe Thr Lys Ala Leu Thr Glu His Leu Val Ala Glu Asn 210 215 220 Gln Ala Tyr Val Pro Thr Ile Ile Val Arg Pro Ser Val Val Ala Ala 225 230 235 240 Ile Lys Asp Glu Pro Ile Lys Gly Trp Leu Gly Asn Trp Tyr Gly Ala 245 250 255 Thr Gly Leu Thr Val Phe Thr Ala Lys Gly Leu Asn Arg Val Ile Tyr 260 265 270 Gly His Ser Ser Asn Ile Val Asp Leu Ile Pro Val Asp Tyr Val Ala 275 280 285 Asn Leu Val Ile Ala Ala Gly Ala Lys Ser Ser Lys Ser Thr Asp Leu 290,295,300 Lys Val Tyr Asn Cys Ser Ser Ala Cys Asn Pro Ile Thr Ile Gly 305 310 315 320 Lys Leu Met Ser Met Phe Ala Glu Asp Ala Ile Lys Gln Lys Ser Tyr 325 330 335 Only Met Pro Leu Pro Gly Trp Tyr Ile Phe Thr Lys Tyr Lys Trp Leu 340 345 350 Val Leu Leu Thr And Leu Phe Gln Val Leu Pro Ala Tyr Thr 355 360 365 Asp Leu Tyr Arg His Lew With Gly Lys Asn Pro Arg Tyr With Lew 370 375 380 Gln Ser Leu Val Asn Gln Thr Arg Ser Ser Ile Asp Phe Phe Thr Ser 385 390 395 400 His Ser Trp Val Met Lys Ala Asp Arg Val Arg Glu Leu Phe Ala Ser 405 410 415 Leu Ser Pro Ala Asp Lys...

Claims

1. A kind of product capable of producing E 8, E Yeast cells containing 10-dodecadienyl coenzyme A, wherein the yeast cells express at least one heterologous desaturase capable of introducing one or more double bonds into a 12-carbon acyl-CoA fatty acid chain, thereby converting the fatty acid-CoA into desaturated fatty acid-CoA, wherein at least a portion of the desaturated fatty acid-CoA is E 8, E 10-Dodecadienylcoenzyme A, wherein: a) The at least one heterologous desaturase is Cpo_CPRQ with the sequence SEQ ID NO: 2; b) The at least one heterologous desaturase is a mutant of Cpo_CPRQ with the sequence SEQ ID NO: 2, wherein the S at position 85 is mutated to A; c) The at least one heterologous desaturase is at least two desaturases, wherein at least one of the two desaturases is Cpo_CPRQ with the sequence SEQ ID NO: 2; and the other of the two desaturases is a desaturase capable of introducing at least one double bond into a fatty acyl-CoA with a carbon chain length of 12. d) The at least one heterologous desaturase is at least two desaturases, wherein at least one of the two desaturases is a mutant of Cpo_CPRQ with the sequence SEQ ID NO: 2, wherein the S at position 85 is mutated to A; and the other of the two desaturases is a desaturase capable of introducing at least one double bond into a fatty acyl-CoA with a carbon chain length of 12; or e) The at least one heterologous desaturase is at least two desaturases, wherein at least one of the two desaturases is Cpo_CPRQ with the sequence SEQ ID NO: 2; and the other of the two desaturases is a mutant of Cpo_CPRQ with the sequence SEQ ID NO: 2 capable of introducing at least one double bond into a fatty acyl-CoA with a carbon chain length of 12, wherein the S at position 85 of the mutant is mutated to A.

2. The yeast cell according to claim 1, wherein the other desaturase is Z9-12 desaturase and / or E 9-12 Desaturase.

3. The yeast cell according to claim 1, wherein the other desaturase is Z9-12 desaturase.

4. The yeast cell according to claim 1, wherein the at least one heterologous desaturase is at least two desaturases, wherein the other desaturase is Cpo_NPVE with the sequence SEQ ID NO: 67 or Cpo_SPTQ with the sequence SEQ ID NO:

69.

5. The yeast cell according to claim 1, wherein the yeast cell belongs to the following genera: *Blavus*, *Candida*, *Cryptococcus*, *Clostridium*, *Oleoma*, *Morchella*, *Mucor*, *Pythium*, *Rhodotorula*, *Rhodotorula*, *Hydrocotyle*, *Yeast*, or *Yersinia*.

6. The yeast cell according to claim 1, wherein the yeast cell belongs to the following species: *Blancium trispora*, *Candida albicans*, ... C. revkaufi Tropical Candida, Cryptococcus curviflora, Microsporum cristatum, Microsporum davidii, Microsporum davidii, Microsporum davidii, S. davidii, S. davidii, M. alpineus, M. davidii, M. ramanii, M. gracilis, Mucor, Brackenella, Pythium terrestris, Rhodotorula buergerianum, Rhodotorula glutinis, Rhodotorula glutinis, Rhodotorula gracilis, Rhodotorula glutinis, Rhodotorula gracilis, Rhodotorula glutinis R. pinicola , Pullulania, Dermoxymycota, Saccharomyces cerevisiae or Yersinia lipolytica.

7. The yeast cell according to claim 1, wherein the yeast cell is a Yeast lipolytica cell or a Saccharomyces cerevisiae cell.

8. The yeast cell according to claim 1, wherein the yeast cell is capable of producing E 8, E 10-Dodecadien-1-ol, the yeast cells also express at least one heterologous fatty acyl-CoA reductase EC 1.2.1.84 capable of converting at least a portion of the desaturated fatty acyl-CoA into desaturated fatty alcohol, wherein the fatty acyl-CoA reductase is capable of converting at least a portion of the... E 8, E 10-Dodecadienyl Coenzyme A is converted to E 8, E 10-Dodecadien-1-ol.

9. The yeast cell according to claim 8, wherein the fatty acyl-CoA reductase is Ase_FAR with the sequence of SEQ ID NO: 10, Aip_FAR with the sequence of SEQ ID NO: 61, Hs_FAR with the sequence of SEQ ID NO: 71, Has_FAR with the sequence of SEQ ID NO: 73, Hv_FAR with the sequence of SEQ ID NO: 75, Har_FAR with the sequence of SEQ ID NO: 12, or Cpo_FAR with the sequence of SEQ ID NO:

76.

10. The yeast cell according to claim 9, wherein the fatty acyl-CoA reductase is a mutant of Ase_FAR.

11. The yeast cell of claim 10, wherein the Ase_FAR mutant has a mutation at position 198 or 413.

12. The yeast cell of claim 11, wherein the Ase_FAR mutant has a T198A mutation or an S413A mutation.

13. The yeast cell according to claim 1, further comprising one or more of the following: - Expressing heterologous cytochrome b5, - Expresses heterologous cytochrome b5 reductase EC 1.6.2.

2. - Expressing hemoglobin, - Mutations in one or more genes encoding elongase that result in partial or complete loss of elongase activity. - Mutations that include one or more genes encoding thioesterases that result in partial or complete loss of thioesterase activity. - Contains at least one mutation that reduces the activity of at least one of Hfd1, Hfd2, Hfd3, Hfd4, Fao1, GPAT, and Pex10. - Expressing a fatty acyl synthase variant having a modified ketone synthase domain, wherein the fatty acyl synthase variant is a variant of Fas1 with the sequence SEQ ID NO: 16 or a variant of Fas2 with the sequence SEQ ID NO:

18. - Expressing thioesterase, - Expresses a fusion protein of truncated fatty acyl synthase and truncated thioesterase.

14. The yeast cell of claim 13, wherein the heterologous cytochrome b5 is cytochrome b5 derived from a lepidopteran species.

15. The yeast cell according to claim 14, wherein the heterologous cytochrome b5 is cytochrome b5 derived from the cotton bollworm.

16. The yeast cell according to claim 14, wherein the heterologous cytochrome b5 is cytochrome b5 HarCyb5 with the sequence SEQ ID NO:

4.

17. The yeast cell of claim 13, wherein the heterologous cytochrome b5 reductase EC 1.6.2.2 is a cytochrome b5 reductase derived from a lepidopteran species.

18. The yeast cell of claim 17, wherein the heterologous cytochrome b5 reductase EC 1.6.2.2 is derived from the cotton bollworm.

19. The yeast cell according to claim 17, wherein the heterologous cytochrome b5 reductase EC 1.6.2.2 is a cytochrome b5 reductase derived from cotton bollworm with the sequence SEQ ID NO:

24.

20. The yeast cell of claim 13, wherein the hemoglobin is hemoglobin derived from *Vibrio fecalis*.

21. The yeast cell of claim 20, wherein the hemoglobin is the hemoglobin of *Vibrio fecalis* with the sequence SEQ ID NO:

6.

22. The yeast cell of claim 13, wherein the mutation in one or more genes encoding the elongase results in partial or complete loss of Elo1 activity. ELO1 Gene mutation, the ELO1 The gene sequence is SEQ ID NO:

13.

23. The yeast cell of claim 22, wherein the mutation is a deletion.

24. The yeast cell of claim 13, wherein the mutation in one or more genes encoding thioesterase is the sequence of SEQ ID NO:

19. YAL10_F14729g The gene mutation, sequence number SEQ ID NO: 54 YALI0_ E18876g The gene mutation or sequence is SEQ ID NO:

55. YALI0_D03597g Mutations.

25. The yeast cell of claim 24, wherein the mutation is a deletion.

26. The yeast cell of claim 13, wherein the fatty acyl synthase variant is a mutant Fas1 with a mutation at position 123, or a mutant Fas2 with a mutation at position 1220.

27. The yeast cell of claim 26, wherein the mutation at position 123 is the L123V mutation.

28. The yeast cell of claim 26, wherein the mutation at position 1220 is an I1220F or I1220W mutation.

29. The yeast cell according to claim 13, wherein the thioesterase is a heterothioesterase.

30. The yeast cell of claim 29, wherein the thioesterase is expressed at a high level.

31. The yeast cell according to claim 29, wherein the thioesterase is a thioesterase from *Euphorbia tirucalli* with the sequence SEQ ID NO: 33, a thioesterase from *Euphorbia tirucalli* with the sequence SEQ ID NO: 57, a thioesterase from *Cinnamomum camphora* with the sequence SEQ ID NO: 35, or a thioesterase from *Escherichia coli* with the sequence SEQ ID NO:

26.

32. The yeast cell according to claim 29, wherein the thioesterase is a thioesterase derived from camphor tree with the sequence SEQ ID NO: 35 or a thioesterase derived from Escherichia coli with the sequence SEQ ID NO:

26.

33. The yeast cell according to claim 13, wherein the sequence of the fusion protein is SEQ ID NO:

59.

34. The yeast cell of claim 1, further comprising at least one mutation that reduces the activity of at least one of Hfd1, Hfd2, Hfd3, Hfd4, Fao1, GPAT and Pex10.

35. The yeast cell of claim 1, wherein the yeast cell is capable of producing a titer of at least 0.5 mg / L. E 8, E 10-Dodecadien-1-ol.

36. The yeast cell of claim 35, wherein the titer is at least 0.6 mg / L.

37. The yeast cell of claim 35, wherein the titer is at least 0.7 mg / L.

38. The yeast cell of claim 35, wherein the titer is at least 0.8 mg / L.

39. The yeast cells of claim 35, wherein the titer is at least 0.9 mg / L.

40. The yeast cell of claim 35, wherein the titer is at least 1 mg / L.

41. The yeast cells of claim 35, wherein the titer is at least 1.5 mg / L.

42. The yeast cells of claim 35, wherein the titer is at least 2.5 mg / L.

43. The yeast cells of claim 35, wherein the titer is at least 5.0 mg / L.

44. The yeast cells of claim 35, wherein the titer is at least 10 mg / L.

45. The yeast cells of claim 35, wherein the titer is at least 15 mg / L.

46. ​​The yeast cells of claim 35, wherein the titer is at least 20 mg / L.

47. The yeast cells of claim 35, wherein the titer is at least 25 mg / L.

48. The yeast cells of claim 35, wherein the titer is at least 50 mg / L.

49. The yeast cells of claim 35, wherein the titer is at least 100 mg / L.

50. The yeast cells of claim 35, wherein the titer is at least 250 mg / L.

51. The yeast cells of claim 35, wherein the titer is at least 500 mg / L.

52. The yeast cells of claim 35, wherein the titer is at least 750 mg / L.

53. The yeast cell of claim 35, wherein the titer is at least 1 g / L.

54. The yeast cell of claim 35, wherein the titer is at least 2 g / L.

55. The yeast cell of claim 35, wherein the titer is at least 3 g / L.

56. The yeast cell of claim 35, wherein the titer is at least 4 g / L.

57. The yeast cell of claim 35, wherein the titer is at least 5 g / L.

58. The yeast cell of claim 35, wherein the titer is at least 6 g / L.

59. The yeast cell of claim 35, wherein the titer is at least 7 g / L.

60. The yeast cell of claim 35, wherein the titer is at least 8 g / L.

61. The yeast cell of claim 35, wherein the titer is at least 9 g / L.

62. The yeast cell of claim 35, wherein the titer is at least 10 g / L.

63. The yeast cell of claim 8, wherein the yeast cell further expresses an expression capable of at least partially expressing the... E 8, E 10-Dodecadien-1-ol converted to E 8, E Acetyltransferase EC2.3.1.84 of 10-dodecadienyl acetate, thereby enabling the yeast cells to produce E 8, E 10-Dodecadienylacetate.

64. The yeast cell of claim 63, wherein the acetyltransferase is a heterologous acetyltransferase (AcT) expressed from the yeast cell or a natural acetyltransferase overexpressed from the yeast cell.

65. The yeast cell according to claim 63, wherein the acetyltransferase is Sc_Atf1 with the sequence SEQ ID NO:

37.

66. The yeast cell of claim 8, wherein the yeast cell further expresses an expression capable of at least partially expressing the... E 8, E 10-Dodecadien-1-ol converted to E 8, E Aldehyde-forming fatty acyl-CoA reductase EC1.2.1.50, alcohol dehydrogenase EC1.1.1.2 and / or fatty alcohol oxidase EC1.1.3.20 for 10-dodecadienal.

67. The yeast cell according to claim 1, wherein the yeast cell further comprises: i) Having one or more mutations that result in reduced activity of one or more natural acyl-CoA oxidases; and ii) Expressing at least one set of enzymes comprising at least one acyl-CoA oxidase capable of oxidizing fatty acyl-CoA, wherein the set of enzymes is capable of shortening fatty acyl-CoA of a first carbon chain length X to a shortened fatty acyl-CoA having a second carbon chain length X', wherein X' ≤ X-2.

68. The yeast cell according to claim 67, wherein X' = 12.

69. The yeast cell of claim 67, further expressing a desaturase capable of introducing at least one double bond into fatty acyl-CoA of said carbon chain length X.

70. The yeast cell according to claim 69, wherein the desaturase is CroZ11 desaturase with sequence SEQ ID NO: 63 or CpaE11 desaturase with sequence SEQ ID NO:

65.

71. A method for producing in yeast cells E 8, E A method for 10-dodecadienyl coenzyme A, the method comprising the steps of providing yeast cells and incubating the yeast cells in a culture medium, wherein the yeast cells express: i) at least one heterologous desaturase capable of introducing one or more double bonds into a fatty acyl-CoA with a carbon chain length of 12, thereby converting said fatty acyl-CoA into desaturated fatty acyl-CoA, wherein at least a portion of said desaturated fatty acyl-CoA is E 8, E 10-Dodecadienylcoenzyme A; wherein: a) The at least one heterologous desaturase is Cpo_CPRQ with the sequence SEQ ID NO: 2; b) The at least one heterologous desaturase is a mutant of Cpo_CPRQ with the sequence SEQ ID NO: 2, wherein the S at position 85 is mutated to A; c) The at least one heterologous desaturase is at least two desaturases, wherein at least one of the two desaturases is Cpo_CPRQ with the sequence SEQ ID NO: 2; and the other of the two desaturases is a desaturase capable of introducing at least one double bond into a fatty acyl-CoA with a carbon chain length of 12. d) The at least one heterologous desaturase is at least two desaturases, wherein at least one of the two desaturases is a mutant of Cpo_CPRQ with the sequence SEQ ID NO: 2, wherein the S at position 85 is mutated to A; and the other of the two desaturases is a desaturase capable of introducing at least one double bond into a fatty acyl-CoA with a carbon chain length of 12; or e) The at least one heterologous desaturase is at least two desaturases, wherein at least one of the two desaturases is Cpo_CPRQ with the sequence SEQ ID NO: 2; and the other of the two desaturases is a mutant of Cpo_CPRQ with the sequence SEQ ID NO: 2, capable of introducing at least one double bond into a 12-carbon acyl-CoA fatty acid chain, wherein the S at position 85 of the mutant is mutated to A. Thus generating E 8, E 10-Dodecadienylcoenzyme A.

72. The method of claim 71, wherein the yeast cells further express at least one heterologous fatty acyl-CoA reductase EC 1.2.1.84, which is capable of converting at least a portion of the desaturated fatty acyl-CoA into desaturated fatty alcohols, wherein the fatty acyl-CoA reductase is capable of converting at least a portion of the... E 8, E 10-Dodecadienyl Coenzyme A is converted to E 8, E 10-Dodecadien-1-ol, thereby producing E 8, E 10-Dodecadien-1-ol.

73. The method according to claim 71, wherein the other desaturase is a Z9-12 desaturase and / or an E9-12 desaturase.

74. The method according to claim 71, wherein the other desaturase is a Z9-12 desaturase.

75. The method of claim 71, wherein the method further comprises: E 8, E 10-Dodecadienylcoenzyme A is converted into lipids or free fatty acids, the lipids or free fatty acids are recovered, and the lipids or free fatty acids are converted into... E 8, E The steps for 10-dodecadien-1-ol.

76. The method of claim 75, wherein the lipid is a triglyceride.

77. The method of claim 72, wherein the method further comprises recycling the E 8, E The steps for 10-dodecadien-1-ol.

78. The method of claim 71, wherein the yeast cell is defined as any one of claims 2 to 44.

79. The method of claim 72, further comprising the step of: At least part of the above can be expressed by acetyltransferase or by chemical transformation. E 8, E 10-Dodecadien-1-ol converted to E 8, E 10-Dodecadienyl acetate, thereby further producing E 8, E 10-Dodecadienylacetate.

80. The method of claim 79, further comprising recycling the E 8, E The steps for 10-dodecadienyl acetate.

81. The method of claim 72, further comprising expressing the ability to at least partially [describe / transmit / distribute] the [method / process]. E 8, E 10-Dodecadien-1-ol converted to E 8, E Aldehyde formation of 10-dodecadienal using fatty acyl-CoA reductase EC 1.2.1.50, alcohol dehydrogenase EC 1.1.1.2, and / or fatty alcohol oxidase EC 1.1.3.20, or by chemical conversion, at least partially comprising the aforementioned enzymes. E 8, E 10-Dodecadien-1-ol converted to E 8, E The step of 10-dodecadienal, thereby further producing E 8, E 10-Dodecadienal.

82. The method of claim 81, further comprising recycling the... E 8, E The steps for 10-dodecadienal.

83. The method of claim 75, further comprising expressing the ability to at least partially [describe / transmit / distribute] the [method / process]. E 8, E 10-Dodecadien-1-ol converted to E 8, E Aldehyde formation of 10-dodecadienal using fatty acyl-CoA reductase EC 1.2.1.50, alcohol dehydrogenase EC 1.1.1.2, and / or fatty alcohol oxidase EC 1.1.3.20, or by chemical conversion, at least partially comprising the aforementioned enzymes. E 8, E 10-Dodecadien-1-ol converted to E 8, E The step of 10-dodecadienal, thereby further producing E 8, E 10-Dodecadienal.

84. The method of claim 83, further comprising recycling the... E 8, E The steps for 10-dodecadienal.

85. The method of claim 71, wherein the culture medium comprises an amount of an extractant equal to or greater than its turbidity concentration in an aqueous solution, wherein the extractant is a nonionic ethoxylated surfactant, and wherein the turbidity concentration refers to the concentration of the surfactant in the solution above which, at a given temperature, the mixture of the surfactant and the solution begins to separate into two phases and thus becomes turbid.

86. The method of claim 85, wherein the aqueous solution is a culture medium at the culture temperature.

87. The method of claim 85, wherein the nonionic ethoxylated surfactant is an antifoaming agent or a polyethoxylated surfactant.

88. The method of claim 85, wherein the nonionic ethoxylated surfactant is: a mixture of polyoxyethylene polyoxypropylene ether, a polyether dispersion, an antifoaming agent comprising polyethylene glycol monostearate, a fatty alcohol alkoxylate, a polyethoxylated surfactant, or ethoxylated and propoxylated C 16 -C 18 Alcohol-based defoamers or combinations thereof.

89. The method of claim 88, wherein the defoamer comprising polyethylene glycol monostearate is dimethicone.

90. The method of claim 85, wherein the culture medium comprises an amount of extractant greater than at least 50% of its turbidity concentration, and / or wherein the culture medium comprises an amount of extractant at least twice its turbidity concentration.

91. The method of claim 90, wherein the culture medium comprises an amount of extractant greater than its turbidity concentration of at least 100%, wherein the turbidity concentration is measured in the culture medium.

92. The method of claim 90, wherein the culture medium comprises an amount of extractant greater than its turbidity concentration by at least 150%, wherein the turbidity concentration is measured in the culture medium.

93. The method of claim 90, wherein the culture medium comprises an amount of extractant greater than its turbidity concentration by at least 200%, wherein the turbidity concentration is measured in the culture medium.

94. The method of claim 90, wherein the culture medium comprises an amount of extractant greater than its turbidity concentration by at least 250%, wherein the turbidity concentration is measured in the culture medium.

95. The method of claim 90, wherein the culture medium comprises an amount of extractant greater than its turbidity concentration by at least 300%, wherein the turbidity concentration is measured in the culture medium.

96. The method of claim 90, wherein the culture medium comprises an amount of extractant greater than its turbidity concentration by at least 350%, wherein the turbidity concentration is measured in the culture medium.

97. The method of claim 90, wherein the culture medium comprises an amount of extractant greater than its turbidity concentration of at least 400%, wherein the turbidity concentration is measured in the culture medium.

98. The method of claim 90, wherein the culture medium comprises an amount of extractant greater than its turbidity concentration by at least 500%, wherein the turbidity concentration is measured in the culture medium.

99. The method of claim 90, wherein the culture medium comprises an amount of extractant greater than at least 750% of its turbidity concentration, wherein the turbidity concentration is measured in the culture medium.

100. The method of claim 90, wherein the culture medium comprises an amount of extractant greater than its turbidity concentration of at least 1000%, wherein the turbidity concentration is measured in the culture medium.

101. The method of claim 90, wherein the culture medium comprises an extractant in an amount at least three times its turbidity concentration, wherein the turbidity concentration is measured in the culture medium.

102. The method of claim 90, wherein the culture medium comprises an extractant in an amount at least four times its turbidity concentration, wherein the turbidity concentration is measured in the culture medium.

103. The method of claim 90, wherein the culture medium comprises an extractant in an amount at least five times its turbidity concentration, wherein the turbidity concentration is measured in the culture medium.

104. The method of claim 90, wherein the culture medium comprises an extractant in an amount at least six times its turbidity concentration, wherein the turbidity concentration is measured in the culture medium.

105. The method of claim 90, wherein the culture medium comprises an extractant in an amount at least seven times its turbidity concentration, wherein the turbidity concentration is measured in the culture medium.

106. The method of claim 90, wherein the culture medium comprises an extractant in an amount at least eight times its turbidity concentration, wherein the turbidity concentration is measured in the culture medium.

107. The method of claim 90, wherein the culture medium comprises an extractant in an amount at least nine times its turbidity concentration, wherein the turbidity concentration is measured in the culture medium.

108. The method of claim 90, wherein the culture medium comprises an extractant in an amount at least 10 times its turbidity concentration, wherein the turbidity concentration is measured in the culture medium.

109. The method of claim 90, wherein the culture medium comprises an extractant in an amount at least 12.5 times its turbidity concentration, wherein the turbidity concentration is measured in the culture medium.

110. The method of claim 90, wherein the culture medium comprises an extractant in an amount at least 15 times its turbidity concentration, wherein the turbidity concentration is measured in the culture medium.

111. The method of claim 90, wherein the culture medium comprises an extractant in an amount at least 17.5 times its turbidity concentration, wherein the turbidity concentration is measured in the culture medium.

112. The method of claim 90, wherein the culture medium comprises an extractant in an amount at least 20 times its turbidity concentration, wherein the turbidity concentration is measured in the culture medium.

113. The method of claim 90, wherein the culture medium comprises an extractant in an amount at least 25 times its turbidity concentration, wherein the turbidity concentration is measured in the culture medium.

114. The method of claim 90, wherein the culture medium comprises an extractant in an amount at least 30 times its turbidity concentration, wherein the turbidity concentration is measured in the culture medium.

115. The method of claim 90, wherein the turbidity concentration is measured at the culture temperature.

116. The method of claim 85, further comprising: E 8, E The step of converting 10-dodecadienyl coenzyme A into lipids or free fatty acids, wherein the lipids or free fatty acids produced by the yeast cells or the... E 8, E 10-Dodecadien-1-ol is present in an emulsion in the fermentation broth. The method further includes the step of disrupting the emulsion to obtain a composition comprising a product phase, the product phase comprising the extractant and the lipid or free fatty acid or the... E 8, E 10-Dodecadien-1-ol.

117. The method of claim 116, wherein the yeast cells produce the... E 8, E 10-Dodecadienylacetate and / or the aforementioned E 8, E 10-Dodecadienal is present in an emulsion in the fermentation broth. The method further includes the step of disrupting the emulsion to obtain a composition comprising a product phase, the product phase comprising the extractant and the... E 8, E 10-Dodecadienylacetate and / or the aforementioned E 8, E 10-Dodecadienal.

118. The method of claim 116, wherein the step of disrupting the emulsion comprises or consists of the following: The phase separation step of the fermentation broth yields a composition consisting of three phases: an aqueous phase, a phase containing cells and cell debris, and a product phase comprising the extractant and the lipids or free fatty acids. E 8, E 10-Dodecadien-1-ol.

119. The method of claim 118, wherein the phase separation step is a centrifugation step.

120. The method of claim 118, wherein the product phase comprises E 8, E 10-Dodecadienylacetate and / or E 8, E 10-Dodecadienal.

121. The method of claim 116, wherein the product phase comprises the lipids or free fatty acids initially present in the fermentation broth and / or E 8, E At least 50% of 10-dodecadien-1-ol.

122. The method of claim 121, wherein the product phase comprises at least 50% of the product initially present in the fermentation broth. E 8, E 10-Dodecadienylacetate and / or the aforementioned E 8, E 10-Dodecadienal.

123. The method of claim 121, wherein the product phase comprises at least 55% of the fat or free fatty acids initially present in the fermentation broth and / or E 8, E 10-Dodecadien-1-ol.

124. The method of claim 121, wherein the product phase comprises at least 60% of the fat or free fatty acids initially present in the fermentation broth and / or E 8, E 10-Dodecadien-1-ol.

125. The method of claim 121, wherein the product phase comprises at least 65% of the fat or free fatty acids initially present in the fermentation broth and / or E 8, E 10-Dodecadien-1-ol.

126. The method of claim 121, wherein the product phase comprises at least 70% of the fat or free fatty acids initially present in the fermentation broth and / or E 8, E 10-Dodecadien-1-ol.

127. The method of claim 121, wherein the product phase comprises at least 75% of the fat or free fatty acids initially present in the fermentation broth and / or E 8, E 10-Dodecadien-1-ol.

128. The method of claim 121, wherein the product phase comprises at least 80% of the fat or free fatty acids initially present in the fermentation broth and / or E 8, E 10-Dodecadien-1-ol.

129. The method of claim 121, wherein the product phase comprises at least 85% of the fat or free fatty acids initially present in the fermentation broth and / or E 8, E 10-Dodecadien-1-ol.

130. The method of claim 121, wherein the product phase comprises at least 90% of the fat or free fatty acids initially present in the fermentation broth and / or E 8, E 10-Dodecadien-1-ol.

131. The method of claim 121, wherein the product phase comprises at least 95% of the fat or free fatty acids initially present in the fermentation broth and / or E 8, E 10-Dodecadien-1-ol.

132. The method of claim 121, wherein the product phase comprises at least 55% of the product initially present in the fermentation broth. E 8, E 10-Dodecadienylacetate and / or the aforementioned E 8, E 10-Dodecadienal.

133. The method of claim 121, wherein the product phase comprises at least 60% of the product initially present in the fermentation broth. E 8, E 10-Dodecadienylacetate and / or the aforementioned E 8, E 10-Dodecadienal.

134. The method of claim 121, wherein the product phase comprises at least 65% of the product initially present in the fermentation broth. E 8, E 10-Dodecadienylacetate and / or the aforementioned E 8, E 10-Dodecadienal.

135. The method of claim 121, wherein the product phase comprises at least 70% of the product initially present in the fermentation broth. E 8, E 10-Dodecadienylacetate and / or the aforementioned E 8, E 10-Dodecadienal.

136. The method of claim 121, wherein the product phase comprises at least 75% of the product initially present in the fermentation broth. E 8, E 10-Dodecadienylacetate and / or the aforementioned E 8, E 10-Dodecadienal.

137. The method of claim 121, wherein the product phase comprises at least 80% of the product initially present in the fermentation broth. E 8, E 10-Dodecadienylacetate and / or the aforementioned E 8, E 10-Dodecadienal.

138. The method of claim 121, wherein the product phase comprises at least 85% of the product initially present in the fermentation broth. E 8, E 10-Dodecadienylacetate and / or the aforementioned E 8, E 10-Dodecadienal.

139. The method of claim 121, wherein the product phase comprises at least 90% of the product initially present in the fermentation broth. E 8, E 10-Dodecadienylacetate and / or the aforementioned E 8, E 10-Dodecadienal.

140. The method of claim 121, wherein the product phase comprises at least 95% of the product initially present in the fermentation broth. E 8, E 10-Dodecadienylacetate and / or the aforementioned E 8, E 10-Dodecadienal.

141. The method of claim 116, further comprising the step of: - Recover the lipids or free fatty acids or the E 8, E 10-Dodecadien-1-ol, - will at least partially describe E 8, E 10-Dodecadien-1-ol chemical conversion E 8, E 10-Dodecadienal and / or E 8, E 10-Dodecadienylacetate.

142. The method of claim 141, wherein the recovery is achieved by a distillation step or by column purification.

143. The method of claim 142, wherein the distillation step is a vacuum distillation step.

144. The method of claim 141, further comprising recycling E 8, E 10-Dodecadienal and / or E 8, E The steps for 10-dodecadienyl acetate.

145. The method of claim 144, wherein the recovery is achieved by a distillation step or by column purification.

146. The method of claim 145, wherein the distillation step is a vacuum distillation step.

147. The method of claim 77, further comprising disposing of the recovered... E 8, E 10-Dodecadien-1-ol, E 8, E 10-Dodecadienylacetate and / or E 8, E Steps for preparing a pheromone composition from 10-dodecadienal.

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