Biopesticide composition

Abstract

The present disclosure relates to a biopesticide composition comprising a target compound selected from (Z,E)-9,11-hexadecadienal and, optionally, one or more compounds selected from (Z)-9-hexadecenal, (Z)-11-hexadecenal and / or hexadecanal, in combination with one or more carriers, agents, additives, adjuvants and / or excipients. The production of the target compound is achieved by using an E11 desaturase derived from the sugarcane borer, which catalyzes the formation of a double bond in the E configuration at position 11 of (Z)-9-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA.

Description

Technical Field

[0001] The present disclosure describes a bio-based biopesticide composition containing a sex pheromone component of the sugarcane borer (Diatraea saccharalis), a pest of sugarcane, and a method for producing such a biopesticide composition. Further, this specification also describes the genetically engineered cells and enzymes that express them, and a method for applying such a biopesticide composition for pest control.

Background Art

[0002] Integrated pest management (IPM) is playing an increasingly important role in both increasing crop yields and minimizing environmental impacts and organic food production. In IPM, alternative pest control methods such as the use of pheromones for mating disruption or mass trapping and attracting beneficial insects are employed.

[0003] Pheromones consist of diverse groups of chemical compounds that insects (like other organisms) use for communication between individuals of the same species in various situations such as attracting mating partners, warning, leaving trails, and aggregating. Insect pheromones related to long-distance partner search are already used in the fields of agriculture and forestry for pest monitoring and control, and are used as a safe and environmentally friendly method alternative to pesticides. The biological production of pheromones used for pest control is superior to chemical synthesis in terms of price, specificity, and environmental impact.

[0004] Pheromones and pheromone precursors can be produced by genetically engineered cell factories modified to contain a pathway that expresses the enzymes necessary to convert intracellular precursor metabolites into the desired pheromones and pheromone precursors, as described in International Publication Nos. 2021078452 and 2021123128.

[0005] Known pheromones include fatty acyl alcohols, aldehydes, and acetates having one or more double bonds with specific Z or / and E orientations at specific positions in the carbon skeleton. The sugarcane borer (D. saccharalis), a pest of sugarcane, is a major pest of sugarcane in Central and South America. The main component of the sex pheromone of this pest is (Z,E)-9,11-hexadecadienal (Svatos, et al., 2001). Minor components of the pheromone are (Z)-11-hexadecenal, (Z)-9-hexadecenal, and hexadecanal (Kalinova, Kindl, Hovorka, Hoskovec, & Svatos, 2005)(Da Silva, et al., 2021).

[0006] Zhao et al. (2004) studied the formation of compounds produced when labeled fatty acids were topically applied to the glands of the moth Dendrolimus punctatus with respect to its sex pheromones (Z)-5-dodecenol and (Z,E)-5,7-dodecadienol. The paper further speculates that the pathway of the sex pheromone of D. punctatus involves the formation of an intermediate compound (Z,E)-9,11-hexadecadienoyl-CoA, but such a compound has been searched for and not detected in any of the reported experiments. Lienard et al. (2010) named yeast cells expressing a desaturase from D. punctatus as Dpu_APSQ. The authors speculated that the desaturase Dpu_APSQ introduces an E11-unsaturation into the (Z)-9-hexadecenoic acid naturally produced by yeast that produces the diunsaturated C16 fatty acid (Z,E)-9,11-hexadecadienoic acid. However, the inventors have discovered that Dpu_APSQ produces another unknown diunsaturated hexadecadienoic acid that elutes slightly later than (Z,E)-9,11-hexadecadienoic acid rather than (Z,E)-9,11-hexadecadienoic acid.

[0007] Therefore, to date, no bio-based method for generating the pheromone of the sugarcane borer has been available, and thus, there is a need to identify the enzymes capable of performing the biosynthesis of the pheromone precursor and to develop recombinant strains and methods for such pheromone production. SUMMARY OF THE INVENTION

[0008] Until the present disclosure, there has been no enzyme available for expression in a genetically modified host cell for the introduction of an E11 double bond into (Z)-9-hexadecenoyl-CoA (Z9-16:CoA), which is an essential function for the production of the major sex pheromone component of the sugarcane borer. However, disclosed herein is, surprisingly, a novel enzyme that catalyzes the E11 desaturation of the (Z)-9-hexadecenoyl-CoA substrate and produces (Z,E)-9,11-hexadecadienoyl-CoA, which is a precursor of the sugarcane borer pheromone (Z,E)-9,11-hexadecadienal (Z9,E11-16:Ald), in the presence of the (Z)-9-hexadecenoyl-CoA substrate. Further, the present disclosure provides a Δ9-desaturase capable of introducing a Z9 double bond into (E)-11-hexadecenoyl-CoA to obtain (Z,E)-9,11-hexadecadienoyl-CoA. Accordingly, the inventors have successfully prepared for the first time a bio-based biopesticide composition containing the pheromone component of the sugarcane borer formulated for the control of pests such as the sugarcane borer. Accordingly, in a first aspect, provided herein is a biopesticide composition comprising (Z,E)-9,11-hexadecadienal, and optionally, one or more compounds selected from (Z)-9-hexadecenal, (Z)-11-hexadecenal and / or hexadecanal, in combination with one or more carriers, agents, additives, adjuvants and / or excipients.

[0009] In a further aspect, provided herein is a method for controlling pests comprising spraying the composition described herein in the habitat of the pests, wherein the target compound enables the control of the pests.

[0010] In yet a further aspect, the present disclosure describes a method for producing a biopesticide composition of the present disclosure, including the following: (I) Culturing genetically modified yeast cells that produce hexadecanoyl-CoA and express the following: a) A Δ9 desaturase that catalyzes the formation of a Z-configured double bond at position 9 of hexadecanoyl-CoA, thereby producing (Z)-9-hexadecenoyl-CoA; b) An E11 desaturase that catalyzes the formation of an E-configured double bond at position 11 of (Z)-9-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA; c) An alcohol-forming fatty acyl-CoA reductase (FAR) that converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadiene; (II) Enzymatically or chemically converting (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal, and (III) Recovering and / or isolating (Z,E)-9,11-hexadecadienal and, optionally, one or more of its precursors, as needed.

[0011] In yet a further aspect, the present disclosure describes genetically modified yeast cells that produce (Z,E)-9,11-hexadecadienoyl-CoA and (Z,E)-9,11-hexadecadien-1-ol, said cells producing hexadecanoyl-CoA and expressing the following: a. A Δ9 desaturase that catalyzes the formation of a Z-configured double bond at position 9 of hexadecanoyl-CoA, thereby producing (Z)-9-hexadecenoyl-CoA; b. An E11 desaturase that catalyzes the formation of an E-configured double bond at position 11 of (Z)-9-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA; and c. An alcohol-forming fatty acyl-CoA reductase (FAR) that converts (Z,E)-9,11-hexadecadienyl-CoA to (Z,E)-9,11-hexadecadien-1-ol.

[0012] In yet a further aspect, the present specification describes a cell culture comprising the genetically modified microbial cell described herein and a growth medium.

[0013] In yet a further aspect, the present specification describes an E11 desaturase having an amino acid sequence contained in the E11 desaturase of SEQ ID NO: 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0014]

Figure 1

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Figure 5

[0015] Incorporation by reference All publications, patents, and patent applications mentioned herein are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of any conflict between the terms of this specification and the terms incorporated by reference, the terms of this specification shall prevail and apply.

Modes for Carrying Out the Invention

[0016] Definitions Throughout this disclosure, when referring to, for example, a pheromone component or precursor, (Z,E)-9,11-hexadecadienal refers to an aliphatic aldehyde with a 16-carbon chain, having an aldehyde at C1 and a double bond in the Z configuration at C9 and a double bond in the E configuration at C11, and alternative terms such as Z9,E11-16:Ald or (Z9,E11)-hexadecadienal may be used interchangeably. Similar nomenclature may be used for corresponding fatty acids, CoA derivatives, alcohols, acids, or other pathway compounds such as acetic acid.

[0017] The term "saturated" refers to a compound that does not have a carbon-carbon double or triple bond.

[0018] The term "unsaturated", which is interchangeable with the term "desaturated" as used herein, is used for a compound that contains one or more carbon-carbon double or triple bonds, preferably carbon-carbon double bonds. Throughout this specification, the following nomenclature is used: A Δi unsaturated compound, where i is an integer, refers to a compound having a double or triple carbon-carbon bond at position i of the carbon chain. Thus, the length of the carbon chain is at least equal to i. For example, a Δ12 unsaturated compound refers to a compound having a double or triple carbon-carbon bond at position 12 and a carbon chain length of 13 or more. The double or triple bond can be in the E or Z configuration. Thus, an Ei or Zi unsaturated compound refers to a compound having a carbon-carbon double bond in the E or Z configuration, respectively, at position i of the carbon chain, and the total length of its carbon chain is at least equal to i. For example, an E11 fatty alcohol is unsaturated at position 11 in the E configuration and has a carbon chain length of 12 or more.

[0019] The term "Δ11 desaturase" as used herein refers to a desaturase enzyme that catalyzes the introduction of a double bond at the position between C11 and C12 in a saturated or unsaturated fatty acyl compound such as a saturated or unsaturated fatty acyl coenzyme A (fatty acyl-CoA) having a carbon chain of at least 12 carbons.

[0020] As used herein, the term "E11 desaturase" refers to a Δ11 desaturase enzyme that catalyzes the introduction of a double bond in the E configuration at position 11 between C11 and C12 in a saturated or unsaturated fatty acyl compound such as fatty acyl coenzyme A (fatty acyl-CoA) having a carbon chain of at least 12 carbons.

[0021] As used herein, the term "Δ9 desaturase" refers to a desaturase enzyme that catalyzes the introduction of a double bond at the position between C9 and C10 in a saturated or unsaturated fatty acyl compound such as fatty acyl coenzyme A (fatty acyl-CoA) having a carbon chain of at least 10 carbons.

[0022] As used herein, the term "Z9 desaturase" refers to a Δ9 desaturase enzyme that catalyzes the introduction of a double bond in the Z configuration at the position between C9 and C10 in a saturated or unsaturated fatty acyl compound such as fatty acyl coenzyme A (fatty acyl-CoA) having a carbon chain of at least 10 carbons.

[0023] As used herein, the term "biopesticide" is a shortened form of "biological pesticide" and refers to several types of pest management interventions through predation, parasitism, or chemical relationships. In the EU, biopesticides are defined as "a form of pesticides based on microorganisms or natural products." In the United States, the EPA defines them as "pesticide substances of natural origin (biochemical pesticides) that control pests, microorganisms (microbial pesticides) that control pests, pesticide substances produced by plants with added genetic material (plant-incorporated protectants) or those containing PIP." More specifically, the present disclosure relates to biopesticides containing natural products or substances of natural origin. In this context, these are produced by culturing and concentrating naturally occurring organisms and / or their metabolites, including bacteria and other microorganisms, fungi, nematodes, proteins, etc. These compounds are considered an important element of integrated pest management (IPM) programs and have received significant attention in practical terms as alternatives to synthetic chemical plant protection products (PPP). The Biocontrol Agents Manual (2009: formerly the Biopesticide Manual) provides an overview of available biological insecticide (and other biology-based control) products.

[0024] As used herein, the term "bio-based" is used to characterize bio-based products, (I) the total carbon content of the product is at least 30%, and (II) the carbon content of the renewable raw material (bio-based) is at least 20%.

[0025] As recognized by the Circular Bio-based Europe Joint Undertaking (CBE Joint Undertaking) established in 2021, the development of bio-based materials is essential for the EU to achieve the climate goals set in the European Green Deal. The present disclosure provides a method for efficiently obtaining fatty alcohols and aliphatic aldehydes with a high bio-based carbon content (%).

[0026] Both fossil and renewable raw materials are mainly composed of carbon (C). There are several isotopes of carbon. Isotopes 14C is radioactive and naturally present in all living organisms (plants, animals, etc.) at a certain rate. This rate is approximately the same as the rate of 14 C in the atmosphere. At this rate, 14 the radioactivity level of C is 100%. When an organism dies, this concentration, and thus the radiation dose rate, decays with a half-life of approximately 5700 years. Therefore, by measuring the radioactive 14 C level of an unknown substance, the age of the carbon contained in that substance can be determined.

[0027] "Young" carbon (0 - 10 years) derived from renewable raw materials such as plants and animals has a relative isotope 14 C concentration approximately the same as that in the atmosphere, and the radioactivity 14 of such young carbon 14 C level is approximately 100%.

[0028] "Old" carbon (millions of years) derived from synthesis or fossil (petrochemical products) has an isotope 14 C that has undergone a much longer time than its half-life (about 5700 years), so the isotope 14 C has significantly decreased. Therefore, carbon derived from synthesis or fossil fuel sources has a relative concentration of isotope 14 C that is approximately 0%, and the radioactivity 14 C level of such old carbon is approximately 0%.

[0029] In one embodiment, the term "radioactive 14 C level" refers to the total radioactive 14 C level of a given substance, product, or composition, as described above.

[0030] The isotope 14 C method can be used to determine the concentration of young (renewable) substances compared to the concentration of old (fossil) resources. The carbon content of renewable raw materials is called the "bio-based carbon content". The carbon content of renewable raw materials or the "bio-based carbon content" can be determined as described below.

[0031] When measuring the bio-based carbon content, the results may be reported as the "bio-based carbon content ratio". This indicates the ratio of "natural" (carbon sources derived from plants or animal by-products) to "synthetic" or "fossil" (petrochemical) carbon sources. For reference, 100% bio-based carbon indicates that the material is obtained entirely from plant or animal by-products, and 0% bio-based carbon indicates that the material contains no carbon obtained from plant or animal by-products. Intermediate values represent mixtures of natural and fossil resources.

[0032] Example: If a product has a radioactive 14 C level of 80%, it means that 80% of the product is composed of renewable carbon and 20% is composed of fossil carbon (C). In other words, it means that 80% of the product is bio-based.

[0033] Analytical measurement values may be cited as "percent modern carbon (pMC)". This is the ratio of 14 C measured in a sample relative to the current modern standard sample (NIST 4990C). The bio-derived carbon content ratio is calculated from pMC by applying a small adjustment factor for 14 C in the current atmospheric carbon dioxide. 14 It is important to note that in all internationally recognized standards that use

[0034] The term "fatty acyl compound" as used herein refers to an aliphatic compound having a long aliphatic chain, i.e., usually an aliphatic chain having 12 to 28 carbon atoms, for example, an aliphatic chain having 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 are either saturated or unsaturated. Fatty acyl compounds can contain various functional group end groups.

[0035] As used herein, the term "fatty acyl-CoA" is used interchangeably with "fatty acyl-CoA ester" and refers to a compound of the general formula R-CO-SCoA, where R is a fatty acid carbon chain having 12 to 28 carbon atoms, for example, a fatty acid carbon chain consisting of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 carbon atoms. The fatty acid carbon chain is attached to the -SH group of CoA by a thioester bond. Fatty acyl CoA is saturated or unsaturated depending on whether the fatty acid from which it is derived is saturated or unsaturated.

[0036] As used herein, the term "fatty alcohol" refers to an alcohol having a carbon chain length of 13 to 28 carbon atoms, for example, 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 unsaturated.

[0037] As used herein, the term "fatty alcohol acetate" refers to an acetate having an aliphatic carbon chain, i.e., an aliphatic chain of 13 to 28 carbon atoms, for example, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 carbon atoms. Fatty acyl acetates can be saturated or unsaturated.

[0038] As used herein, the term "fatty aldehyde" refers to an aldehyde having a carbon chain length of 13 to 28 carbon atoms, for example, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 carbon atoms. Fatty aldehydes can be saturated or unsaturated.

[0039] As used herein, the term "functional variant" refers to a functional variant of an enzyme that retains at least a portion of the activity of the parent enzyme. Thus, a functional variant of a desaturase or other pathway enzyme catalyzes a reaction similar to that of the parent enzyme, but the efficiency and specificity of the reaction may be different. For example, the efficiency may be decreased or increased compared to the parent enzyme.

[0040] As used herein, the terms "heterologous", "recombinant", or "genetically modified", and their grammatical equivalents, are used interchangeably with respect to nucleotides, polypeptides, and cells, and refer to entities "derived from different species or cells". For example, a heterologous or recombinant polynucleotide gene is a gene within a host cell that does not naturally contain that gene, i.e., the gene is derived from a species or cell type different from the host cell. A heterologous or recombinant polypeptide is a polypeptide produced within a host cell that does not naturally contain that polypeptide, i.e., the polypeptide is derived from a species or cell type different from the host cell. When the terms are used herein with respect to a host cell, they refer to a host cell that contains and expresses a heterologous or recombinant polynucleotide.

[0041] As used herein, the term "% identity" is used herein with respect to the relatedness between two amino acid sequences, or two nucleotide sequences, using standard alignment software well known in the art, applying the settings (including gaps) directed by the software, and, where appropriate, considering any conservative substitutions as part of the sequence identity in accordance with the NCIUB rules (hftp: / / www.chem.qmul.ac.uk / iubmb / misc / naseq.html; NC-IUB, Eur J Biochem (1985)) to achieve the maximum percent identity / similarity / homology. 5' or 3' extensions, or insertions (in the case of nucleic acids), or N' or C' extensions, or insertions (in the case of polypeptides) typically do not result in a decrease in identity, similarity, or homology when using such standard software.

[0042] As used herein, the term "degenerate" with respect to the genetic code reflects that multiple different variant nucleotide sequences can encode the same polypeptide because there are two or more nucleotide triplets that function as codons corresponding to a particular amino acid. Thus, the codons of the coding sequence of a particular polypeptide can be altered using an appropriate codon bias table for a particular host cell so as to obtain optimal expression in the particular host.

[0043] As used herein, the term "pest" refers to organisms that are considered harmful to humans, animals, or cultivated crops, especially animals such as insects, particularly in the context of agriculture and animal husbandry. A pest refers to any organism that is invasive, highly fecund, harmful, troublesome, toxic, or destructive to crops or animals, humans or things associated with humans, livestock, human buildings, wild ecosystems, etc. In particular, the pests used herein are used with respect to the sugarcane borer, a sugarcane pest, and its previous life stage (larva).

[0044] As used herein, the term "pheromone" is used with respect to signal transduction compounds of natural origin that are used in chemical communication between individuals of the same species in nature. For example, lepidopteran insect pheromones are unbranched aliphatic chains ending in an alcohol, aldehyde, or acetate functional group (9 to 18 carbon atoms, e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbon atoms), ending in an alcohol, aldehyde, or acetate functional group and containing up to three double bonds in the aliphatic backbone. Thus, unsaturated fatty alcohols, unsaturated fatty aldehydes, and unsaturated fatty alcohol acetates are usually included in pheromones. Pheromone compositions can be produced chemically or biochemically, for example, as described herein. Thus, pheromones include unsaturated fatty alcohols, unsaturated fatty aldehydes, and / or unsaturated fatty alcohol acetates such as can be obtained by the methods and cells described herein.

[0045] As used herein, the titer of a compound refers to the concentration at which the compound is produced. When the compound is produced by cells, this term refers to the total concentration produced by the cells, i.e., the total amount of the compound divided by the volume of the medium. This means that especially in the case of volatile compounds, the titer includes the portion of the compound that may have evaporated from the medium, and thus is determined by recovering the produced compound from the fermentation broth and potential off-gases from the culture vessel.

[0046] The terms "pathway", "biosynthetic pathway", or "metabolic pathway" used interchangeably herein refer to one or more enzymes that act in concert within a living cell to convert one or more substrate precursors into a chemical product. A pathway can include one enzyme or multiple enzymes that act in sequence or in combination. A pathway that includes only one enzyme may also be referred to as "biotransformation", which is particularly relevant to embodiments where a precursor or substrate is exogenously supplied to the host cell and converted by the enzyme into the desired final product. Enzymes are characterized by having catalytic activity, by which the chemical structure of the substrate can be changed. Enzymes can have two or more substrates and can produce two or more products. Enzymes may be dependent on cofactors, which are inorganic or organic compounds (cofactors and / or coenzymes), and may or may not be considered part of the pathway.

[0047] The term "in vivo" as used herein refers to within a living cell or organism such as an animal, a plant, a microorganism, etc.

[0048] The term "in vitro" as used herein refers to outside of a living cell or organism and includes, but is not limited to, for example, microwell plates, tubes, flasks, beakers, tanks, reactors, etc.

[0049] As used herein, the terms "substrate" or "precursor" refer to any compound that can be converted into another compound. For the sake of clarity, substrates and / or precursors include both compounds that are generated in situ by enzymatic reactions within cells and exogenous compounds such as exogenous organic molecules that can be metabolized by the host cell into the desired compound.

[0050] The term "expression" includes all steps involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0051] The term "expression vector" refers to a single-stranded or double-stranded, linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operably linked to control sequences that enable its expression. Expression vectors include expression cassettes for integrating genes into host cells, and plasmids and / or chromosomes containing such genes.

[0052] The term "host cell" refers to any cell type that is sensitive to transformation, transfection, transduction, etc. by a nucleic acid construct or expression vector containing a polynucleotide to be expressed within the host cell. Host cells include all progeny of the parental cell, including those that are not identical to the parental cell due to mutations that occur during replication.

[0053] The term "polynucleotide construct" refers to a single-stranded or double-stranded polynucleotide that has been isolated from a gene of natural origin, or modified or synthesized to contain segments of nucleic acid in a manner not found in nature, and includes a polynucleotide encoding a polypeptide and one or more control sequences.

[0054] The term "operably linked" refers to a configuration in which a control sequence is positioned at an appropriate location relative to the coding polynucleotide such that the control sequence directs the expression of the coding polynucleotide.

[0055] The terms "nucleotide sequence" and "polynucleotide" are used interchangeably herein.

[0056] The terms "comprise" and "include" and their variants such as "comprises", "comprising", "includes", "including", as used throughout this specification, and the appended claims, are to be construed inclusively. These terms are intended to convey that, where the context allows, they may include other elements or integers not specifically recited.

[0057] As used herein, the articles "a" and "an" are used to refer to one or more (i.e., one or at least one) of the grammatical objects of the article. For example, "an element" may mean one element or more than two elements.

[0058] Terms such as "preferably", "generally", "particularly", and "typically" are not intended to limit the scope of the claimed invention or to suggest that a particular feature is important, essential, or critical to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that may or may not be utilized in particular embodiments of the invention.

[0059] As used herein, the term "cell culture medium" refers to a medium containing a plurality of host cells as described herein. The cell culture may contain a single host cell line or may contain two or more different host cell lines. The culture medium may be any medium, for example, a liquid medium (i.e., culture broth) or a semi-solid medium containing a recombinant host, and may further contain additional components such as, for example, a carbon source; a nitrogen source; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases; and the like.

[0060] As used herein, the terms "endogenous" or "native" refer to a gene or polypeptide within a host cell that is derived from the same host cell.

[0061] As used herein, the term "deletion" refers to manipulating a gene so that it is not expressed in a host cell.

[0062] As used herein, the term "disruption" refers to manipulating either a gene or a mechanism involved in gene expression so that it is not expressed in a host cell.

[0063] As used herein, the term "attenuation" refers to manipulating either a gene or a mechanism involved in gene expression, and the gene expression decreases as compared to the expression without manipulation.

[0064] All methods described herein can be performed in any suitable order of steps, unless otherwise indicated herein or clearly contradicted by the context. The use of all examples, or exemplary expressions (e.g., "for example") provided herein is merely intended to more clearly illustrate the invention and does not limit the scope of the claimed invention. No expression in this specification shall be construed as indicating an essential, non-claimed element for the practice of the invention.

[0065] All percentages, ratios, and proportions herein are by weight unless otherwise specified. Components by weight percent (wt.%, also written as weight %) are based on the total weight of the composition in which the component is included (e.g., the total amount of the reaction mixture), unless otherwise noted.

[0066] As used herein, the terms "substantially", "nearly", or "about" refer to a reasonable deviation of a value or parameter such that the value or parameter is not significantly changed. Terms regarding deviations from these values should be construed to include deviations of values where the deviation does not negate the meaning of the value from which the deviation occurred. For example, in relation to a reference numerical value, terms of degree can include values within a range of plus or minus 10% from that numerical value. For example, a deviation from a certain value can include values that are plus or minus 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, etc., from that value, i.e., values that are plus or minus a specific percentage from that value.

[0067] As used herein, the term "and / or" is intended to represent an inclusive "or". The expression "X and / or Y" is intended to mean X or Y and both X and Y. Further, the expression "X, Y and / or Z" is intended to mean only X, Y, and Z, or any combination of X, Y, and Z.

[0068] As used herein, the term "isolated" when referring to a compound means a compound that has been placed by human intervention in a form or environment different from that in which it is found in nature. Isolated compounds include, but are not limited to, those in which the ratio of the compound to other components with which it is associated in nature has been increased or decreased. In an important embodiment, the amount of the compound is increased as compared to other components with which it is associated in nature. In an embodiment, the compounds of the present disclosure may be isolated in pure or substantially pure form. In this context, a substantially pure compound means that the compound is separated from other unwanted or undesirable substances that are present at the start of the manufacture of the compound or that are produced in the manufacturing process. Such a substantially pure compound preparation, when represented in native or recombinant form, contains less than 10% by weight, such as less than 8% by weight, such as less than 6% by weight, such as less than 5% by weight, such as less than 4% by weight, such as less than 3% by weight, such as less than 2% by weight, such as less than 1% by weight, such as less than 0.5% by weight of other foreign or unwanted substances normally associated with the compound. In certain embodiments, the isolated compound is at least 90% pure by weight, such as at least 91% pure by weight, such as at least 92% pure by weight, such as at least 93% pure by weight, such as at least 94% pure by weight, such as at least 95% pure by weight, such as at least 96% pure by weight, such as at least 97% pure by weight, such as at least 98% pure by weight, such as at least 99% pure by weight, such as at least 99.5% pure by weight, such as 100% pure by weight.

[0069] The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a mature, spliced mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks the intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor of mRNA that is processed by a series of steps including splicing before it appears as a mature, spliced mRNA.

[0070] The term "coding sequence" refers to a nucleotide sequence that directly specifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. A coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0071] As used herein, the term "control sequence" refers to a nucleotide sequence required for the expression of a polynucleotide encoding a polypeptide. A control sequence may be native (i.e., from the same gene) or heterologous or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide. Control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide coding sequences, promoter sequences, signal peptide coding sequences, translation termination (stop) sequences, transcription termination (stop) sequences, etc. For functionality, a control sequence typically must include a promoter sequence, a transcription termination signal, and a translation termination signal. A control sequence may be provided together with a linker for the purpose of introducing specific restriction sites to facilitate ligation of the control sequence to the coding region of the polynucleotide encoding the polypeptide.

[0072] Δ9 desaturase The present disclosure further provides a Δ9 desaturase comprising an amino acid sequence that is a desaturase included in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82, or a functional variant that is at least 50% identical to a desaturase included in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82. In some embodiments, the Δ9 desaturase is 50% to 100% identical to the Δ9 desaturase included in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82, for example, 50% to 60%, for example 60% to 70%, for example 70% to 80%, for example 80% to 90%, for example 90% to 92%, for example 92% to 94%, for example 94% to 96%, for example 96% to 98%, for example 98% to 99%, for example 100% identical. The Δ9 desaturase of the present disclosure refers to a desaturase enzyme that catalyzes the introduction of a double bond at the position between C9 and C10 of a saturated or unsaturated fatty acyl compound such as fatty acyl coenzyme A (fatty acyl-CoA) having a carbon chain of at least 10 carbon atoms.

[0073] Δ9 gene In a further aspect, a polynucleotide sequence encoding a Δ9 desaturase (Δ9 desaturase gene) is provided, and the polynucleotide sequence is at least 50% identical to the coding sequence of the Δ9 desaturase included in SEQ ID NO: 83. In some embodiments, the Δ9 desaturase gene is 50% to 100% identical to the Δ9 desaturase gene included in SEQ ID NO: 83, for example, 50% to 60%, for example 60% to 70%, for example 70% to 80%, for example 80% to 90%, for example 90% to 92%, for example 92% to 94%, for example 94% to 96%, for example 96% to 98%, for example 98% to 99%, for example 100% identical. In a preferred embodiment, the Δ9 desaturase gene encodes the Δ9 desaturase of SEQ ID NO: 82, or the described functional variant. In other embodiments, the Δ9 desaturase gene has codons optimized for heterologous expression in a microorganism, particularly yeast.

[0074] E11 desaturase One aspect provides an E11 fatty acyl-CoA desaturase (E11 desaturase) comprising an amino acid sequence that is the E11 desaturase contained in SEQ ID NO: 1 or a functional variant that is at least 50% identical to the E11 desaturase contained in SEQ ID NO: 1. In some embodiments, the E11 desaturase is 50% to 100% identical to the E11 desaturase contained in SEQ ID NO: 1, such as 50% to 60%, such as 60% to 70%, such as 70% to 80%, such as 80% to 90%, such as 90% to 92%, such as 92% to 94%, such as 94% to 96%, such as 96% to 98%, such as 98% to 99%, such as 100% identical. In further particular embodiments, the E11 desaturase catalyzes the introduction of a double bond at the E configuration at position 11 in the (Z9)-9-hexadecenoyl-CoA substrate, providing (Z,E)-9,11-hexadecadienoyl-CoA having double bonds at position 9 in the Z configuration and at position 11 in the E configuration.

[0075] A further aspect provides an E11 fatty acyl-CoA desaturase (E11 desaturase) comprising an amino acid sequence that is the E11 desaturase contained in SEQ ID NO: 1 or 80 or a functional variant that is at least 50% identical to the E11 desaturase contained in SEQ ID NO: 1 or 80. In some embodiments, the E11 desaturase is 50% to 100% identical to the E11 desaturase contained in SEQ ID NO: 1 or 80, such as 50% to 60%, such as 60% to 70%, such as 70% to 80, such as 80% to 90%, such as, 90% to 92%, such as 92% to 94%, such as 94% to 96%, such as 96% to 98%, such as 98% to 99%, such as, 100% identical. In further particular embodiments, the E11 desaturase catalyzes the introduction of a double bond at the E configuration at position 11 in the (Z9)-9-hexadecenoyl-CoA substrate, providing (Z,E)-9,11-hexadecadienoyl-CoA having double bonds at position 9 in the Z configuration and at position 11 in the E configuration.

[0076] A further aspect provides an E11 fatty acyl-CoA desaturase (E11 desaturase) comprising an amino acid sequence that is the E11 desaturase contained in SEQ ID NO: 80 or a functional variant that is at least 50% identical to the E11 desaturase contained in SEQ ID NO: 80. In some embodiments, the E11 desaturase is 50% to 100% identical to the E11 desaturase contained in SEQ ID NO: 80, such as 50% - 60%, such as 60% - 70%, such as 70% - 80%, such as 80% - 90%, such as 90% - 92%, such as 92% - 94%, such as 94% - 96%, such as 96% - 98%, such as 98% - 99%, such as 100% identical. In further specific embodiments, the E11 desaturase catalyzes the introduction of a double bond to the E configuration at position 11 in the (Z9)-9-hexadecenoyl-CoA substrate, providing (Z,E)-9,11-hexadecadienoyl-CoA having a double bond at position 9 in the Z configuration and at position 11 in the E configuration.

[0077] In some embodiments, there is provided an E11 fatty acyl-CoA desaturase (E11 desaturase) comprising an amino acid sequence that has at least 50% identity to the E11 desaturase contained in SEQ ID NO: 1, SEQ ID NO: 80, SEQ ID NO: 90, or SEQ ID NO: 92, such as 50% - 60%, such as 60% - 70%, such as 70% - 80%, such as 80% - 90%, such as 90% - 92%, such as 92% - 94%, such as 94% - 96%, such as 96% - 98%, such as 98% - 99%, such as 100% identical.

[0078] In some embodiments, an E11 fatty acyl-CoA desaturase (E11 desaturase) is provided that has at least 50% identity with the E11 desaturase contained in SEQ ID NO: 1, SEQ ID NO: 80, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, or SEQ ID NO: 104, for example 50% - 60%, for example 60% - 70%, for example 70% - 80%, for example 80% - 90%, for example 90% - 92%, for example 92% - 94%, for example 94% - 96%, for example 96% - 98%, for example 98% - 99%, for example 100% identical.

[0079] E11 gene In a further aspect, a polynucleotide sequence encoding E11 desaturase (E11 desaturase gene) is provided, and the polynucleotide sequence is at least 50% identical to the E11 desaturase coding sequence contained in SEQ ID NO: 2. In some embodiments, the E11 desaturase gene is 50% - 100% identical to the E11 desaturase gene contained in SEQ ID NO: 2 or 93, for example 50% - 60%, for example 60% - 70%, for example 70% - 80%, for example 80% - 90%, for example 90% - 92%, for example 92% - 94%, for example 94% - 96%, for example 96% - 98%, for example 98% - 99%, for example 100% identical. In a preferred embodiment, the E11 desaturase gene encodes the E11 desaturase of SEQ ID NO: 1 or the described functional variant. In other embodiments, the E11 desaturase gene is codon-optimized for heterologous expression, particularly in microorganisms such as yeast.

[0080] In a further aspect, a polynucleotide sequence encoding E11 desaturase (E11 desaturase gene) is provided, and the polynucleotide sequence is at least 50% identical to the coding sequence of E11 desaturase contained in SEQ ID NO: 2 or 81. In some embodiments, the E11 desaturase gene is 50% to 100% identical to the E11 desaturase gene contained in SEQ ID NO: 2, 81, or 93, such as 50% to 60%, such as 60% to 70%, such as 70% to 80%, such as 80% to 90%, such as 90% to 92%, such as 92% to 94%, such as 94% to 96%, such as 96% to 98%, such as 98% to 99%, such as 100% identical. In a preferred embodiment, the E11 desaturase gene encodes the E11 desaturase of SEQ ID NO: 1 or 80, or a described functional variant. In other embodiments, the E11 desaturase gene is codon-optimized for heterologous expression, particularly in microorganisms such as yeast.

[0081] In a further aspect, a polynucleotide sequence encoding E11 desaturase (E11 desaturase gene) is provided, and the polynucleotide sequence is at least 50% identical to the coding sequence of E11 desaturase contained in SEQ ID NO: 81. In some embodiments, the E11 desaturase gene is 50% to 100% identical to the E11 desaturase gene contained in SEQ ID NO: 81, such as 50% to 60%, such as 60% to 70%, such as 70% to 80%, such as 80% to 90%, such as 90% to 92%, such as 92% to 94%, such as 94% to 96%, such as 96% to 98%, such as 98% to 99%, such as 100% identical. In a preferred embodiment, the E11 desaturase gene encodes the E11 desaturase of SEQ ID NO: 80, or a described functional variant. In other embodiments, the E11 desaturase gene is codon-optimized for heterologous expression, particularly in microorganisms such as yeast.

[0082] In some embodiments, a polynucleotide sequence optimized for codons for heterologous expression encoding the E11 desaturase disclosed herein is provided and has a DNA sequence contained in SEQ ID NO: 2, SEQ ID NO: 81, SEQ ID NO: 91, or SEQ ID NO: 93, or a homolog thereof containing mutations due to the degeneracy of the genetic code. In some embodiments, the E11 desaturase gene is 50% to 100% identical to the E11 desaturase gene contained in SEQ ID NO: 2, SEQ ID NO: 81, SEQ ID NO: 91, or SEQ ID NO: 93, such as 50% to 60%, such as 60% to 70%, such as 70% to 80%, such as 80% to 90%, such as 90% to 92%, such as 92% to 94%, such as 94% to 96%, such as 96% to 98%, such as 98% to 99%, such as 100% identical.

[0083] In some embodiments, a polynucleotide sequence optimized for codons for heterologous expression encoding the E11 desaturase disclosed herein is provided and has a DNA sequence contained in SEQ ID NO: 2, SEQ ID NO: 81, SEQ ID NO: 91, or SEQ ID NO: 93, or a homolog thereof containing mutations due to the degeneracy of the genetic code. In some embodiments, the E11 desaturase gene is 50% to 100% identical to the E11 desaturase gene contained in SEQ ID NO: 2, SEQ ID NO: 81, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, or SEQ ID NO: 105, such as 50% to 60%, such as 60% to 70%, such as 70% to 80%, such as 80% to 90%, such as 90% to 92%, such as 92% to 94%, such as 94% to 96%, such as 96% to 98%, such as 98% to 99%, such as 100% identical.

[0084] Z11 desaturase In some embodiments, the present disclosure provides a Z11 desaturase having at least 70% sequence identity with the amino acid sequence contained in the Z11 desaturase of SEQ ID NO: 72, 74, 76, or 78. In some embodiments, the Z11 desaturase is expressed in the genetically modified host cell of the present disclosure. In some embodiments, a Z11 desaturase having at least 70% sequence identity with the amino acid sequence contained in the Z11 desaturase of SEQ ID NO: 72, 74, 76, or 78 is employed in the methods disclosed herein. In some embodiments, the Z11 desaturase has 70% to 100% sequence identity with the amino acid sequence contained in the Z11 desaturase of SEQ ID NO: 72, 74, 76, or 78, such as 70% to 80%, such as 80% to 90%, such as 90% to 92%, such as 92% to 94%, such as 94% to 96%, such as 96% to 98%, such as 98% to 99%, such as 100% sequence identity.

[0085] Gene construct A further aspect provides a polynucleotide construct comprising the described E11 desaturase gene operably linked to one or more control sequences. Such control sequences can be native or heterologous to the E11 desaturase gene. The polynucleotide construct can be further incorporated into an expression vector for expression of the E11 desaturase gene in a host cell.

[0086] Genetically modified host cell A further aspect provides a genetically modified microbial cell that produces (Z,E)-9,11-hexadecadienoyl-CoA, wherein the cell heterologously expresses the E11 desaturase of the present disclosure. This cell introduces a double bond of the E configuration at position 11 in the (Z)-9-hexadecenoyl-CoA substrate in the presence of the (Z)-9-hexadecenoyl-CoA substrate, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA having a double bond of the E configuration at position 11. Additionally, the present disclosure provides a genetically modified microbial cell as defined herein that expresses a Δ9-desaturase capable of introducing a Z9 double bond into (E)-11-hexadecenoyl-CoA to produce (Z,E)-9,11-hexadecadienoyl-CoA.

[0087] The cells provided herein can further include an effective biosynthetic pathway for converting (Z,E)-9,11-hexadecadienoyl-CoA into a) (Z,E)-9,11-hexadecadien-1-ol; b) (Z,E)-9,11-hexadecadienal; and / or c) (Z,E)-9,11-hexadecadienyl acetate, selected target compounds, wherein the pathway includes a) an alcohol-forming fatty acyl-CoA reductase (FAR) that converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol; b) an acetyltransferase that converts (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienyl acetate; c) an alcohol dehydrogenase that converts (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal; and / or d) a fatty alcohol oxidase that converts (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal, and expresses one or more pathway polypeptides selected therefrom.

[0088] In some embodiments, the cells provided herein convert (Z,E)-9,11-hexadecadienoyl-CoA into a) (Z,E)-9,11-hexadecadien-1-ol; b) (Z,E)-9,11-hexadecadienal; and / or c) (Z,E)-9,11-hexadecadienyl acetate, and further comprise a biosynthetic pathway effective to convert said (Z,E)-9,11-hexadecadienoyl-CoA into a target compound selected from: d) an alcohol-forming fatty acyl-CoA reductase (FAR) that converts (Z,E)-9,11-hexadecadienoyl-CoA into (Z,E)-9,11-hexadecadien-1-ol; e) an acetyltransferase that converts (Z,E)-9,11-hexadecadien-1-ol into (Z,E)-9,11-hexadecadienyl acetate; f) a fatty alcohol oxidase that converts (Z,E)-9,11-hexadecadien-1-ol into (Z,E)-9,11-hexadecadienal, expressing one or more pathway polypeptides selected from:

[0089] In some embodiments, cells are provided, a) the FAR is at least 70% identical to a FAR contained in SEQ ID NOs: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64, 66, 88, or 95; b) the acetyltransferase is at least 70% identical to an acetyltransferase contained in SEQ ID NO: 106; c) the fatty alcohol oxidase is at least 70% identical to a fatty alcohol oxidase contained in SEQ ID NO: 70.

[0090] When the cells express such pathway enzymes, (I) FAR is preferably at least 70% identical to the FAR contained in SEQ ID NO: 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65, for example at least 80%, for example at least 90%, for example at least 95%, for example 100% identical; (II) Acetyltransferase is preferably at least 70% identical to the acetyltransferase contained in SEQ ID NO: 71, for example at least 80%, for example at least 90%, for example at least 95%, for example 100% identical; (III) Alcohol dehydrogenase is preferably at least 70% identical to the alcohol dehydrogenase contained in SEQ ID NO: 68, and (IV) Fatty alcohol oxidase is preferably at least 70% identical to the fatty alcohol oxidase contained in SEQ ID NO: 69 or 70, for example at least 80%, for example at least 90%, for example at least 95%, for example 100% identical.

[0091] In some embodiments, (I) FAR is at least 70% identical to the FAR contained in SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64, 66, 88, or 95; (II) Acetyltransferase is at least 70% identical to the acetyltransferase contained in SEQ ID NO: 71; (III) Alcohol dehydrogenase is at least 70% identical to the alcohol dehydrogenase contained in SEQ ID NO: 68; and (IV) Fatty alcohol oxidase is at least 70% identical to the fatty alcohol oxidase contained in SEQ ID NO: 69 or 70.

[0092] The genetically modified host cell may further comprise a functional biosynthetic pathway for generating a (Z)-9-hexadecenoyl-CoA substrate, in particular a pathway that expresses one or more heterologous Δ9 desaturases that introduce a double bond in the Z configuration at position 9 in the hexadecenoyl-CoA substrate. Such Δ9 desaturases are suitably at least 70% identical to the Δ9 desaturases included in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82, such as at least 80%, such as at least 90%, such as at least 95%, such as 100% identical.

[0093] The genetically modified host cell may further comprise a functional biosynthetic pathway for generating a (Z)-9-hexadecenoyl-CoA substrate, in particular a pathway that expresses one or more heterologous Δ9 desaturases that introduce a double bond in the Z configuration at position 9 in the hexadecenoyl-CoA substrate. Such Δ9 desaturases are suitably at least 70% identical to the Δ9 desaturases included in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, or 22, such as at least 80%, such as at least 90%, such as at least 95%, or such as 100% identical.

[0094] In further embodiments, the host cell is further modified such that one or more native or endogenous genes are attenuated, disrupted and / or deleted. Other modifications include overexpression of one or more pathway genes, or modifications that provide an increased amount of a substrate for at least one enzyme of the pathways described herein. The genetically modified host cell can also be further modified to increase resistance to one or more substrates, intermediates, or product molecules from the pathways described herein, and / or the host cell can contain at least two copies of one or more genes of such pathways. In some embodiments, the host cell can contain at least two copies of one or more genes of the (Z,E)-9,11-hexadecadien-1-ol pathway.

[0095] The host cells provided herein are suitably fungal cells such as yeast cells. Preferred yeasts include those belonging to a genus selected from Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, Lipomyces, and optionally, the yeast cells belong to a species selected from Saccharomyces cerevisiae, Saccharomyces boulardi, Pichia pastoris, Kluyveromyces marxianus, Cryptococcus albidus, Lipomyces lipofera, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulan, and Yarrowia lipolytica.

[0096] However, for example, filamentous fungal cells selected from species consisting of the following may also be useful: Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicolalanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium pururogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.

[0097] In alternative or additional embodiments provided herein, genetically modified yeast cells that produce (Z,E)-9,11-hexadecadienoyl-CoA and (Z,E)-9,11-hexadecadien-1-ol, said cells producing hexadecanoyl-CoA, and a) expressing a Δ9 desaturase that catalyzes the formation of a double bond of the Z configuration (Z) at position 9 of hexadecanoyl-CoA, thereby producing (Z)-9-hexadecenoyl-CoA; b) expressing an E11 desaturase that catalyzes the formation of a double bond of the E configuration at position 11 of (Z)-9-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA; and c) expressing an alcohol-forming fatty acyl-CoA reductase (FAR) that converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol.

[0098] In further embodiments, the yeast cells may express one or more enzymes selected from the following: a) a Z11 desaturase that catalyzes the formation of a double bond of the Z configuration at position 11 of hexadecanoyl-CoA, thereby producing (Z)-11-hexadecenoyl-CoA; b) One or more alcohol-forming fatty acyl-CoA reductases (FAR) that convert hexadecanoyl-CoA to hexadecan-1-ol, (Z)-9-hexadecenoyl-CoA to (Z)-9-hexadecen-1-ol, and (Z)-11-hexadecenoyl-CoA to (Z)-11-hexadecen-1-ol, respectively.

[0099] In some embodiments, genetically modified yeast cells are provided that produce (Z,E)-9,11-hexadecadienoyl-CoA and (Z,E)-9,11-hexadecadien-1-ol, said yeast cells producing hexadecanoyl-CoA, and (I) expressing an E11 desaturase that catalyzes the formation of a double bond in the E configuration at position 11, thereby producing E-11-hexadecenoyl-CoA; (II) expressing a Δ9 desaturase that catalyzes the formation of a double bond in the Z configuration at position 9 of (E)-11-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA; (III) expressing an alcohol-forming fatty acyl-CoA reductase (FAR) that converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol.

[0100] As demonstrated in the examples herein, including Example 18, the cells of the present disclosure can produce (Z,E)-9,11-hexadecadienoyl-CoA and (Z,E)-9,11-hexadecadien-1-ol by introducing unsaturations in any order, i.e., first introducing a double bond in the E configuration at position 11 and then introducing a double bond in the Z configuration at position 9, or vice versa.

[0101] In yet further embodiments, a) The E11 desaturase has an amino acid sequence that is at least 50% identical to the E11 desaturase contained in SEQ ID NO: 1, for example 50% to 100%, for example 50% - 60%, for example 60% - 70%, for example 70% - 80%, for example 80% - 90%, for example 90% - 92%, for example 92% - 94%, for example 94% - 96%, for example 96% - 98%, for example 98% - 99%, for example 100% identical to the E11 desaturase contained in SEQ ID NO: 1 of the E11 desaturase. b) The Δ9 desaturase has an amino acid sequence that is at least 70% identical to the Δ9 desaturase contained in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 82, for example at least 80%, for example at least 90%, for example at least 95%, for example 100% identical; c) The Z11 desaturase has an amino acid sequence that is at least 70% identical to the Z11 desaturase contained in SEQ ID NO: 72, 74, 76, or 78, for example at least 80%, for example at least 90%, for example at least 95%, for example 100% identical; and d) The alcohol - forming fatty acyl - CoA reductase (FAR) has an amino acid sequence that is at least 70% identical to the FAR contained in SEQ ID NO: 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65, for example at least 80%, for example at least 90%, for example at least 95%, for example 100% identical.

[0102] In this embodiment, the yeast cell is preferably of the species Saccharomyces cerevisiae or Yarrowia lipolytica.

[0103] In some embodiments, a yeast cell is provided, (I) The E11 desaturase has at least 70% sequence identity with the amino acid sequence contained in the E11 desaturase of SEQ ID NO: 1, 80, 90, 92, 96, 98, 100, 102, or 104; (II) The Δ9 desaturase has at least 70% sequence identity with the amino acid sequence contained in the Δ9 desaturase of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82; (III) The Z11 desaturase has at least 70% sequence identity with the amino acid sequence contained in the Z11 desaturase of SEQ ID NO: 72, 74, 76, or 78; and / or (IV) The alcohol-forming fatty acyl-CoA reductase (FAR) has at least 70% sequence identity with the amino acid sequence contained in the FAR of SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 88, or 95.

[0104] In some embodiments, yeast cells are provided, (I) The E11 desaturase has the amino acid sequence contained in the E11 desaturase of SEQ ID NO: 1, 80, 90, 92, 96, 98, 100, 102, or 104; (II) The Δ9 desaturase has the amino acid sequence contained in the Δ9 desaturase of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82; (III) The Z11 desaturase has the amino acid sequence contained in the Z11 desaturase of SEQ ID NO: 72, 74, 76, or 78; and / or (IV) The alcohol-forming fatty acyl-CoA reductase (FAR) has the amino acid sequence contained in the FAR of SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 88, or 95.

[0105] Cultivation A further aspect provides a cell culture comprising the host cell and a growth medium described herein. Growth media suitable for eukaryotic and prokaryotic cells are well known in the art.

[0106] A method for producing the compounds of the present disclosure. A further aspect provides a method for producing a target compound selected from: (I) (Z,E)-9,11-hexadecadienoyl-CoA; (II) (Z,E)-9,11-hexadecadien-1-ol; (III) (Z,E)-9,11-hexadecadienal; and / or (IV) (Z,E)-9,11-hexadecadienyl acetate; the method comprising culturing the cell culture described herein under conditions capable of producing the target compound by cell culture; and optionally recovering and / or isolating the target compound.

[0107] The cell culture can be cultured in a nutrient medium using methods known in the art under conditions suitable for the production of the target compound and / or its precursor and / or for the growth of the cell number. For example, the culture can be by shake flask culture or by small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentation) in a laboratory or industrial fermenter in a suitable medium under conditions in which the host cell can grow and / or proliferate and can be recovered and / or isolated if desired.

[0108] Cultivation can be carried out in a suitable nutrient medium containing a carbon source, a nitrogen source, and inorganic salts using procedures known in the art. Suitable media are available from commercial vendors or can be prepared according to published recipes (e.g., the catalog of the American Type Culture Collection). The selection of a suitable medium may be based on the selection of the host cell and / or on regulatory requirements for the host cell. Such media are available in the art. Optionally, the medium may contain additional components that favor the transformed expression host over other potentially contaminating microorganisms. Thus, in certain embodiments, a suitable nutrient medium contains a carbon source (e.g., glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellulosic biomass hydrolysate, etc.), a nitrogen source (e.g., ammonium sulfate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (e.g., yeast extract, malt extract, peptone, etc.), and an inorganic nutrient source (e.g., phosphate, magnesium, potassium, zinc, iron, etc.).

[0109] Cultivation of the host cells may be carried out over a period of from about 0.5 days to about 30 days. The cultivation process may be a batch process, a continuous process, or a fed-batch process and is suitably carried out at a temperature in the range of 0 to 100 °C or 10 to 80 °C, e.g., about 20 °C to about 50 °C and / or at a pH of about 2 to about 10. Preferred fermentation conditions for yeast and filamentous fungi are a temperature in the range of about 25 °C to about 55 °C and a pH of about 3 to about 9. Suitable conditions are usually selected based on the selection of the host cell. Thus, in certain embodiments, the method of the present disclosure further comprises one or more elements selected from: (I) culturing a cell culture in a nutrient medium; (II) culturing a cell culture under aerobic or anaerobic conditions; (III) culturing a cell culture with agitation; (IV) culturing a cell culture at a temperature of 25 to 50 °C; (V) culturing a cell culture at a pH of 3 to 9; and (VI) culturing a cell culture for 10 hours to 30 days.

[0110] The cell cultures of the present disclosure can be recovered and / or isolated using methods known in the art. For example, the target compound can be recovered from the nutrient medium by conventional methods including, but not limited to, centrifugation, filtration, spray drying, or lyophilization. In certain embodiments, the method includes a recovery and / or isolation step of separating the liquid phase of the cell or cell culture from the solid phase of the cell or cell culture to obtain a supernatant containing the target compound and / or subjecting the supernatant to one or more of the following steps: (I) disrupting the cells of the cell culture to release the target compound intracellularly into the supernatant; (II) separating the supernatant from the solid phase of the cell culture by, for example, filtration or gravity separation; (III) contacting the supernatant with one or more adsorption resins to obtain at least a portion of the generated target compound; (IV) contacting the supernatant with one or more ion exchange or reverse phase chromatography columns to obtain at least a portion of the target compound; (V) extracting the target compound; and / or (VI) precipitating the E11 fatty acyl compound by crystallization or evaporating the solvent of the liquid phase; and optionally isolating the target compound by filtration or gravity separation; Thereby, the target compound is recovered and / or isolated.

[0111] The methods described herein may include one or more in vitro steps during the process of manufacturing the target compound. Thus, in one embodiment, the method further includes supplying to the cell culture one or more precursors or substrates in the pathway of the target compound. If the target compound is not the desired final product, additional steps of chemically or biologically / enzymatically modifying the target compound, such as oxidation and / or acetylation, may be added to the methods of the present disclosure. Thus, in an attractive embodiment, the step performed in vitro includes chemically or enzymatically reducing (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol (Z9,E11-16:OH), preferably using a reductase enzyme (FAR). In another attractive embodiment, the step performed in vitro includes chemically or enzymatically reducing (Z,E)-9,11-hexadecadienoic acid to (Z,E)-9,11-hexadecadien-1-ol. In a further attractive embodiment, the step performed in vitro includes chemically or enzymatically oxidizing (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal. In a further attractive embodiment, the step performed in vitro includes chemically or enzymatically acetylating (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienyl acetate.

[0112] The method may further include recovering the target compound and mixing it with one or more carriers, agents, additives, adjuvants, and / or excipients to produce a biopesticide composition.

[0113] In this method, one or more carriers, drugs, additives, adjuvants and / or excipients preferably include a protecting agent containing conjugated sulfur that protects the target compound from being converted into an acid. Such protecting agents preferably include compounds selected from zinc pyrithione, 5-amino-1,3,4-thiadiazole-2-thiol, 2-thiazoline-2-thiol, 5-methyl-1,3,4-thiadiazole-2-thiol, 2-mercaptobenzimidazole, 2-mercapto-1-methylimidazole, and sodium pyrithione, and have been shown to have a significantly high effect on stabilizing fatty aldehydes from oxidation. In some embodiments, the method further includes mixing at least 10 mg of the protecting agent per gram of aldehyde and / or alcohol. One or more carriers, drugs, additives, adjuvants and / or excipients may include a carrier that promotes the sustained release of the target compound, and this carrier may be, as necessary, (i) a polymeric substrate selected from plastics, wax emulsions, oil emulsions, or microcapsules and / or (ii) zeolite.

[0114] In a preferred embodiment, the target compound is (Z,E)-9,11-hexadecadien-1-ol, (Z,E)-9,11-hexadecadienal, or (Z,E)-9,11-hexadecadienyl acetate (Z9,E11-16:OAc). In another aspect, a method for producing the biopesticide composition described herein is provided, comprising: (II) generating hexadecanoyl-CoA and culturing genetically modified yeast cells that express: a) a Δ9 desaturase that catalyzes the formation of a Z-configured double bond at position 9 of hexadecanoyl-CoA, thereby generating (Z)-9-hexadecenoyl-CoA; b) an E11 desaturase that catalyzes the formation of an E-type double bond at position 11 of (Z)-9-hexadecenoyl-CoA, thereby generating (Z,E)-9,11-hexadecadienoyl-CoA; c) An alcohol-forming fatty acyl-CoA reductase (FAR) that converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol; (IV) enzymatically or chemically converting (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal; and (V) optionally, recovering and / or isolating (Z,E)-9,11-hexadecadienal and, optionally, one or more of its precursors.

[0115] In some embodiments of this alternative aspect, the genetically modified yeast cells further express one or more enzymes selected from the following: a) A Z11 desaturase that catalyzes the formation of a double bond at the Z configuration (Z) at position 11 of hexadecanoyl-CoA, thereby producing (Z)-11-hexadecenoyl-CoA; b) One or more alcohol-forming fatty acyl-CoA reductases (FAR) that convert hexadecanoyl-CoA to hexadecan-1-ol, (Z)-9-hexadecenoyl-CoA to (Z)-9-hexadecen-1-ol, and (Z)-11-hexadecenoyl-CoA to (Z)-11-hexadecen-1-ol, respectively; And the method further comprises enzymatically or chemically converting hexadecan-1-ol to hexadecanal, (Z)-9-hexadecen-1-ol to (Z)-9-hexadecenal, and (Z)-11-hexadecen-1-ol to (Z)-11-hexadecenal, and optionally recovering and / or isolating hexadecanal, (Z)-9-hexadecenal, and (Z)-11-hexadecenal, and, optionally, one or more of its precursors.

[0116] In some embodiments, an alternative method for producing a biopesticide composition is provided, the method comprising the following steps: (I) culturing genetically modified yeast cells that produce hexadecanoyl-CoA and express the following: a. an E11 desaturase that catalyzes the formation of a double bond in the E configuration at position 11 of hexadecanoyl-CoA, thereby producing E-11-hexadecenoyl-CoA; b. a Δ9 desaturase that catalyzes the formation of a double bond in the Z configuration at position 9 of (E)-11-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA; c. an alcohol-forming fatty acyl-CoA reductase (FAR) that converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol (II) enzymatically or chemically converting (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal; and (III) optionally, recovering and / or isolating (Z,E)-9,11-hexadecadienal and, optionally, one or more of its precursors.

[0117] The success of the use of this alternative method is shown in Example 18, demonstrating the possibility of introducing an E11 double bond prior to the Z9 double bond in hexadecanoyl-CoA.

[0118] In some embodiments, there is provided a method wherein the genetically modified yeast cells further express one or more enzymes selected from the following: a. a Z11 desaturase that catalyzes the formation of a double bond in the Z configuration at position 11 of hexadecanoyl-CoA, thereby producing (Z)-11-hexadecenoyl-CoA; b. one or more alcohol-forming fatty acyl-CoA reductases (FAR) that convert hexadecanoyl-CoA to hexadecan-1-ol, (Z)-9-hexadecenoyl-CoA to (Z)-9-hexadecen-1-ol, and (Z)-11-hexadecenoyl-CoA to (Z)-11-hexadecen-1-ol, respectively; Furthermore, the method further comprises enzymatically or chemically converting hexadecan-1-ol to hexadecanal, (Z)-9-hexadecen-1-ol to (Z)-9-hexadecenal, and (Z)-11-hexadecen-1-ol to (Z)-11-hexadecenal, and / or recovering and / or isolating, optionally, hexadecanal, (Z)-9-hexadecenal and (Z)-11-hexadecenal, and, optionally, one or more of their precursors.

[0119] In other embodiments in this alternative aspect, the method further comprises mixing the recovered (Z,E)-9,11-hexadecadienal, (Z)-9-hexadecenal, (Z)-11-hexadecenal, hexadecanal, and, optionally, one or more of their precursors with one or more carriers, agents, additives, adjuvants and / or excipients to produce a biopesticide composition. These carriers, agents, additives, adjuvants and / or excipients preferably comprise one or more compounds selected from the following compounds: a) A conjugate sulfur compound selected from zinc pyrithione, 5-amino-1,3,4-thiadiazole-2-thiol, 2-thiazoline-2-thiol, 5-methyl-1,3,4-thiadiazole-2-thiol, 2-mercaptobenzimidazole, 2-mercapto-1-methylimidazole and sodium pyrithione, a protective agent that prevents the target compound from being converted to an acid; and / or b) A carrier that promotes slow release from a mixture of (Z,E)-9,11-hexadecadienal, (Z)-9-hexadecenal, (Z)-11-hexadecenal and / or hexadecanal, optionally a polymeric substrate selected from (i) plastics, wax emulsions, oil emulsions, or microcapsules and / or (ii) zeolite.

[0120] Composition In a further aspect, there is provided a biopesticide composition comprising a target compound selected from the following: a) (Z,E)-9,11-Hexadecadien-1-ol (Z9,E11-16:OH); b) (Z,E)-9,11-Hexadecadienal (Z9,E11-16: Ald); and / or c) (Z,E)-9,11-Hexadecadienyl acetate (Z9,E11-16:OAc); and one or more carriers, drugs, additives, adjuvants and / or excipients.

[0121] Yet another aspect provides a biopesticide composition comprising a target compound selected from (Z,E)-9,11-hexadecadienal and, optionally, one or more compounds selected from (Z)-9-hexadecena l, (Z)-11-hexadecena l and / or hexadecanal, in combination with one or more carriers, agents, additives, adjuvants and / or excipients. In certain embodiments, the biopesticide composition further comprises at least trace amounts of one or more compounds selected from hexadecan-1-ol, (Z)-9-hexadecen-1-ol, (Z)-11-hexadecen-1-ol, and (Z,E)-9,11-hexadecadien-1-ol. In other embodiments, the biopesticide composition further comprises (Z,E)-9,11-hexadecadienyl acetate. In a preferred embodiment, in the biopesticide composition, at least one or more of hexadecan-1-ol, (Z)-9-hexadecen-1-ol, (Z)-11-hexadecen-1-ol, and (Z,E)-9,11-hexadecadien-1-ol are obtained from the culture of the genetically modified host cell described herein, and optionally, the composition comprises one or more additional compounds or metabolites from the cell culture. Such compounds and / or metabolites of the cell culture include precursors of the target compound, as well as compounds selected from trace metals, vitamins, salts, yeast nitrogen base, carbon source, YNB, and / or amino acids for fermentation. In particular, the biopesticide composition comprises a concentration of the target compound of at least 1 mg / kg of the composition, such as at least 5 mg / kg, such as at least 10 mg / kg, such as at least 20 mg / kg, such as at least 50 mg / kg, such as at least 100 mg / kg, at least 500 mg / kg, at least 1,000 mg / kg, at least 5,000 mg / kg, at least 10,000 mg / kg, at least 50,000 mg / kg of the composition.

[0122] The composition may also advantageously include one or more protecting agents containing conjugated sulfur that prevent the further conversion of the target compound into an acid. Such protecting agents include compounds selected from zinc pyrithione, 5-amino-1,3,4-thiadiazole-2-thiol, 2-thiazoline-2-thiol, 5-methyl-1,3,4-thiadiazole-2-thiol, 2-mercaptobenzimidazole, 2-mercapto-1-methylimidazole, and sodium pyrithione. The composition preferably contains at least 10 mg of the protecting agent per gram of the target compound.

[0123] In some embodiments, the composition further comprises a carrier that promotes the sustained release of the target compound, and optionally, (i) a polymeric substrate selected from plastics, wax emulsions, oil emulsions, or microcapsules, and / or (ii) a carrier that is a zeolite.

[0124] In some embodiments, the biopesticide composition contains at least 50% bio-based carbon, 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%, such as at least 99%, such as at least 100% bio-based carbon.

[0125] In some embodiments, the biopesticide composition contains at least 20% bio-based carbon, such as at least 30% bio-based carbon, such as at least 40% bio-based carbon, such as at least 50% bio-based carbon, such as at least 60% bio-based carbon, such as at least 70 bio-based carbon, such as at least 75% bio-based carbon, such as at least 80% bio-based carbon, such as at least 85% bio-based carbon, such as at least 90% bio-based carbon, such as at least 95% bio-based carbon, such as 100% bio-based carbon.

[0126] In some embodiments, the biopesticide composition comprises 20% to 100% bio-based carbon, such as 30% to 100% bio-based carbon, such as 40% to 100% bio-based carbon, such as 50% to 100% bio-based carbon, such as 60% to 100% bio-based carbon, such as 70% to 100% bio-based carbon, such as 75% to 100% bio-based carbon, such as 80% to 100% bio-based carbon, such as 85% to 100% bio-based carbon, such as 90% to 100% bio-based carbon, such as 95% to 100% bio-based carbon, such as 100% bio-based carbon.

[0127] In some embodiments, the biopesticide composition comprises 50% or less fossil-based carbon, such as 45% or less, such as 40% or less, such as 35% or less, such as 30% or less, such as 25% or less, such as 20% or less, such as 15% or less, such as 10% or less, such as 5% or less, such as 1% or less fossil-based carbon.

[0128] In some embodiments, the biopesticide composition comprises 90% bio-based carbon, 91% bio-based carbon, 92% bio-based carbon, 93% bio-based carbon, 94% bio-based carbon, 95% bio-based carbon, 96% bio-based carbon, 97% bio-based carbon, 98% bio-based carbon, 99% bio-based carbon, or 100% bio-based carbon, such as 94% bio-based carbon.

[0129] In some embodiments, the biopesticide composition comprises Z9,E11-16:OH, which is at least 90% bio-based, such as at least 92% bio-based carbon, such as at least 94% bio-based carbon, such as 96% bio-based carbon, such as 98% bio-based carbon, such as 100% bio-based carbon, such as 94% bio-based.

[0130] Use A further aspect provides a method of controlling or monitoring pests, comprising applying the compositions described herein to the pests' habitat, enabling the target compounds to control the pests. The target compounds described herein are also in this case particularly effective against the sugarcane borer, and according to a preferred embodiment, the habitat is a sugarcane field and the pest is the sugarcane borer.

[0131] Sequence Listing This application includes a Sequence Listing created by PatentIn, which is included below, and is also electronically filed in ST26 format, the entire contents of which are hereby incorporated by reference into this specification. [Table 1] TIFF2025521689000002.tif242156TIFF2025521689000003.tif120159

[0132] Examples Example 1 - Construction of Biobricks and Plasmids All heterologous genes were synthesized for Y. lipolytica by GeneArt (Life Technologies) in codon-optimized versions. The genes were amplified by PCR using Phusion U Hot Start DNA Polymerase (ThermoFisher) or obtained by restriction enzyme digestion to obtain fragments for cloning into yeast expression vectors. The primers are listed in Table 1 and the resulting DNA fragments (biobricks) are listed in Table 2. The PCR products or restriction enzyme digestion reaction products were separated on a 1% agarose gel containing Midori Green Advance (Nippon Genetics Europe GmbH). The appropriately sized PCR / restriction enzyme digestion products were excised from the gel and purified using a Nucleospin Gel and PCR Clean-up kit (Macherey-Nagel).

[0133] The yeast vector containing the USER cassette was linearized with FastDigest SfaAI (ThermoFisher) at 37 °C for 2 hours and then nicked with Nb.Bsml (New England Biolabs) at 65 °C for 1 hour. The vector containing the resulting sticky ends was separated by gel electrophoresis, excised from the gel, and gel purified using the Nucleospin Gel and PCR Clean-up kit (Macherey-Nagel). The DNA fragment was cloned into the vector by USER cloning as described in (Holkenbrink, et al., 2018)(Jensen, et al., 2014). The USER reaction was transformed into chemically competent E. coli DHα cells, and the cells were seeded on lysogeny broth (LB) agar plates containing 100 mg / L ampicillin. The plates were incubated overnight at 37 °C, and the resulting colonies were screened by colony PCR. The plasmid was purified from an overnight E. coli liquid culture, and correct cloning was confirmed by sequencing. The constructed vectors are shown in Table 3.

Table 2

Table 3

Table 4

[0134] Example 2 - Construction of Yeast Strains The yeast strains were constructed by transformation of DNA vectors as described in (Holkenbrink, et al., 2018)(Jensen, et al., 2014). The strains were selected on yeast peptone dextrose (YPD) agar medium containing the appropriate antibiotic selection agent or on synthetic dropout medium (Sigma-Adrich) lacking specific amino acids. The correct genotype was confirmed by colony PCR and, if necessary, by sequencing. The resulting strains are shown in Table 4.

[0135] The strains marked with "***" were constructed as follows: The indicated genes were amplified with gene-specific primers containing a forward primer with a 5' overhang "ACTTTTTGCAGTACUAACCGCAG" and a reverse primer with a 3' overhang "CACGCGAU". The first "ATG" of the target gene sequence was omitted. These PCR products were cloned into an integration vector or an episomal vector together with BB9454 as described in (Holkenbrink, et al., 2018).

[0136] The strains marked with "****" were constructed as follows: The indicated genes were cloned into an integration vector or an episomal vector together with BB10398 and a synthetic minimal terminator (SEQ ID NO: 86) (ST13043) or the Y. lipolytica native LIP2 terminator (ST13149, ST13247) according to the "Golden-Gate Assembly" described in (Pryor, J.M., et al., 2020) or the user manual of the NEBridge Golden Gate Assembly Kit (New England BioLabs, Inc.). The first "ATG" of the target gene sequence was omitted. The episomal vector pBP10995 is derived from pCfB3405 (Holkenbrink et al. 2018) and is employed in the golden gate assembly.

Table 5

[0137] Example 3 - Cultivation of Strains and Analysis of Fatty Alcohols and Fatty Acid Methyl Esters (FAME) The Y. lipolytica 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) to 2.5 mL of YPG medium (10 g / L yeast extract, 10 g / L peptone, 40 g / L glycerol) in a 24-well plate (EnzyScreen) so that the initial OD600 was 0.2. The plate was incubated at 28 °C with shaking at 300 rpm. After 24 hours, the plate was centrifuged at 3,000 xg for 5 minutes at 4 °C. The supernatant was discarded and the cells were resuspended in 1.25 mL of production medium per well (50 g / L glycerol, 5 g / L yeast extract, 4 g / L KH2PO4, 1.5 g / L MgSO4, 0.2 g / L NaCl, 0.265 g / L CaCl2·2H2O, 2 mL / L trace element solution: 4.5 g / L CaCl2·2H2O, 4.5 g / L ZnSO4·7H2O, 3 g / L FeSO4·7H2O, 1 g / L H3BO3, 1 g / L MnCl2·4H2O, 0.4 g / L Na2MoO4·2H2O, 0.3 g / L CoCl2·6H2O, 0.1 g / L CuSO4·5H2O, 0.1 g / L KI, 15 g / L EDTA). Antibiotics were supplemented to the medium as needed. The plate was incubated at 28 °C with shaking at 300 rpm for 26 hours.

[0138] For fatty acid analysis, 1 mL was taken from each vial and centrifuged at 3,000 xg for 5 minutes at 4°C. Each pellet was extracted with 1 M HCl in 1000 μL of anhydrous methanol. The sample was vortexed for 20 seconds and placed in a water bath at 70°C for 2 hours. The sample was vortexed for 10 seconds every 30 minutes. After the sample was cooled to room temperature, 1000 μL of 1 M NaOH in anhydrous methanol, 500 μL of saturated NaCl aqueous solution, 990 μL of hexane, and 10 μL of 19:Me (10 mg / mL) as an internal standard were added. The sample was vortexed and centrifuged at 3,000 xg for 5 minutes at 21°C. The upper organic phase was analyzed by gas chromatography-mass spectrometry (GC-MS). For fatty alcohol analysis, 1 mL from each vial was collected by centrifuging at 3,000 xg for 5 minutes at 4°C. Each cell pellet was extracted with 1 ml of ethyl acetate:ethanol (84:15), and 10 μL of 19:Me (10 mg / mL) was added as an internal standard. The sample was vortexed for 20 seconds, incubated at room temperature for 1 hour, and then vortexed for 5 minutes. 300 μL of H2O was added to each sample. The sample was vortexed and centrifuged at 3,000 xg for 5 minutes at 21°C. The upper organic phase was analyzed by gas chromatography-mass spectrometry (GC-MS). The GC-MS analysis was performed on an Agilent 7820A GC combined with a mass selective detector Agilent 5977B. The GC was equipped with a DB Fatwax column (30 m × 0.25 mm × 0.25 μm), and helium was used as the carrier gas. The MS was operated in electron impact mode (70 eV), scanned from m / z: 30 to 400, and the injector was set at 220°C in split mode 20:1. The oven temperature was set at 80°C for 1 minute, then increased to 210°C at a rate of 20°C / min, held at 210°C for 7 minutes, and then increased to 230°C at a rate of 20°C / min. Compounds were identified by comparison with the retention times and mass spectra of reference compounds. The data was analyzed by Agilent Masshunter software.

[0139] Example 4 - Production of Z9, E11-16:CoA in yeast Y. lipolytica The newly identified desaturase Ds12389 from sugarcane borer was expressed in the Y. lipolytica strain ST6629 (Holkenbrink et al., 2020) to generate the ST12028 strain. The empty expression vector (pBP9002) was transformed into the same parental strain to obtain the ST10444 as a control strain. The cultivation of both strains and the extraction of fatty acids were carried out according to the method described in Example 3.

[0140] The FAME extract of strain ST12028 expressing Ds12389 (Figure 2A, dashed line) contained the double-unsaturated C16 fatty acid methyl ester (16-2:Me), which was not observed in the FAME extract of the control strain ST10444 (Figure 2A, dotted line). This 16-2:Me eluted at the same retention time (12.954 minutes) as the certified standard of Z9,E11-16:Me (Figure 2A, solid line), and the mass spectrum was consistent with the Z9,E11-16:Me standard substance (Figure 3). Furthermore, strain ST12028 produced 16:Me and Z9-16:Me (Figure 2C). The titer of Z9,E11-16:Me is shown in Table 5.

Table 6

[0141] Example 5 - Expression of D. punctatus desaturase in Y. lipolytica The desaturase Dpu_APSQ gene from D. punctatus was expressed in the Y. lipolytica strain ST6629 (Holkenbrink et al., 2020) to generate the ST10746 strain. The strain ST10444 having only the empty expression vector was used as a control strain.

[0142] The FAME extract of strain ST10746 expressing desaturase Dpu_APSQ did not contain a compound eluting at 12.954 minutes. Instead, a trace amount of an unknown double-unsaturated C16 fatty acyl CoA with a retention time of 12.985 minutes was detected (Figure 2B).

[0143] Example 6 - Production of Z9,E11-16:OH in Y. lipolytica Desaturase Ds12389 derived from Ostrinia furnacalis is co-expressed with fatty acyl-CoA reductase in Y. lipolytica. When this strain is cultured and samples are analyzed as described in Example 3, the fatty alcohol Z9,E11-16:OH is detected.

[0144] Example 7 - Production of (Z9,E11)-hexadecadienal A mixture of primary fatty alcohols containing 96 wt% (Z,E)-9,11-hexadecadien-1-ol (Z9,E11-16:OH) was used as a representative sample for the conversion to aldehyde. Z9,E11-16:OH (560 mg) was dissolved in 1 mL of acetonitrile in a 10 mL round-bottom flask equipped with a magnetic stir bar. Next, 39.0 mg of copper(I) triflate tetrakis(acetonitrile) (5 mol%), 16.0 mg of 2,2'-bipyridine (Bipy) (5 mol%), 9.0 mg of 4-hydroxy TEMPO (2.5 mol%), and 8.5 mg of N-methylimidazole (5 mol%) were added to the reaction mixture, and the mixture was stirred at 30 °C for 2 hours. The reaction mixture was extracted with 10 mL of heptane, and the acetonitrile layer was discarded. The heptane phase was washed with 5 mL of citric acid solution (0.15 wt% aqueous solution). Then, the upper heptane phase was evaporated under reduced pressure until a clear residue was formed, and 493 mg of a product containing 88.0 wt% (Z9,E11)-hexadecadienal (Z9,E11-16:Ald) was obtained.

[0145] Example 8 - Production of Z9,E11-16: acid and Z9,E11-16:OH in Saccharomyces cerevisiae The Ds12389 desaturase (SEQ ID NO: 1) derived from Saccharum officinarum moth is cloned into an S. cerevisiae gene expression vector, either alone or in combination with a fatty acyl reductase, and transformed into S. cerevisiae as described in Jensen, et al., 2014. Culturing of the strains and sample extraction are performed as in Example 3. Strains expressing only Ds12389 desaturase produce Z9,E11-16:Me, while strains additionally expressing the fatty acyl reductase gene produce Z9,E11-16:OH.

[0146] Example 9 - Conversion of Z9,E11-16:Me to Z9,E11-16:OH in Y. lipolytica by various fatty acyl reductases Fatty acyl-CoA reductases from various organisms were expressed in Y. lipolytica. The strains were cultured and fatty alcohol samples were extracted as described in Example 3. However, the culturing time in YPG medium was extended from 24 hours to 47 hours, and 0.2 μl of Z9,E11-16:Me was added to the production medium.

[0147] Extracts of strains ST12118 - ST12123, ST12125, ST12126, ST12129, ST12131 - ST12133, ST12138, ST12140, ST12141, ST12146, ST12151, ST12154, ST12156, ST12159, and ST12560 produced Z9,E11-16:OH. As an example, the GC-MS chromatograms and mass spectra of the extract of strain ST12118 and a pure standard of Z9,E11-16:OH are shown in FIGS. 4A and 4B,C, respectively. The extract of strain ST12118 contained 5 mg / L of Z9,E11-16:OH.

[0148] Example 10 - Production of a bio-based pheromone precursor mixture The main pheromone of sugarcane borer is (Z,E)-9,11-hexadecadienal. Minor components are (Z)-11-hexadecenal, (Z)-9-hexadecenal, and hexadecanal. The fatty alcohol precursors of both the main and minor components can be produced in a single yeast cell. To do this, co-express ΔE11 desaturase Ds12389 (SEQ ID NO: 1) with any of the ΔZ11-16 desaturases such as Desat16 from Amelyois transitella (SEQ ID NO: 72), Desat51 from Helicoverpa zea (SEQ ID NO: 78), Desat37 from Spodoptera exigua (SEQ ID NO: 74), and Desat38 from Spodoptera litura (SEQ ID NO: 76), as well as a suitable fatty acyl-CoA reductase known in the art, for example SEQ ID NO: 46. Culture the strain and extract the fatty alcohol sample as described in Example 3.

[0149] The extract of the strain contains (Z,E)-9,11-hexadecadien-1-ol, hexadecan-1-ol, (Z)-9-hexadecen-1-ol, and (Z)-11-hexadecen-1-ol.

[0150] Chemically oxidize the extract to obtain a composition containing (Z,E)-9,11-hexadecadienal, hexadecanal, (Z)-9-hexadecenal, and (Z)-11-hexadecenal.

[0151] The bio-based carbon content of this composition is determined by C14 radiocarbon dating.

[0152] Example 11 - Co-expression of ΔZ9-16 desaturase and Ds12389 (SEQ ID NO: 1) The ΔE11 desaturase Ds12389 is co-expressed with the ΔZ9-16 desaturase and, if necessary, co-expressed with a fatty acyl-CoA reductase. The strain is cultured and FAME and fatty alcohol samples are extracted as described in Example 3. The strain expressing Ds12389 and the ΔZ9-16 desaturase produces methyl (Z,E)-9,11-hexadecadienoate. Further expression of the fatty acyl CoA reductase results in the further production of (Z,E)-9,11-hexadecadien-1-ol.

[0153] Production of Z9,E11-16:CoA in Y. lipolytica by expression of Example 12-DsaDes1 (SEQ ID NO: 80) The newly identified desaturase DsaDes1 and Ds12389 from Spodoptera frugiperda were expressed in the Y. lipolytica strain ST12834, generating strains ST13043 and ST13042, respectively. The strain ST12834 is derived from ST6629 (Holkenbrink et al., 2020) and further expresses the heterologous NAD(P)H cytochrome b5 oxidoreductase Ncb5or (SEQ ID NO: 84) from Cydia pomonella (International Publication No. WO 2022 / 238404 A1). The empty expression vector (pBP9002) was transformed into the same parental strain ST12834 to obtain ST13046, which was used as a control strain. Cultivation of both strains and extraction of fatty acids were carried out as described in Example 3. The FAME extracts of strains ST13043 and ST13042 expressing DsaDes1 and Ds12389, respectively (Figure 5), contained a diunsaturated C16 fatty acid methyl ester (16-2:Me) that eluted at the same retention time as the Z9,E11-16:Me standard (Figure 5). The mass spectrum of this diunsaturated fatty acid 16-2:Me was consistent with the Z9,E11-16:Me standard (Figure 5). Z9,E11-16:Me was not detected in the FAME extract of the control strain ST13046 (Figure 5). The titer of Z9,E11-16:Me is shown in Table 6.

Table 7

[0154] Example 13 - Production of Z9,E11 - 16:OH in Y. lipolytica by co - expression of DsaDes1 and fatty acyl - CoA reductase Desaturase DsaDes1 derived from the sugarcane borer is co - expressed with fatty acyl - CoA reductase in Y. lipolytica. The strain is cultured and the samples are analyzed as described in Example 3, and the fatty alcohol Z9,E11 - 16:OH is detected.

[0155] Example 14 - Co - expression of ΔZ9 - 16 desaturase and DsaDes1 (SEQ ID NO: 80) ΔE11 desaturase DsaDes1 is co - expressed with ΔZ9 - 16 desaturase and, if necessary, with fatty acyl - CoA reductase. The strain is cultured and FAME and fatty alcohol samples are extracted as described in Example 3. Strains expressing DsaDes1 and ΔZ9 - 16 desaturase produce methyl (Z,E) - 9,11 - hexadecadienoate. When fatty acyl CoA reductase is expressed, furthermore, (Z,E) - 9,11 - hexadecadien - 1 - ol is produced.

[0156] Example 15 - Production of Z9,E11 - 16:Acid and Z9,E11 - 16:OH in Saccharomyces cerevisiae The desaturases Ds12389 (SEQ ID NO: 1) and DsaDes1 (SEQ ID NO: 80) derived from Saccharum officinarum borer were cloned into the S. cerevisiae gene expression vector and transformed into the S. cerevisiae CEN.PK strain as described in Jensen, et al., 2014. The Saccharomyces cerevisiae strain was inoculated from a synthetic dropout agar plate (lacking uracil, leucine, and histidine) into 2.5 mL of synthetic dropout medium (lacking uracil, leucine, and histidine) supplemented with 2% glucose in a 24-well plate (EnzyScreen) to an initial OD600 of 0.1 - 0.2. Sample extraction was performed as in Example 3. The derivatized FAME samples of the strains ST13093 and ST13150 expressing desaturases Ds12389 and DsaDes1, respectively, both contained Z9,E11-16:Me (Table 7), while the derivatized sample of the control strain ST12515 having only the empty expression vector did not contain any Z9,E11-16:Me.

Table 8

[0157] Example 16 - Production of Z9,E11-16:OH in Y. lipolytica by co-expression of DsaDes1 and fatty acyl-CoA reductase The desaturase DsaDes1 derived from sugarcane borer was co-expressed in the Y. lipolytica strain ST6629 (Holkenbrink et al., 2020) together with fatty acyl reductases from different insect species. The empty expression vector (pBP9002) was transformed into the strain ST13146 expressing only DsaDes1 to generate the control strain ST13151. The strains were cultured and the samples were analyzed as described in Example 3. The control strain ST13151 did not produce any fatty alcohol as expected. The strains ST13147, ST13152, ST13162, and ST13251 expressing the newly identified fatty acyl reductase DsaFAR1 from sugarcane borer (SEQ ID NO: 88), FAR1 from Helicoverpa armigera (SEQ ID NO: 46), FAR16 from Spodoptera exigua (SEQ ID NO: 56), and FAR25 from Tyta alba (SEQ ID NO: 95) produced 16:OH, Z11-16:OH, Z9-16:OH, and the target compound Z9,E11-16:OH, respectively (Table 8). The strain ST13147 expressing DsaFAR1 produced much less 16:OH than the strain expressing FAR1 (ST13152), the strain expressing FAR16 (ST13162), or the strain expressing FAR25 (ST13251). This property is advantageous for producing Z9,E11-16:OH in higher purity.

Table 9

[0158] Example 17 - Co-expression of ΔZ9-16 desaturase and DsaDes1 ΔE11 desaturase DsaDes1 (SEQ ID NO: 80) was co-expressed with the newly identified ΔZ9-16 desaturase DsaDes7 from sugarcane borer (SEQ ID NO: 82) to generate ST13149 in the Y. lipolytica strain ST6629 (Holkenbrink et al., 2020). The empty expression vector (pBP9002) was transformed into the strain ST13146 that expresses only DsaDes1, and ST13151 was obtained as a control strain. The strains were cultured, and FAME samples were extracted as described in Example 3. When DsaDes1 was expressed alone, the derivatized sample of the control strain ST13151 contained 2.0% of Z9,E11-16:Me and 10.1% of Z9-16:Me (Table 9). On the other hand, the derivatized FAME sample of the strain ST13149 that co-expresses DsaDes1 and ΔZ9-16 desaturase DsaDes7 contained 3.3% of Z9,E11-16:Me and 15.2% of Z9-16:Me. Due to the presence of DsaDes7, the purity of Z9-16:Me was improved by 50% and that of Z9,E11-16:Me was improved by 65%, respectively. Further expression of fatty acyl-CoA reductase can generate (Z,E)-9,11-hexadecadien-1-ol. This property is advantageous for achieving the production of Z9,E11-16:OH with higher purity.

Table 10

[0159] Example 18 - Conversion of E11-16:Me to Z9,E11-16:CoA in Y. lipolytica by ΔZ9 desaturase The ΔZ9 desaturase DsaDes7 (SEQ ID NO: 82) derived from Saccharum officinarum borer was expressed in the Y. lipolytica strain ST6629 (Holkenbrink et al., 2020) to generate the strain ST13247. The empty expression vector (pBP9002) was transformed into the same parental strain to obtain the control strain ST10444. Both strains were cultured as described in Example 3, and the cultures were carried out either with or without the additional addition of 0.2 g / L of E11-16:Me. After culturing, the samples were extracted and converted to fatty acid methyl esters as described in Example 3. When E11-16:Me was not added to the culture medium, the derivatized FAME samples of the strains ST10444 and ST13247 did not contain Z9,E11-16:Me. However, when the derivatized samples of both strains were supplemented with E11-16:Me, Z9,E11-16:Me was detected (Table 10). The sample of the ST13247 strain had a higher purity of Z9,E11-16:Me than the control strain ST10444. These results confirm that both the Saccharum officinarum borer ΔZ9 desaturase DsaDes7 and the native Y. lipolytica ΔZ9 desaturase OLE1 (SEQ ID NO: 17) can convert E11-16:CoA to Z9,E11-16:CoA.

Table 11

[0160] Example 19 - Production of Z9,E11-16:CoA in Yarrowia lipolytica using alternative E11 desaturases A synthetic protein variant of ΔE11 desaturase DsaDes1 was designed and expressed in the Y. lipolytica strain ST6629 (Holkenbrink et al., 2020), generating strains ST13284, ST13285, ST13286, ST13287, and ST13288. An empty expression vector (pBP9002) was transformed into the same parental strain to obtain the control strain ST10444. Cultivation of both strains and extraction of fatty acids were performed as described in Example 3. As expected, Z9,E11-16:Me was not detected in the FAME extract of the control strain ST10444. The FAME extracts of strains ST13284, ST13285, ST13286, ST13287, and ST13288 expressing the engineered variants Desat87 (SEQ ID NO: 96), Desat88 (SEQ ID NO: 98), Desat89 (SEQ ID NO: 100), Desat90 (SEQ ID NO: 102), and Desat91 (SEQ ID NO: 102), respectively, all contained the target compound Z9,E11-16:Me (Table 11). The sequence identities of the synthetic protein variants are shown in the table (Table 12). This example shows that a wide range of E11 desaturase sequence variants can be used to provide the desired E-alkene.

Table 12

Table 13

[0161] Example 20 - Measurement of Bio-based Carbon Content The bio-based carbon content of the fatty alcohol pheromone precursor was measured using an analytical measurement method that can be cited as "percent modern carbon (pMC)". This is the ratio of the isotope 14C in the sample compared to a modern standard sample (NIST 4990C). The bio-based carbon content percentage is calculated by applying a small adjustment factor to the pMC for the isotope 14C in today's atmospheric carbon dioxide. The genetically engineered strain STSCB for the production of Z9,E11-16:OH was cultured and the product was recovered. The product sample was composed of a mixture of fatty alcohols containing Z9,E11-16:OH. The "percent modern carbon (pMC)" of the product sample was analyzed by the standard test method "ASTM D6866". The modern carbon rate was measured to be 93.80 ± 0.33 pMC, corresponding to 94% of the bio-based carbon content (Table 13). This means that the pMC of Z9,E11-16:OH contained in the product sample is also 94%.

Table 14

[0162] Example 21 - Production of Z9,E11-16:OH in Saccharomyces cerevisiae As described in Maury, et al., 2016 and Jensen, et al., 2014, the E11 desaturase DsaDes1 (SEQ ID NO: 80) and fatty acyl-CoA reductase DsaFAR1 (SEQ ID NO: 88) from the sugarcane borer were cloned into an S. cerevisiae gene expression vector and transformed into the S. cerevisiae CEN.PK strain. The Saccharomyces cerevisiae strain was inoculated from a synthetic dropout agar plate (lacking uracil, leucine, and histidine) into 2.5 mL of synthetic dropout medium (lacking uracil, leucine, and histidine) supplemented with 2% glucose in a 24-well plate (EnzyScreen) such that the initial OD600 was 0.1 - 0.2. Sample extraction was performed as in Example 3. Strain ST13490, which expresses both DsaDes1 and DsaFAR1, produced Z9,E11-16:OH (Table 14). In contrast, control strain ST13491, which expresses only DsaDes1, did not produce any Z9,E11-16:OH. **Table 15**

[0163] References Holkenbrink, C., Dam, M.I., Kildegaard, K., Beder, J., Domenech, D.B., & Borodina, I. (2018). EasyCloneYALI: CRISPR / Cas9-Based Synthetic Toolbox for Engineering of the Yeast Yarrowia lipolytica. Biotechnol J. Holkenbrink, C., Ding, B.-J., Wang, H.-l., Dam, M.I., Petkevicius, K., Kildegaard, K.R., Borodina, I. (2020). Production of moth sex pheromones for pest control by yeast fermentation. Metab Eng., 312 - 321. Jensen, N., Strucko, T., Kildegaard, K., David, F., Maury, J., Mortensen, U., Borodina, I. (2014). EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae. FEMS Yeast Research, 238 - 48. Lienard, M., Lassance, J.-M., Wang, H.-L., Zhao, C.-H., Piskur, J., Johansson, T., & Lofstedt, C. (2010). Elucidation of the sex-pheromone biosynthesis producing 5,7-dodecadienes in Dendrolimus punctatus reveals 11- and 9-desaturases with unusual catalytic properties. Insect Biochemistry and Molecular Biology, 440 - 452. Zhao, C.-H., Adolf, R., & Lofstedt, C. (2004). Sex pheromone biosynthesis in the pine caterpillar moth, Dendrolimus punctatus: pathways leading to Z5-monoene and 5,7-conjugated diene components. Insect Biochemistry and Molecular Biology, 261 - 271. Da Silva, M., Cortes, A., Svensson, G., Lofstedt, C., Lima, E., & Zarbin, P. (2021). Identification of two additional behaviorally active gland constituents of female Diatraea saccharalis (Fabricius) (Lepidoptera Crambidae). Journal of the Brazilian Chemical Society. Holkenbrink, C., Dam, M. I., Kildegaard, K., Beder, J., Domenech, D. B., & Borodina, I. (2018). EasyCloneYALI: CRISPR / Cas9-Based Synthetic Toolbox for Engineering of the Yeast Yarrowia lipolytica. Biotechnol J. Holkenbrink, C., Ding, B.-J., Wang, H.-l., Dam, M. I., Petkevicius, K., Kildegaard, K. R., Borodina, I. (2020). Production of moth sex pheromones for pest control by yeast fermentation. Metab Eng., 312 - 321. Jensen, N., Strucko, T., Kildegaard, K., David, F., Maury, J., Mortensen, U.,... Borodina, I. (2014). EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae. FEMS Yeast Research, 238 - 48. Kalinova, B., Kindl, J., Hovorka, O., Hoskovec, M., & Svatos, A. (2005). (11Z)-hexadec-11-enal enhances the attractiveness of Diatraea saccharalis main pheromone component in wind tunnel experiments. Journal of Applied Entomology. Lienard, M., Lassance, J.-M., Wang, H.-L., Zhao, C.-H., Piskur, J., Johansson, T., & Lofstedt, C. (2010). Elucidation of the sex-pheromone biosynthesis producing 5,7-dodecadienes in Dendrolimus punctatus reveals 11- and 9-desaturases with unusual catalytic properties. Insect Biochemistry and Molecular Biology, 440 - 452. Svatos, A., Kalinova, B., Kindl, J., Kuldova, J., Hovorka, O., Rufino Do Nascimento, R., & Oldham, N. (2001). Chemical Characterization and Synthesis of the Major Component of the Sex Pheromone of the Sugarcane Borer Diatraea saccharalis. Collect. Czech. Chem. Commun., 1682 - 1690. Zhao, C.-H., Adolf, R., & Lofstedt, C. (2004). Sex pheromone biosynthesis in the pine caterpillar moth, Dendrolimus punctatus: pathways leading to Z5-monoene and 5,7-conjugated diene components. Insect Biochemistry and Molecular Biology, 261-271. Pryor, J.M., Potapov, V., Kucera, R.B., Bilotti, K., Cantor, E.J., Lohman, G.J.S. (2020). Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design. PloS ONE 15(9): e0238592.

[0164] Disclosed items The present disclosure further provides the following embodiments and items. Item 1. An E11 fatty acyl-CoA desaturase (E11 desaturase) comprising an amino acid sequence having at least 50% identity with the amino acid sequence of the E11 desaturase contained in SEQ ID NO: 1 or SEQ ID NO: 80. Item 2. The E11 desaturase according to Item 1, which introduces a double bond in the E configuration at position 11 in the presence of a (Z)-9-hexadecenoyl-CoA substrate, thereby generating (Z,E)-9,11-hexadecadienoyl-CoA. Item 3. A polynucleotide sequence codon-optimized for heterologous expression, or a homolog thereof containing mutations resulting from the degeneracy of the genetic code, encoding the E11 desaturase of Item 1 having the DNA sequence contained in SEQ ID NO: 2 or SEQ ID NO: 81. Item 4. A polynucleotide construct comprising the polynucleotide sequence of Item 3 operably linked to one or more control sequences. The polynucleotide construct according to item 4, wherein the control array is heterologous to the polynucleotide. Item 6. A vector comprising the polynucleotide construct according to item 4 or 5. Item 7. A genetically modified microbial cell that produces (Z,E)-9,11-hexadecadienoyl-CoA, heterologously expresses the E11 desaturase of item 1 or 2, and in the presence of a (Z)-9-hexadecenoyl-CoA substrate, introduces a double bond at position 11 in the (Z)-9-hexadecenoyl-CoA substrate in the E configuration, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA having a double bond in the E configuration at position 11. Item 8. (Z,E)-9,11-Hexadecadienoyl-CoA is a) (Z,E)-9,11-Hexadecadien-1-ol; b) (Z,E)-9,11-Hexadecadienal; and / or c) (Z,E)-9,11-Hexadecadienyl acetate, further comprising an effective biosynthetic pathway for converting to a target compound selected from, and said pathway is a) Alcohol-forming fatty acyl-CoA reductase (FAR) that converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol; b) An acetyltransferase that converts (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienyl acetate; c) Alcohol dehydrogenase that converts (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal; and / or d) A fatty alcohol oxidase that converts (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal, and the cell according to item 7, which expresses one or more pathway polypeptides selected from. Item 9. a) The FAR is at least 70% identical to the FAR contained in SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65; b) The acetyltransferase is at least 70% identical to the acetyltransferase contained in SEQ ID NO: 71; c) The alcohol dehydrogenase is at least 70% identical to the alcohol dehydrogenase contained in SEQ ID NO: 68; and d) The fatty alcohol oxidase is at least 70% identical to the fatty alcohol oxidase contained in SEQ ID NO: 69 or 70, the cell according to item 8. Item 10. Further comprising an effective biosynthetic pathway for generating (Z)-9-hexadecenoyl-CoA substrate, said pathway expressing one or more heterologous Δ9 desaturases that introduce a double bond in the Z configuration at position 9 into the hexadecenoyl-CoA substrate in the presence of the hexadecenoyl-CoA substrate, the cell according to items 7-9. Item 11. The Δ9 desaturase is at least 70% identical to the Δ9 desaturase contained in SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82, the cell according to item 10. Item 12. One or more native or endogenous genes are attenuated, disrupted and / or deleted, the cell according to items 7-11. Item 13. One or more pathway genes are overexpressed, the cell according to items 7 to 12. Item 14. Further genetically modified to effect an increase in the amount of substrate for at least one enzyme of the (Z,E)-9,11-hexadecadienal pathway, the cell according to items 7-13. Item 15. Further genetically modified to exhibit increased tolerance to one or more substrates, intermediates, or product molecules from the (Z,E)-9,11-hexadecadienal pathway, the cell according to items 7-14. Item 16. The cell according to any one of Items 7 to 15, comprising at least two copies of one or more genes in the (Z,E)-9,11-hexadecadienal pathway. Item 17. The host cell according to any one of Items 7 to 16, wherein the host cell is a fungal cell. Item 18. The host cell according to Item 17, wherein the fungal cell is a yeast cell. Item 19. The yeast cell belongs to a genus selected from Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces, and optionally, the yeast cell belongs to a species selected from Saccharomyces cerevisiae, Saccharomyces boulardii, Pichia pastoris, Kluyveromyces marxianus, Cryptococcus albidus, Lipomyces lipofera, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulan, and Yarrowia lipolytica. The host cell according to Item 18. Item 20. The fungal cells are Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, MucorThe cell according to item 17, which is a filamentous fungal cell selected from the species consisting of *miehei*, *Myceliophthora thermophila*, *Neurospora crassa*, *Penicillium purpurogenum*, *Phanerochaete chrysosporium*, *Phlebia radiata*, *Pleurotus eryngii*, *Thielavia terrestris*, *Trametes villosa*, *Trametes versicolor*, *Trichoderma harzianum*, *Trichoderma koningii*, *Trichoderma longibrachiatum*, *Trichoderma reesei*, and *Trichoderma viride*. Item 21. A genetically modified yeast cell that produces (Z,E)-9,11-hexadecadienoyl-CoA and (Z,E)-9,11-hexadecadien-1-ol, produces hexadecanoyl-CoA, and a. A Δ9 desaturase that catalyzes the formation of a double bond with a Z configuration at position 9 of hexadecanoyl-CoA, thereby producing (Z)-9-hexadecenoyl-CoA; b. An E11 desaturase that catalyzes the formation of a double bond with an E configuration at position 11 of (Z)-9-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA; and c. A genetically modified yeast cell that expresses an alcohol-forming fatty acyl-CoA reductase (FAR) that converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol. Item 22. a. A Z11 desaturase that catalyzes the formation of a double bond with a Z configuration at position 11 of hexadecanoyl-CoA, thereby producing (Z)-11-hexadecenoyl-CoA; b. The yeast cell according to item 21, further expressing one or more enzymes selected from one or more alcohol-forming fatty acyl-CoA reductases (FARs) that convert hexadecanoyl-CoA to hexadecan-1-ol, (Z)-9-hexadecenoyl-CoA to (Z)-9-hexadecen-1-ol, and (Z)-11-hexadecenoyl-CoA to (Z)-11-hexadecen-1-ol, respectively. Item 23.a) The E11 desaturase has the amino acid sequence contained in the E11 desaturase of SEQ ID NO: 1 or 80, b) The Δ9 desaturase has the amino acid sequence contained in the Δ9 desaturase of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82, c) The Z11 desaturase has the amino acid sequence contained in the Z11 desaturase of SEQ ID NO: 72, 74, 76, or 78, and d) The alcohol-forming fatty acyl-CoA reductase (FAR) has the amino acid sequence contained in SEQ ID NO: 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65, the yeast cell according to item 21 or 22. Item 24. The yeast cell according to item 21 or 23, wherein the yeast cell is Saccharomyces cerevisiae or Yarrowia lipolytica. Item 25. A cell culture composed of the genetically modified microbial cell according to items 7 to 24 and a growth medium. Item 26.a) (Z,E)-9,11-Hexadecadienoyl-CoA; b) (Z,E)-9,11-Hexadecadien-1-ol; c) (Z,E)-9,11-Hexadecadienal; and / or d) (Z,E)-9,11-Hexadecadienyl acetate; A method for producing a target compound selected from, culturing the cell culture according to item 25 under conditions that enable the production of the target compound, and, if necessary, recovering and / or isolating the target compound. Item 27. The method according to item 26, comprising externally supplying one or more substrates or precursors of the target compound pathway to the cell culture. Item 28. The method according to item 26 or 27, wherein one or more steps for producing the target compound are carried out in vitro. Item 29. The method according to items 26 to 28, wherein the step carried out in vitro comprises reducing (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol using a reductase enzyme (FAR). Item 30. The method according to items 26 to 28, wherein the step carried out in vitro comprises chemically or enzymatically reducing (Z,E)-9,11-hexadecadienoic acid to (Z,E)-9,11-hexadecadien-1-ol. Item 31. The method according to items 26 to 29, wherein the step carried out in vitro comprises chemically or enzymatically oxidizing (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal. Item 32. The method according to items 26 to 29, wherein the step carried out in vitro comprises chemically or enzymatically acetylating (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienyl acetate. Item 33. The method according to any one of items 26 to 32, further comprising recovering the target compound and mixing it with one or more carriers, drugs, additives, adjuvants and / or excipients to produce a biopesticide composition. Item 34. The method according to item 33, wherein the one or more carriers, drugs, additives, adjuvants and / or excipients comprise a protective agent containing conjugated sulfur that prevents the target compound from being converted to an acid. Item 35. The method according to item 34, wherein the protective agent comprises a compound selected from zinc pyrithione, 5-amino-1,3,4-thiadiazole-2-thiol, 2-thiazoline-2-thiol, 5-methyl-1,3,4-thiadiazole-2-thiol, 2-mercaptobenzimidazole, 2-mercapto-1-methylimidazole, and sodium pyrithione. Item 36. The method according to items 34 to 35, comprising mixing at least 10 mg of a protective agent per gram of aldehyde and / or alcohol. Item 37. The method according to items 33 to 37, wherein one or more carriers, agents, additives, adjuvants, and / or excipients comprise a carrier that promotes the sustained release of the target compound, and optionally, (i) a polymeric substrate selected from plastics, wax emulsions, oil emulsions, or microcapsules and / or (ii) a carrier that is a zeolite. Item 38. A method for producing a biopesticide composition, (I) generating hexadecanoyl-CoA, a. a Δ9 desaturase that catalyzes the formation of a Z-configuration double bond at position 9 of hexadecanoyl-CoA, thereby generating (Z)-9-hexadecenoyl-CoA; b. an E11 desaturase that catalyzes the formation of an E-configuration double bond at position 11 of (Z)-9-hexadecenoyl-CoA, thereby generating (Z,E)-9,11-hexadecadienoyl-CoA; c. culturing a genetically modified yeast cell that expresses an alcohol-forming fatty acyl-CoA reductase (FAR) that converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol: (II) converting (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal enzymatically or chemically; and (III) optionally, recovering and / or isolating (Z,E)-9,11-hexadecadienal and optionally one or more of its precursors. Item 39. The genetically modified yeast cell is a. A Z11 desaturase that catalyzes the formation of a double bond with a Z configuration at position 11 of hexadecanoyl-CoA, thereby generating (Z)-11-hexadecenoil-CoA; b. One or more enzymes selected from one or more alcohol-forming fatty acyl-CoA reductases (FARs) that convert hexadecanoyl-CoA to hexadecan-1-ol, (Z)-9-hexadecenoil-CoA to (Z)-9-hexadecen-1-ol, and (Z)-11-hexadecenoil-CoA to (Z)-11-hexadecen-1-ol, respectively; further expressing and enzymatically or chemically converting hexadecan-1-ol to hexadecanal, (Z)-9-hexadecen-1-ol to (Z)-9-hexadecenal, and (Z)-11-hexadecen-1-ol to (Z)-11-hexadecenal, and / or, if necessary, recovering and / or isolating hexadecanal, (Z)-9-hexadecenal and (Z)-11-hexadecenal, and, if necessary, one or more of their precursors; the method according to item 38, further comprising Item 40. Further, mixing the recovered (Z,E)-9,11-hexadecadienal, (Z)-9-hexadecenal, (Z)-11-hexadecenal, hexadecanal, and, if necessary, one or more of their precursors with one or more carriers, agents, additives, adjuvants, and / or excipients to produce a biopesticide composition; the method according to item 38 or 39, comprising Item 41. The carrier, agent, additive, adjuvant, and / or excipient is a) A conjugate sulfur compound selected from zinc pyrithione, 5-amino-1,3,4-thiadiazole-2-thiol, 2-thiazoline-2-thiol, 5-methyl-1,3,4-thiadiazole-2-thiol, 2-mercaptobenzimidazole, 2-mercapto-1-methylimidazole, and sodium pyrithione, a protective agent that prevents the target compound from being converted to an acid; and / or b) A carrier that promotes the sustained release from a mixture of (Z,E)-9,11-hexadecadienal, (Z)-9-hexadecenal, (Z)-11-hexadecenal and / or hexadecanal, optionally a polymer matrix selected from (i) plastics, wax emulsions, oil emulsions, or microcapsules and / or (ii) zeolites, the method according to item 40, comprising one or more compounds selected from the carriers. Item 42. A biopesticide composition comprising a target compound selected from (Z,E)-9,11-hexadecadienal and, optionally, one or more compounds selected from (Z)-9-hexadecenal, (Z)-11-hexadecenal and / or hexadecanal, in combination with one or more carriers, agents, additives, adjuvants and / or excipients. Item 43. The biopesticide composition according to item 42, further comprising at least a trace amount of one or more compounds selected from hexadecan-1-ol, (Z)-9-hexadecen-1-ol, (Z)-11-hexadecen-1-ol, and (Z,E)-9,11-hexadecadien-1-ol, and optionally other metabolites of cell culture. Item 44. A biopesticide composition further comprising (Z,E)-9,11-hexadecanediyl acetate. Item 45. One or more of hexadecanoyl-CoA, (Z)-9-hexadecenoyl-CoA, (Z)-11-hexadecenoyl-CoA, (Z,E)-9,11-hexadecadienoyl-CoA, hexadecan-1-ol, (Z)-9-hexadecen-1-ol, (Z)-11-hexadecen-1-ol, and (Z,E)-9,11-hexadecadien-1-ol are obtained from the methods described in items 26 to 41, and optionally one or more additional compounds or metabolites obtained from the cell culture described in item 25, the biopesticide composition according to items 42 to 44. Item 46. The biopesticide composition according to items 42 to 45, wherein the concentration of the target compound is at least 1 mg per kg of the composition. Item 47. The composition according to any one of Items 42 to 46, further comprising a protecting agent containing conjugated sulfur that prevents the target compound from being converted into an acid. Item 48. The composition according to Item 47, wherein the protecting agent contains a compound selected from zinc pyrithione, 5-amino-1,3,4-thiadiazole-2-thiol, 2-thiazoline-2-thiol, 5-methyl-1,3,4-thiadiazole-2-thiol, 2-mercaptobenzimidazole, 2-mercapto-1-methylimidazole, and sodium pyrithione. Item 49. The composition according to any one of Items 47 to 48, containing at least 10 mg of the protecting agent per gram of aldehyde and / or alcohol. Item 50. The composition according to any one of Items 42 to 49, further comprising a carrier that promotes the sustained release of the target compound, and optionally (i) a polymer substrate selected from plastics, wax emulsions, oil emulsions, or microcapsules and / or (ii) a carrier that is a zeolite. Item 51. A method for controlling or monitoring pests, comprising spraying the composition according to any one of Items 42 to 50 on the habitat of the pests to cause the target compound to control the pests. Item 52. The method according to Item 51, wherein the habitat is a sugarcane field and the pest is the sugarcane borer.

Claims

1. An E11 fatty acyl-CoA desaturase (E11 desaturase) comprising an amino acid sequence having at least 50% identity with the E11 desaturase contained in Accession No. 1, Accession No. 80, Accession No. 90, Accession No. 92, Accession No. 96, Accession No. 98, Accession No. 100, Accession No. 102, or Accession No.

104.

2. The E11 desaturase according to claim 1, wherein in the presence of a (Z)-9-hexadecenoyl-CoA substrate, the E11 desaturase introduces a double bond of the E configuration at position 11, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA.

3. A codon-optimized polynucleotide sequence for heterologous expression, encoding the E11 desaturase according to claim 1, having a DNA sequence contained in Accession No. 2, Accession No. 81, Accession No. 91, Accession No. 93, Accession No. 97, Accession No. 99, Accession No. 101, Accession No. 103, Accession No. 105, or a homolog thereof including mutations resulting from degeneracy of the genetic code.

4. A polynucleotide construct comprising the polynucleotide sequence according to claim 3 operably linked to one or more control sequences.

5. The polynucleotide construct according to claim 4, wherein the control sequence is heterologous to the polynucleotide.

6. A vector comprising the polynucleotide construct according to claim 4 or 5.

7. A genetically modified microbial cell that produces (Z,E)-9,11-hexadecadienoyl-CoA, which, in the presence of a (Z)-9-hexadecenoyl-CoA substrate, introduces a double bond of the E configuration at position 11 in the (Z)-9-hexadecenoyl-CoA substrate, thereby producing the (Z,E)-9,11-hexadecadienoyl-CoA having a double bond of the E configuration at position 11, and heterologously expresses the E11 desaturase according to claim 1 or 2.

8. The (Z,E)-9,11-hexadecadienoyl-CoA is a) (Z,E)-9,11-hexadecadien-1-ol; b) (Z,E)-9,11-hexadecadienal; and / or c) (Z,E)-9,11-hexadecadienyl acetate, and further comprises an effective biosynthetic pathway for converting to a target compound selected from the group consisting of, and the pathway is d) An alcohol-forming fatty acyl-CoA reductase (FAR) that converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol; e) An acetyltransferase that converts (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienyl acetate; f) A fatty alcohol oxidase that converts (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal, the cell according to claim 7, which expresses one or more pathway polypeptides selected from the group consisting of:

9. a) The FAR is at least 70% identical to a FAR contained in SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64, 66, 88, or 95; b) The acetyltransferase is at least 70% identical to the acetyltransferase contained in SEQ ID NO: 106; c) The fatty alcohol oxidase is at least 70% identical to the fatty alcohol oxidase contained in SEQ ID NO: 70, the cell according to claim 8.

10. The cell according to claims 7-9, further comprising an effective biosynthetic pathway for generating a (Z)-9-hexadecenoyl-CoA substrate, said pathway expressing one or more heterologous Δ9 desaturases that introduce a double bond in the Z configuration at position 9 of the hexadecenoyl-CoA substrate in the presence of the hexadecenoyl-CoA substrate.

11. The cell according to claim 10, wherein the Δ9 desaturase is at least 70% identical to a Δ9 desaturase contained in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82.

12. The cell according to claims 7-11, wherein one or more native or endogenous genes are attenuated, disrupted and / or deleted.

13. The cell according to claims 7-12, wherein one or more pathway genes are overexpressed.

14. The cell according to claims 7-13, further genetically modified to provide an increased amount of substrate for at least one enzyme of the (Z,E)-9,11-hexadecadienal pathway.

15. The cell according to claims 7 to 14, which is further genetically modified to show increased resistance to one or more substrates, intermediates, or product molecules from the (Z,E)-9,11-hexadecadienal pathway.

16. The cell according to claims 7 to 15, comprising at least two copies of one or more genes of the (Z,E)-9,11-hexadecadienal pathway.

17. The cell according to claims 7 to 15, comprising at least two copies of one or more genes of the (Z,E)-9,11-hexadecadien-1-ol pathway.

18. The host cell according to claims 7 to 17, wherein the host cell is a fungal cell.

19. The host cell according to claim 18, wherein the fungal cell is a yeast cell.

20. The yeast cell belongs to a genus selected from Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces, and optionally, the yeast cell belongs to a species selected from Saccharomyces cerevisiae, Saccharomyces boulardii, Pichia pastoris, Kluyveromyces marxianus, Cryptococcus albidus, Lipomyces lipofera, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulan, and Yarrowia lipolytica. The host cell according to claim 19.

21. The fungal cells are Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bacterioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium tricothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, MucorThe cell according to claim 18, which is a filamentous fungal cell selected from species consisting of miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpureogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.

22. A genetically modified yeast cell that produces (Z,E)-9,11-hexadecadienoyl-CoA and (Z,E)-9,11-hexadecadien-1-ol, produces hexadecanoyl-CoA, and a. A Δ9 desaturase that catalyzes the formation of a double bond with a Z configuration at position 9 of the hexadecanoyl-CoA, thereby producing (Z)-9-hexadecenoyl-CoA. b. An E11 desaturase that catalyzes the formation of an E-type double bond at position 11 of said (Z)-9-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA; c. A genetically modified yeast cell that expresses an alcohol-forming fatty acyl-CoA reductase (FAR) that converts said (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol. **Claim 23** a. A Z11 desaturase that catalyzes the formation of a Z-configured double bond at position 11 of said hexadecanoyl-CoA, thereby producing (Z)-11-hexadecenoyl-CoA; b. The yeast cell according to claim 22, further expressing one or more enzymes selected from one or more alcohol-forming fatty acyl-CoA reductases (FAR) that convert said hexadecanoyl-CoA to hexadecan-1-ol, said (Z)-9-hexadecenoyl-CoA to (Z)-9-hexadecen-1-ol, and said (Z)-11-hexadecenoyl-CoA to (Z)-11-hexadecen-1-ol, respectively. **Claim 24** a) The E11 desaturase has at least 70% sequence identity with the amino acid sequence contained in the E11 desaturase of SEQ ID NO: 1, 80, 90, 92, 96, 98, 100, 102, or 104; b) The Δ9 desaturase has at least 70% sequence identity with the amino acid sequence contained in the Δ9 desaturase of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82; c) The Z11 desaturase has at least 70% sequence identity with the amino acid sequence contained in the Z11 desaturase of SEQ ID NO: 72, 74, 76, or 78; and / or d) The alcohol-forming fatty acyl-CoA reductase (FAR) has at least 70% sequence identity with the amino acid sequence contained in the FAR of SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64, 66, 88, or 95. The yeast cell according to claim 22 or 23. **Claim 25** a) The E11 desaturase has the amino acid sequence contained in the E11 desaturase of SEQ ID NO: 1, 80, 90, 92, 96, 98, 100, 102, or 104; b) The Δ9 desaturase has an amino acid sequence contained in the Δ9 desaturase of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82; c) The Z11 desaturase has an amino acid sequence contained in the Z11 desaturase of SEQ ID NO: 72, 74, 76, or 78, and / or d) The alcohol-forming fatty acyl-CoA reductase (FAR) has an amino acid sequence contained in the FAR of SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64, 66, 88, or 95, the yeast cell according to claims 22-24.

26. The yeast cell according to claims 22-25, wherein the yeast cell is the species Saccharomyces cerevisiae or Yarrowia lipolytica.

27. A cell culture composed of the genetically modified microbial cell according to claims 7-26 and a growth medium.

28. a) (Z,E)-9,11-hexadecadienoyl-CoA b) (Z,E)-9,11-hexadecadien-1-ol; c) (Z,E)-9,11-hexadecadienal; and / or d) (Z,E)-9,11-hexadecadienyl acetate; A method for producing a target compound selected from the group consisting of culturing the cell culture according to claim 27 under conditions under which the cell culture can produce the target compound, and, if necessary, recovering and / or isolating the target compound.

29. The method according to claim 28, comprising externally supplying one or more substrates or precursors of the target compound pathway to the cell culture.

30. The method according to claim 28 or 29, wherein one or more steps of producing the target compound are carried out in vitro.

31. The step carried out in vitro comprises reducing (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol using a reductase enzyme (FAR), the method according to claims 28-30.

32. The step carried out in vitro comprises chemically or enzymatically reducing (Z,E)-9,11-hexadecadienoic acid to (Z,E)-9,11-hexadecadien-1-ol, the method according to claims 28-30.

33. The step carried out in vitro comprises a chemical or enzymatic oxidation of (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal, the method according to claims 28 to 31.

34. The step carried out in vitro comprises a chemical or enzymatic acetylation of (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienyl acetate, the method according to claims 28 to 31.

35. The method according to any one of claims 28 to 34, further comprising recovering the target compound and mixing it with one or more carriers, agents, additives, adjuvants and / or excipients to produce a biopesticide composition.

36. One or more of said carriers, agents, additives, adjuvants and / or excipients comprise a protective agent comprising conjugated sulfur that prevents the target compound from being converted to an acid, the method according to claim 35.

37. The protective agent comprises a compound selected from zinc pyrithione, 5-amino-1,3,4-thiadiazole-2-thiol, 2-thiazoline-2-thiol, 5-methyl-1,3,4-thiadiazole-2-thiol, 2-mercaptobenzimidazole, 2-mercapto-1-methylimidazole and sodium pyrithione, the method according to claim 36.

38. The method according to claim 36 or 37, comprising mixing at least 10 mg of the protective agent per gram of aldehyde and / or alcohol.

39. One or more of said carriers, agents, additives, adjuvants and / or excipients comprise a carrier that promotes the sustained release of the target compound, optionally a polymeric substrate selected from (i) plastics, wax emulsions, oil emulsions, or microcapsules and / or (ii) a carrier that is a zeolite, the method according to claims 35 to 38.

40. (I) generating hexadecanoyl-CoA, a. a Δ9 desaturase that catalyzes the formation of a double bond with a Z configuration at position 9 of the hexadecanoyl-CoA, thereby producing (Z)-9-hexadecenoic-CoA; b. An E11 desaturase that catalyzes the formation of a double bond with the E configuration at position 11 of the (Z)-9-hexadecenoyl-CoA, thereby generating (Z,E)-9,11-hexadecadienoyl-CoA; c. Culturing a genetically modified yeast cell that expresses an alcohol-forming fatty acyl-CoA reductase (FAR) that converts the (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol; (II) Converting the (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal, either enzymatically or chemically; and (III) Optionally, recovering and / or isolating the (Z,E)-9,11-hexadecadienal and, optionally, one or more of its precursors. A method comprising the steps.

41. The genetically modified yeast cell is a. A Z11 desaturase that catalyzes the formation of a double bond with the Z configuration at position 11 of hexadecanoyl-CoA, thereby generating (Z)-11-hexadecenoyl-CoA; b. One or more enzymes selected from one or more alcohol-forming fatty acyl-CoA reductases (FAR) that convert the hexadecanoyl-CoA to hexadecan-1-ol, the (Z)-9-hexadecenoyl-CoA to (Z)-9-hexadecen-1-ol, and the (Z)-11-hexadecenoyl-CoA to (Z)-11-hexadecen-1-ol, respectively, and Further comprising enzymatically or chemically converting the hexadecan-1-ol to hexadecanal, the (Z)-9-hexadecen-1-ol to (Z)-9-hexadecenal, and the (Z)-11-hexadecen-1-ol to (Z)-11-hexadecenal, and / or, optionally, recovering and / or isolating the hexadecanal, the (Z)-9-hexadecenal, and the (Z)-11-hexadecenal, and, optionally, one or more of their precursors. The method according to claim 40.

42. (I) Generating hexadecanoyl-CoA, a. An E11 desaturase that catalyzes the formation of a double bond with the E configuration at position 11 of the hexadecanoyl-CoA, thereby generating (E)-11-hexadecenoyl-CoA; b. A Δ9 desaturase that catalyzes the formation of a double bond with a Z configuration at position 9 of the (E)-11-hexadecenoil-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA; c. Culturing a genetically modified yeast cell expressing an alcohol-forming fatty acyl-CoA reductase (FAR) that converts the (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol; (II) Converting the (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal enzymatically or chemically; and (III) Optionally, recovering and / or isolating the (Z,E)-9,11-hexadecadienal and, optionally, one or more of its precursors, a method for producing a biopesticide composition.

43. The genetically modified yeast cell is a. A Z11 desaturase that catalyzes the formation of a double bond with a Z configuration at position 11 of the hexadecanoyl-CoA, thereby producing (Z)-11-hexadecenoil-CoA; b. One or more enzymes selected from one or more alcohol-forming fatty acyl-CoA reductases (FAR) that convert the hexadecanoyl-CoA to hexadecan-1-ol, the (Z)-9-hexadecenoil-CoA to (Z)-9-hexadecen-1-ol, and the (Z)-11-hexadecenoil-CoA to (Z)-11-hexadecen-1-ol, respectively, further expressing And further comprising enzymatically or chemically converting the hexadecan-1-ol to hexadecanal, the (Z)-9-hexadecen-1-ol to (Z)-9-hexadecenal, and the (Z)-11-hexadecen-1-ol to (Z)-11-hexadecenal, and / or optionally recovering and / or isolating the hexadecanal, the (Z)-9-hexadecenal, and the (Z)-11-hexadecenal, and, optionally, one or more of their precursors, the method according to claims 40-42.

44. The method according to claims 40 to 43, further comprising mixing the recovered (Z,E)-9,11-hexadecadienal, (Z)-9-hexadecenal, (Z)-11-hexadecenal, hexadecanal, and, optionally, one or more precursors thereof with one or more carriers, agents, additives, adjuvants, and / or excipients to produce the biopesticide composition.

45. The carrier, agent, additive, adjuvant, and / or excipient is a) a protective agent containing a conjugated sulfur compound selected from zinc pyrithione, 5-amino-1,3,4-thiadiazole-2-thiol, 2-thiazoline-2-thiol, 5-methyl-1,3,4-thiadiazole-2-thiol, 2-mercaptobenzimidazole, 2-mercapto-1-methylimidazole, and sodium pyrithione, which prevents the target compound from being converted to an acid; and / or b) one or more compounds selected from carriers that promote the sustained release from a mixture of (Z,E)-9,11-hexadecadienal, (Z)-9-hexadecenal, (Z)-11-hexadecenal, and / or hexadecanal, and optionally, (i) a polymeric substrate selected from plastics, wax emulsions, oil emulsions, or microcapsules and / or (ii) a carrier that is a zeolite, according to the method of claim 44.

46. A biopesticide composition comprising a target compound selected from (Z,E)-9,11-hexadecadienal and, optionally, one or more compounds selected from (Z)-9-hexadecenal, (Z)-11-hexadecenal, and / or hexadecanal, in combination with one or more carriers, agents, additives, adjuvants, and / or excipients.

47. The biopesticide composition according to claim 46, further comprising at least a trace amount of one or more compounds selected from hexadecan-1-ol, (Z)-9-hexadecen-1-ol, (Z)-11-hexadecen-1-ol, and (Z,E)-9,11-hexadecadien-1-ol, and optionally, other metabolites of the cell culture.

48. The biopesticide composition according to claim 46 or 47, further comprising (Z,E)-9,11-hexadecanediyl acetate.

49. One or more of said hexadecanoyl-CoA, (Z)-9-hexadecenoyl-CoA, (Z)-11-hexadecenoyl-CoA, (Z,E)-9,11-hexadecadienoyl-CoA, hexadecan-1-ol, (Z)-9-hexadecen-1-ol, (Z)-11-hexadecen-1-ol, and (Z,E)-9,11-hexadecadien-1-ol are obtained from the method according to claims 26 to 41, and optionally, said composition comprises one or more further compounds or metabolites obtained from the cell culture according to claim 25, the biopesticide composition according to claims 46 to 48.

50. The biopesticide composition according to claims 46 to 49, wherein said biopesticide composition comprises at least 20% bio-based carbon, such as at least 30% bio-based carbon, such as at least 40% bio-based carbon, such as at least 50% bio-based carbon, such as at least 60% bio-based carbon, such as at least 70% bio-based carbon, such as at least 75% bio-based carbon, such as at least 80% bio-based carbon, such as at least 85% bio-based carbon, such as at least 90% bio-based carbon, such as at least 95% bio-based carbon, such as 100% bio-based carbon.

51. The biopesticide composition according to claims 46 to 49, wherein said biopesticide composition comprises 20% to 100% bio-based carbon, such as 30% to 100% bio-based carbon, such as 40% to 100% bio-based carbon, such as 50% to 100% bio-based carbon, such as 60% to 100% bio-based carbon, such as 70% to 100% bio-based carbon, such as 75% to 100% bio-based carbon, such as 80% to 100% bio-based carbon, such as 85% to 100% bio-based carbon, such as 90% to 100% bio-based carbon, such as 95% to 100% bio-based carbon, such as 100% bio-based carbon.

52. The composition according to claims 46 to 49, wherein the composition contains at least 50% bio-based carbon, for example at least 55%, for example at least 60%, for example at least 65%, for example at least 70%, for example at least 75%, for example at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 99%, for example at least 100% bio-based carbon.

53. The composition according to claims 46 to 49, wherein the composition contains 90% bio-based carbon, 91% bio-based carbon, 92% bio-based carbon, 93% bio-based carbon, 94% bio-based carbon, 95% bio-based carbon, 96% bio-based carbon, 97% bio-based carbon, 98% bio-based carbon, 99% bio-based carbon, or 100% bio-based carbon, for example 94% bio-based carbon.

54. The composition according to claims 46 to 52, wherein the composition contains 50% or less fossil-based carbon, for example 45% or less, for example 40% or less, for example 35% or less, for example 30% or less, for example 25% or less, for example 20% or less, for example 15% or less, for example 10% or less, for example 5% or less, for example 1% or less fossil-based carbon.

55. The composition according to claims 46 to 54, wherein the concentration of the target compound is at least 1 mg per kg of the composition.

56. The composition according to claims 46 to 55, further comprising a protecting agent containing conjugated sulfur that protects against decomposition such as decomposition by conversion of the target compound to an acid.

57. The composition according to claim 56, wherein the protecting agent contains a compound selected from zinc pyrithione, 5-amino-1,3,4-thiadiazole-2-thiol, 2-thiazoline-2-thiol, 5-methyl-1,3,4-thiadiazole-2-thiol, 2-mercaptobenzimidazole, 2-mercapto-1-methylimidazole, and sodium pyrithione.

58. The composition according to claims 56 to 57, wherein the composition contains at least 10 mg of the protecting agent per gram of aldehyde and / or alcohol.

59. A carrier that promotes the sustained release of the target compound, and optionally further comprises a carrier that is (i) a polymer substrate selected from plastics, wax emulsions, oil emulsions, or microcapsules, and / or (ii) zeolite, the biopesticide composition according to claims 46 to 58.

60. A method for controlling or monitoring the pest, comprising spraying the biopesticide composition according to claims 46 to 59 on the habitat of the pest, and enabling the control of the pest by the target compound.

61. The method according to claim 60, wherein the habitat is a sugarcane field and the pest is the sugarcane borer.

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