Biopesticide composition

Abstract

The present invention relates to biopesticide compositions comprising a target compound selected from the group consisting of (Z, E)-9, 11-hexadecadienal, and optionally one or more compounds selected from the group consisting of (Z)-9-hexadecenal, (Z)-11-hexadecenal and / or hexadecanal, and one or more carriers, reagents, additives, adjuvants and / or excipients. The production of the target compound is to catalyze position 11 in (Z)-9-hexadecadienoyl coenzyme A to form an E-configuration double bond by using E11 desaturase from small sugarcane borer, thereby producing (Z, E)-9, 11-hexadecadienoyl coenzyme A. The invention further provides a preparation method of the (Z, E)-9, 11-hexadecadienoyl coenzyme A.

Description

Technical Field

[0001] The present disclosure describes bio-based biopesticide compositions comprising a sugarcane borer Diatraea saccharalis mating pheromone component, and methods of producing such biopesticide compositions. Also described herein are engineered cells and the enzymes they express, and methods of using such biopesticide compositions to control pests. Background Art

[0002] Integrated pest management (IPM) plays an increasingly important role in increasing crop yields, minimizing environmental impact and achieving organic food production. IPM employs alternative pest control methods, such as the use of pheromones to disrupt pest mating or mass trapping, or to attract beneficial insects.

[0003] Pheromones constitute a diverse group of chemical compounds that insects (like other organisms) use to communicate between individuals of the same species in a variety of contexts, including mating attraction, alarm, trail marking, and aggregation. Insect pheromones associated with long-distance mate finding have been used in agricultural and forestry applications to monitor and control pests as a safe and environmentally friendly alternative to pesticides. The biological production of pheromones for pest control has advantages over chemical synthesis in terms of price, specificity, and environmental impact.

[0004] Pheromones and pheromone precursors can be produced by genetically engineered cell factories that are modified to include pathways for expressing the enzymes necessary to convert cell precursor metabolites into the desired pheromones and pheromone precursors, as described in WO2021078452 and WO2021123128.

[0005] Known pheromones include fatty acyl alcohols, aldehydes and acetates with one or more double bonds at specific positions of the carbon backbone with specific Z or / and E orientations. The sugarcane borer, C. sciadipsa, is a major pest of sugarcane in Central and South America. The major component of its mating pheromone is (Z,E)-9,11-hexadecadienal (Svatos et al., 2001). Minor pheromone components 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) involved the sex pheromones (Z)-5-dodecenol and (Z,E)-5,7-dodecadienol of the moth Dendrolimus punctatus and studied the formation of the compounds produced when the labeled fatty acids were topically applied to the moth glands. The paper further speculated that the pathway for the sex pheromone of Dendrolimus punctatus involves the formation of an intermediate compound (Z,E)-9,11-hexadecadienoyl-CoA, but in fact, this compound was not detected in any of the experiments reported, despite the fact that it has been sought. Lienard et al. (2010) described yeast cells expressing a desaturase from Dendrolimus punctatus, named Dpu_APSQ. The authors speculated that the desaturase Dpu_APSQ introduces E11 desaturation into the naturally occurring (Z)-9-hexadecenoic acid in yeast, thereby producing the diunsaturated C16 fatty acid (Z,E)-9,11-hexadecadienoic acid. However, the present inventors found that Dpu_APSQ did not produce (Z,E)-9,11-hexadecadienoic acid, but instead produced another unknown di-unsaturated hexadecadienoic acid, which eluted slightly later than (Z,E)-9,11-hexadecadienoic acid.

[0007] Therefore, there is no bio-based method for producing C. serrata pheromones to date, and therefore, there is a need to identify enzymes capable of biosynthesis of pheromone precursors and to develop recombinant strains and methods for producing such pheromones. Summary of the invention

[0008] Until the present disclosure, there were no enzymes available for expression in genetically modified host cells for the introduction of an E11 double bond in (Z)-9-hexadecenoyl-CoA (Z9-16:CoA) - essential for the production of the primary mating pheromone component of the small sugar cane borer. However, disclosed herein is a novel enzyme that surprisingly catalyzes the desaturation of E11 in a (Z)-9-hexadecenoyl-CoA substrate and produces (Z,E)-9,11-hexadecadienoyl-CoA in the presence of this (Z)-9-hexadecenoyl-CoA substrate, which is a precursor of the small sugar cane borer pheromone (Z,E)-9,11-hexadecadienal (Z9,E11-16:Ald). In addition, the present disclosure provides a Δ9-desaturase capable of introducing a Z9 double bond in (E)-11-hexadecenoyl-CoA to provide (Z,E)-9,11-hexadecadienoyl-CoA. Therefore, the inventors have successfully prepared for the first time a bio-based biopesticide composition comprising a pheromone component of the small sugar cane borer, the bio-based biopesticide composition being formulated for controlling pests such as the small sugar cane borer. Therefore, in a first aspect of the present invention, a biopesticide composition is provided, the biopesticide composition comprising (Z,E)-9,11-hexadecadienal, and one or more compounds optionally 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 another aspect described herein, there is provided a method of controlling pests comprising distributing a composition described herein in a habitat of the pests and allowing the target compound to control the pests.

[0010] In another aspect described herein, a method for producing the biopesticide composition of the present disclosure is provided, comprising (I) culturing a genetically engineered yeast cell that produces palmitoyl-CoA and expressing

[0011] a) Δ9 desaturase, which catalyzes the formation of a Z-configuration double bond at position 9 in hexadecanoyl-CoA, thereby producing (Z)-9-hexadecenoyl-CoA;

[0012] b) E11 desaturase, which catalyzes the formation of an E-configuration double bond at position 11 in (Z)-9-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecenoyl-CoA;

[0013] c) Alcohol-forming fatty acyl-CoA reductase (FAR) converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol;

[0014] (II) enzymatically or chemically converting (Z,E)-9,11-hexadecadien-1-ol into (Z,E)-9,11-hexadecadienal; and

[0015] (III) optionally recovering and / or isolating (Z,E)-9,11-hexadecadienal and optionally one or more precursors thereof.

[0016] In yet another aspect described herein, a genetically engineered yeast cell produces (Z,E)-9,11-hexadecadienoyl-CoA and (Z,E)-9,11-hexadecadien-1-ol, the cell producing hexadecanoyl-CoA and expressing

[0017] a. Δ9 desaturase catalyzes the formation of a Z-configuration double bond at position 9 in hexadecanoyl-CoA, thereby producing (Z)-9-hexadecenoyl-CoA;

[0018] b. E11 desaturase catalyzes the formation of an E-configuration double bond at position 11 in (Z)-9-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA; and

[0019] c. Alcohol Formation Fatty acyl-CoA reductase (FAR) converts (Z,E)-9,11-hexadecadienyl-CoA to (Z,E)-9,11-hexadecadien-1-ol.

[0020] In yet another aspect described herein, a cell culture includes the genetically engineered microbial cells described herein and a growth medium.

[0021] In yet another aspect, described herein is an E11 desaturase having an amino acid sequence contained in the E11 desaturase of SEQ ID NO: 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A method for producing the major and minor pheromone components of the sugarcane borer, (Z,E)-9,11-hexadecadienal, is shown. The method includes the biosynthesis of (Z,E)-9,11-hexadecadien-1-ol and the subsequent chemical oxidation to the corresponding aldehyde (Z,E)-9,11-hexadecadienal. Minor components of the sugarcane borer can be produced following the same process shown due to side biochemical reactions.

[0023] Figure 2 shows GC-MS analysis of FAME extracts from Yarrowia lipolytica strains expressing C. caneborer and Dendrolimus punctatus desaturases. (A) Total ion chromatogram of FAME extracts from strains expressing Ds12389 C. caneborer desaturase (dashed line) compared with extracts from control strains transformed with empty vector (dashed line) and authentic standards of (Z,E)-9,11-hexadecadienoic acid methyl ester (Z9,E11-16:Me) (solid line). (B) Total ion chromatogram of FAME extracts from strains expressing Dpu_APSQ (dashed line) compared with extracts from control strains transformed with empty vector (dotted line) and authentic standards of Z9,E11-16:Me (solid line).

[0024] Figure 3 Mass spectra of the detected products from GC-MS chromatograms are shown. (A) Product eluting at 12.954 minutes from a FAME extract of a Yarrowia lipolytica strain expressing Ds12389. (B) Authentic standard of Z9, E11-16:Me.

[0025] Figure 4 shows (A) a representative GC chromatogram of an extract of strain ST12118 (cultivated in medium supplemented with Z9, E11-16:Me), which expresses the fatty acyl-CoA reductase HarFAR (also referred to herein as FAR1), and a Z9, E11-16:OH analytical standard. (B) Mass spectra of the ST12118 extract and (C) the Z9, E11-16:OH analytical standard at retention time 13.6-13.7 minutes.

[0026] Figure 5 shows representative GC chromatograms of extracts of strains ST13046, ST13042, and ST13043 (A) and Z9,E11-16:Me analytical standards. Mass spectra of extracts of ST13046 (B), ST13042 (C), ST13043 (D), and (E) Z9,E11-16:Me analytical standards with a retention time of 11.08 minutes.

[0027] Incorporation by Reference

[0028] All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict in a terminology herein and that of an incorporated reference, the terminology herein controls. DETAILED DESCRIPTION

[0029] definition

[0030] Throughout the present disclosure, when referring to a pheromone component or precursor, such as (Z,E)-9,11-hexadecadienal, it refers to a fatty aldehyde having a 16-carbon chain, an aldehyde at C1, a Z-configuration double bond at C9, and an E-configuration double bond at C11, alternative terms such as Z9,E11-16:Ald or (Z9,E11)-hexadecadienal can be used interchangeably. Similar nomenclature can be used for other pathway compounds, such as the corresponding fatty acids, CoA derivatives, alcohols, acids, or acetates.

[0031] The term "saturated" refers to compounds that have no double or triple carbon bonds.

[0032] The term "desaturated" used interchangeably with the term "unsaturated" herein with respect to compounds refers to compounds containing one or more double carbon-carbon bonds or triple carbon-carbon bonds, preferably double carbon-carbon bonds. The following nomenclature is used throughout this article: Δi desaturated compounds, where i is an integer, refer to compounds having a double carbon-carbon bond or a triple carbon-carbon bond at the i position of the carbon chain. Thus, the carbon chain length is at least equal to i. For example, a Δ12 desaturated compound refers to a compound having a double carbon-carbon bond or a triple carbon-carbon bond at the 12 position and a carbon chain length of 13 or longer. The double or triple bond can be in the E configuration or the Z configuration. Thus, an Ei or Zi desaturated compound refers to a compound having a double carbon-carbon bond in the E configuration or the Z configuration, respectively, at position i of the carbon chain, and the total length is at least equal to i. For example, an E11 fatty alcohol has desaturation at position 11 of the E configuration and a carbon chain length of 12 or longer.

[0033] As used herein, the term "Δ11 desaturase" refers to a desaturase that catalyzes the introduction of a double bond between the C11 and C12 positions of a saturated or desaturated fatty acyl compound, such as a fatty acyl-CoA having a carbon chain of at least 12 carbons.

[0034] As used herein, the term "E11 desaturase" refers to a Δ11 desaturase that catalyzes the introduction of a double bond in the E configuration at position 11 between C11 and C12 of a saturated or desaturated fatty acyl compound, such as a fatty acyl-coenzyme A (fatty acyl-CoA) having a carbon chain of at least 12 carbons.

[0035] As used herein, the term "Δ9 desaturase" refers to a desaturase that catalyzes the introduction of a double bond between the C9 and C10 positions of a saturated or desaturated fatty acyl compound, such as a fatty acyl-coenzyme A (fatty acyl-CoA) having a carbon chain of at least 10 carbons.

[0036] As used herein, the term "Z9 desaturase" refers to a Δ9 desaturase that catalyzes the introduction of a double bond in the Z configuration between the C9 and C10 positions of a saturated or desaturated fatty acyl compound, such as a fatty acyl-coenzyme A (fatty acyl-CoA) having a carbon chain of at least 10 carbons.

[0037] The term "biopesticide" as used herein is short for "biological pesticide" and refers to several types of pest management interventions: by predation, parasitism or chemical relationships. In the European Union, a biopesticide is defined as "a pesticide based on a microorganism or natural product". In the United States, the EPA defines them as "including natural substances that control pests (biochemical pesticides), microorganisms that control pests (microbial pesticides), and insecticidal substances produced by plants containing added genetic material (plant-derived protectants) or PIPs". The present disclosure is more specifically related to biopesticides that contain natural products or naturally occurring substances. In this context, these are made 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 part of integrated pest management (IPM) programs and have received widespread attention as an alternative to synthetic chemical plant protection products (PPPs). The Handbook of Biological Control Agents (2009: formerly the Handbook of Biopesticides) provides a review of available biopesticide (and other biologically based control) products.

[0038] As used herein, the term "biobased" is used to characterize biobased products where:

[0039] (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%.

[0040] As recognized by the Circular Biobased Europe Joint Commitment (CBE Joint Commitment) established in 2021, the development of biobased materials is essential if the EU is to achieve the climate goals set out in the European Green Deal. The present disclosure provides a method for efficiently providing fatty alcohols and fatty aldehydes with a high biobased carbon content (%).

[0041] Both fossil and renewable raw materials consist mainly of carbon (C). Carbon exists in several isotopic forms. 14 C is radioactive and occurs naturally in all living organisms (plants, animals, etc.) at a fixed relative concentration that is almost the same as the relative concentration in the atmosphere. 14 C concentration. At this concentration, 14 The radioactivity level of C is 100%. Once the organism is no longer alive, this concentration, as well as the radioactivity rate, decays, with a half-life of about 5700 years. Therefore, the radioactivity of the unknown substance 14 C levels can help determine the age of the carbon contained in the material.

[0042] "Young" carbon (0 to 10 years old) from renewable feedstocks (such as plants or animals) has relative isotopic14 C concentration, which is almost the same as that in the atmosphere 14 C concentration is the same, so the radioactivity of this young carbon 14 The C level is about 100%.

[0043] Isotopes of "ancient" carbon (millions of years old) from synthetic or fossil (petrified) sources 14 C is greatly reduced, as the ages of such synthetic and fossil sources far exceed the isotopic 14 The half-life of C is about 5700 years. Therefore, carbon from synthetic or fossil sources has a relative isotopic 14 C concentration, and therefore the radioactivity of this old carbon 14 C levels are approximately 0%.

[0044] In one embodiment, the term "radioactive 14 C level" refers to the total radioactivity of a given substance, product or combination 14 C level, as defined above.

[0045] isotope 14 The C method can be used to determine the concentration of young (renewable) materials compared to the concentration of old (fossil) resources. The carbon content of renewable feedstocks is called the "biobased carbon content." The carbon content or "biobased carbon content" of renewable feedstocks can be determined as follows.

[0046] When the biobased carbon content is measured, the result may be reported as "% Biobased Carbon." This represents the percentage of carbon that comes from "natural" (plant or animal byproduct) sources versus the percentage of carbon that comes from "synthetic" or "fossil" (petrochemical) sources. For reference, 100% Biobased Carbon means the material comes entirely from plants or animal byproducts, while 0% Biobased Carbon means the material does not contain any carbon from plants or animal byproducts. Values ​​in between represent a mixture of natural and fossil sources.

[0047] Example: If the product is radioactive 14 A C level of 80% means that the product is composed of 80% renewable carbon and 20% fossil carbon (C). In other words, the product is 80% biobased.

[0048] The analytical measurement may be called "percent modern carbon (pMC)". This is the amount of carbon present in the sample. 14 The % biobased carbon content is calculated by comparing the percentage of carbon dioxide in the air to the current reference standard (NIST 4990C). 14 C is calculated from pMC using a small adjustment factor. It is important to note that all 14Internationally recognized standards for pMC all assume that the plant or biomass raw material comes from the natural environment. pMC can be analyzed by standard test methods, such as "ASTM D6866".

[0049] The term "fatty acyl compound" as used herein refers to a fatty compound having a long fatty chain, i.e., a fatty chain generally having 12 to 28 carbon atoms, such as 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 may be saturated or desaturated. Fatty acyl compounds may include various functional end groups.

[0050] 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, wherein R is an aliphatic carbon chain having a carbon chain length of 12 to 28 carbon atoms, such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 carbon atoms. The aliphatic carbon chain is linked to the -SH group of CoA via a thioester bond. Fatty acyl-CoA can be saturated or desaturated, depending on whether the fatty acid from which it is derived is saturated or desaturated.

[0051] As used herein, the term "fatty alcohol" refers to an alcohol having a carbon chain length of 13 to 28 carbon atoms, such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 carbon atoms. The fatty alcohol can be saturated or unsaturated.

[0052] As used herein, the term "fatty alcohol acetate" refers to an acetate having an aliphatic carbon chain, i.e., an aliphatic chain having between 13 and 28 carbon atoms, such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 carbon atoms. The fatty acyl acetate may be saturated or desaturated.

[0053] As used herein, the term "fatty aldehyde" refers to an aldehyde having a carbon chain length of 13 to 28 carbon atoms, such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 carbon atoms. The fatty aldehyde can be saturated or desaturated.

[0054] As used herein, the term "functional variant" refers to a functional variant of an enzyme that retains at least some activity of the parent enzyme. Thus, a functional variant of a desaturase or other pathway enzyme catalyzes a reaction similar to that of its parent enzyme, although the efficiency and specificity of the reaction may be different, e.g., decreased or increased efficiency compared to the parent enzyme.

[0055] The terms "heterologous" or "recombinant" or "genetically modified" and their grammatical equivalents are used interchangeably herein for nucleotides, polypeptides, and cells to refer to entities "derived from a different species or cell." For example, a heterologous or recombinant polynucleotide gene is a gene that is not naturally present in the host cell, i.e., the gene is from a species or cell type different from the host cell. A heterologous or recombinant polypeptide is a polypeptide produced in a host cell that does not naturally contain the polypeptide, i.e., the polypeptide is from a species or cell type different from the host cell. When the terms herein refer to a host cell, they refer to a host cell that contains and expresses a heterologous or recombinant polynucleotide.

[0056] The term "% identity" is used herein to denote the relatedness between two amino acid sequences or two nucleotide sequences using standard alignment software known in the art and applying the settings indicated by the software, including gaps, to achieve the maximum percentage identity / similarity / homology and, if necessary, taking into account any conservative substitutions as part of sequence identity according to NCIUB rules (hftp: / / www.chem.qmul.ac.uk / iubmb / misc / naseq.html; NC-IUB, Eur J Biochem (1985)). 5' or 3' extensions or insertions (for nucleic acids) or N' or C' extensions or insertions (for polypeptides) generally result in a decrease in identity, similarity or homology using such standard software.

[0057] The term "degeneracy" used herein with respect to the genetic code reflects that many different variant nucleotide sequences can encode the same polypeptide because more than one nucleotide triplet exists as a codon for a given amino acid. Therefore, codons in the coding sequence of a given polypeptide can be modified using a codon bias table suitable for a particular host cell to obtain optimal expression in a particular host.

[0058] The term "pest" as used herein refers to organisms, especially animals, such as insects that are considered harmful to humans or animals or cultivated crops, especially in agricultural or livestock production. Pests are any invasive or prolific, harmful, troublesome, noxious, destructive organisms, whether to crops or animals, humans or human concerns, livestock, human structures, wild ecosystems, etc. Pests as used herein particularly refer to the sugarcane borer, the small sugarcane borer, and any of its previous life stages (larvae).

[0059] As used herein, the term "pheromone" refers to a naturally occurring signal compound used in nature for chemical communication between individuals of a species. For example, lepidopteran pheromones consist of an unbranched aliphatic chain (having between 9 and 18 carbon atoms, such as 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 carbon atoms) terminated with an alcohol, aldehyde or acetate functional group and containing up to 3 double bonds in the aliphatic backbone. Thus, pheromones typically contain desaturated fatty alcohols, desaturated fatty aldehydes and desaturated fatty alcohol acetates. Pheromone compositions can be produced chemically or biochemically, such as described herein. Thus, pheromones containing desaturated fatty alcohols, desaturated fatty aldehydes and / or desaturated fatty alcohol acetates can be obtained, for example, by the methods and cells described herein.

[0060] As used herein, the titer of a compound refers to the concentration of the compound produced. When the compound is produced by cells, the term refers to the total concentration produced by the cells, i.e., the total amount of compound divided by the volume of the culture medium. This means that, particularly for volatile compounds, the titer includes the portion of the compound that may evaporate from the culture medium, so the titer is determined by collecting the produced compound from the fermentation broth and potentially the off-gas of the fermentor.

[0061] The terms "pathway" or "biosynthetic pathway" or "metabolic pathway" used interchangeably herein refer to one or more enzymes acting synergistically in a living cell to convert one or more substrate precursors into chemical products. A pathway may include one or more enzymes that act sequentially or in combination. A pathway comprising only one enzyme may also be referred to herein as a "biotransformation" and is particularly useful in embodiments where a host cell is exogenously fed a precursor or substrate for enzymatic conversion to a desired end product. An enzyme is characterized by having catalytic activity that can change the chemical structure of a substrate. An enzyme may have more than one substrate and produce more than one product. An enzyme may also rely on cofactors, which may be inorganic chemical compounds or organic compounds (cofactors and / or coenzymes), which may or may not be considered part of a pathway.

[0062] As used herein, the term "in vivo" means within a living cell or organism, including, for example, an animal, a plant, or a microorganism.

[0063] As used herein, the term "in vitro" means outside a living cell or organism, including but not limited to, for example, in a microtiter plate, test tube, flask, beaker, tank, reactor, and the like.

[0064] The term "substrate" or "precursor" as used herein refers to any compound that can be converted into a different compound. For clarity, substrates and / or precursors include compounds produced in situ by enzymatic reactions in cells or exogenously provided compounds, such as exogenously provided organic molecules, which host cells can metabolize into desired compounds.

[0065] The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0066] 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 a control sequence that provides its expression. Expression vectors include expression cassettes for integrating genes into host cells and plasmids and / or chromosomes containing such genes.

[0067] The term "host cell" refers to any cell type susceptible to transformation, transfection, transduction, etc. of a nucleic acid construct or expression vector comprising a polynucleotide to be expressed in the host cell. Host cells include any progeny of a parent cell, including progeny that are not identical to the parent cell due to mutations that occur during replication.

[0068] The term "polynucleotide construct" refers to a single-stranded or double-stranded polynucleotide isolated from a naturally occurring gene, or modified to contain a nucleic acid fragment not found in nature or a synthetic polynucleotide, which comprises a polynucleotide encoding a polypeptide and one or more control sequences.

[0069] The term "operably linked" refers to a configuration in which a control sequence is placed at an appropriate position relative to a coding polynucleotide such that the control sequence directs the expression of the coding polynucleotide.

[0070] The terms "nucleotide sequence" and "polynucleotide" are used interchangeably herein.

[0071] The terms "include" and "comprising" and variations such as "including", "comprising", "comprising" and "comprising" used in the specification and appended items should be interpreted as inclusive. These words are intended to convey that other elements or integers not specifically listed may be included as long as the context permits.

[0072] The terms "a" and "an" used herein refer to one or more than one (ie, one or at least one) of the grammatical objects of the term. For example, "an element" can mean one element or more than one element.

[0073] The terms "preferred", "typically", "particularly" and "generally" as used herein are not intended to limit the scope of the invention or to imply that certain features are critical, necessary or even important to the structure or function of the invention. Instead, these terms are merely used to highlight alternative or additional features that may or may not be used in a particular embodiment of the invention.

[0074] The term "cell culture" as used herein refers to a culture medium containing a plurality of host cells described herein. The cell culture may include a single host cell strain or may include two or more different host cell strains. The culture medium may be any culture medium that may include a recombinant host, such as a liquid culture medium (i.e., a culture fluid) or a semi-solid culture medium, and may include other components, such as a carbon source; a nitrogen source; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases, etc.

[0075] As used herein, the term "endogenous" or "native" refers to a gene or polypeptide in a host cell that originates from the same host cell.

[0076] As used herein, the term "deletion" refers to the manipulation of a gene so that it is no longer expressed in the host cell.

[0077] As used herein, the term "disruption" refers to the manipulation of a gene, or any machinery involved in gene expression, so that it is no longer expressed in the host cell.

[0078] As used herein, the term "attenuation" refers to the manipulation of a gene or any mechanism involved in gene expression such that the expression of the gene is reduced compared to the expression that would otherwise occur.

[0079] Unless otherwise specified herein or clearly contradictory to the context, all methods described herein can be performed in any suitable order of steps. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and does not limit the scope of the claimed invention. Any language in the specification should not be construed as indicating any unclaimed element essential to the practice of the present invention.

[0080] Unless otherwise specified, all percentages, ratios and proportions herein are calculated by weight. Unless otherwise expressly specified, the weight percentage (weight %, also referred to as wt.%) of a component is based on the total weight of the composition containing the component (e.g., based on the total amount of the reaction mixture).

[0081] The term "substantially" or "approximately" or "about" as used herein refers to a reasonable deviation around a value or parameter so that the value or parameter does not change significantly. These deviation terms should be interpreted as including deviations of values, wherein the deviation does not negate the meaning of the value of the deviation. For example, relative to a reference value, a degree term can include a series of values ​​of the value plus or minus 10%. For example, deviation from a certain value can include a specified value plus or minus a specific percentage of the value, such as plus or minus 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the specified value.

[0082] As used herein, the term "and / or" is intended to mean an inclusive "or." The phrase X and / or Y is intended to mean X or Y as well as X and Y. Furthermore, the phrase X, Y and / or Z is intended to mean X, Y and Z alone or in any combination of X, Y and Z.

[0083] The term "isolated" used herein with respect to a compound refers to placing any compound in a form or environment different from the form or environment found in nature by human intervention. Isolated compounds include, but are not limited to, compounds of the present disclosure, wherein the compound is increased or decreased relative to the ratio of other components with which it is associated in nature. In an important embodiment, the amount of the compound is increased relative to other components with which the compound is associated in nature. In one embodiment, the compounds of the present disclosure can be separated into pure or substantially pure forms. In this context, substantially pure compounds refer to compounds separated from other foreign or unwanted materials that exist or are produced during the manufacturing process from the beginning of the production of the compound. This substantially pure compound preparation contains less than 10%, for example, less than 8%, for example, less than 6%, for example, less than 5%, for example, less than 4%, for example, less than 3%, for example, less than 2%, for example, less than 1%, for example, less than 0.5% of weight percentage, other foreign or unwanted substances that are usually associated with the compound when naturally or recombinantly expressed. In one embodiment, the isolated compound is at least 90% pure, such as at least 91% pure, such as at least 92% pure, such as at least 93% pure, such as at least 94% pure, such as at least 95% pure, such as at least 96% pure, such as at least 97% pure, such as at least 98% pure, such as at least 99% pure, such as at least 99.5% pure, such as 100% pure (by weight).

[0084] 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 intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before it appears as a mature, spliced ​​mRNA.

[0085] 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 usually determined by an open reading frame, which begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0086] The term "control sequence" as used herein refers to the nucleotide sequence necessary for the expression of a polynucleotide encoding a polypeptide. The control sequence can be a native sequence (i.e., from the same gene) or a heterologous sequence or a foreign sequence (i.e., from a different gene) of a polynucleotide encoding a polypeptide. Control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide coding sequence, a promoter sequence, a signal peptide coding sequence, a translation terminator (stop) sequence, and a transcription terminator (stop) sequence. To work, the control sequence must generally include a promoter sequence, a transcription and translation stop signal. The control sequence can be provided together with a joint to introduce a specific restriction site, thereby facilitating the connection of the control sequence to the coding region of the polynucleotide encoding the polypeptide.

[0087] Δ9 desaturase

[0088] The present disclosure also provides a Δ9 desaturase comprising an amino acid sequence that is a desaturase contained 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 contained 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, 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 to the Δ9 desaturase contained in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 82. The Δ9 desaturase of the present invention refers to a desaturase that catalyzes the introduction of a double bond between C9 and C10 of a saturated or desaturated fatty acyl compound (eg, fatty acyl-CoA having a carbon chain of at least 10 carbon atoms).

[0089] Δ9 gene

[0090] In another aspect, a polynucleotide sequence encoding a Δ9 desaturase (Δ9 desaturase gene) is provided, the polynucleotide sequence being at least 50% identical to the Δ9 desaturase coding sequence contained in SEQ ID NO: 83. In some embodiments, the Δ9 desaturase gene is 50% to 100% identical to the Δ9 desaturase gene contained in SEQ ID NO: 83, 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 Δ9 desaturase gene encodes the Δ9 desaturase of SEQ ID NO: 82 or a functional variant thereof. In other embodiments, the Δ9 desaturase gene is codon-optimized for heterologous expression in a microorganism, particularly yeast.

[0091] E11 desaturase

[0092] In one aspect, an E11 fatty acyl-CoA desaturase (E11 desaturase) is provided, 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 a more specific embodiment, the E11 desaturase catalyzes the introduction of an E-configuration double bond at position 11 in a (Z9)-9-hexadecenoyl-CoA substrate, wherein the (Z,E)-9,11-hexadecadienoyl-CoA has a Z-configuration double bond at position 9 and an E-configuration double bond at position 11.

[0093] In another aspect, there is provided an E11 fatty acyl-CoA desaturase (E11 desaturase) comprising an amino acid sequence, the amino acid sequence being the E11 desaturase comprised in SEQ ID NO: 1 or 80, or being a functional variant that is at least 50% identical to the E11 desaturase comprised in SEQ ID NO: 1 or 80. In some embodiments, the E11 desaturase is 50% to 100% identical to the E11 desaturase comprised 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 to the E11 desaturase comprised in SEQ ID NO: 1 or 80. In a more specific embodiment, the E11 desaturase catalyzes the introduction of a double bond in the E configuration at position 11 in a (Z9)-9-hexadecenoyl-CoA substrate to provide a (Z,E)-9,11-hexadecadienoyl-CoA having a double bond in the Z configuration at position 9 and a double bond in the E configuration at position 11.

[0094] In another aspect, an E11 fatty acyl-CoA desaturase (E11 desaturase) is provided, comprising an amino acid sequence, the amino acid sequence being the E11 desaturase contained in SEQ ID NO: 80, or being 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% 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 to the E11 desaturase contained in SEQ ID NO: 80. In a more specific embodiment, the E11 desaturase catalyzes the introduction of a double bond in the E configuration at position 11 in a (Z9)-9-hexadecenoyl-CoA substrate, thereby providing (Z,E)-9,11-hexadecadienoyl-CoA having a double bond in the Z configuration at position 9 and a double bond in the E configuration at position 11.

[0095] In some embodiments, an E11 fatty acyl-CoA desaturase (E11 desaturase) is provided, comprising an amino acid sequence having at least 50% identity, 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 to the E11 desaturase contained in SEQ ID NO: 1, SEQ ID NO: 80, SEQ ID NO: 90 or SEQ ID NO: 92.

[0096] In some embodiments, an E11 fatty acyl-CoA desaturase (E11 desaturase) is provided, comprising an amino acid sequence having at least 50% identity, 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%, or even 100% identical to 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.

[0097] E11 gene

[0098] In another aspect, a polynucleotide sequence encoding an E11 desaturase (E11 desaturase gene) is provided, the polynucleotide sequence being at least 50% identical to the E11 desaturase coding sequence contained in SEQ NO:2. In some embodiments, the E11 desaturase gene is 50% to 100% identical to the E11 desaturase gene contained in SEQ ID NO:2 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 IN NO:1 or said functional variant. In other embodiments, the E11 desaturase gene is codon optimized for heterologous expression in microorganisms, particularly yeast.

[0099] In another aspect, a polynucleotide sequence encoding an E11 desaturase (E11 desaturase gene) is provided, the polynucleotide sequence being at least 50% identical to the E11 desaturase coding sequence contained in SEQ 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 IN NO: 1 or 80 or said functional variant. In other embodiments, the E11 desaturase gene is codon optimized for heterologous expression in a microorganism, particularly yeast.

[0100] In another aspect, a polynucleotide sequence encoding an E11 desaturase (E11 desaturase gene) is provided, the polynucleotide sequence being at least 50% identical to the E11 desaturase coding sequence contained in SEQ 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 said functional variant. In other embodiments, the E11 desaturase gene is codon optimized for heterologous expression in a microorganism, particularly yeast.

[0101] In some embodiments, a polynucleotide sequence encoding an E11 desaturase disclosed herein that is codon-optimized for heterologous expression is provided, having a DNA sequence contained in SEQ NO:2, SEQ ID NO:81, SEQ ID NO:91 or SEQ ID NO:93, or a homolog thereof including variations 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 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.

[0102] In some embodiments, a polynucleotide sequence encoding an E11 desaturase disclosed herein that is codon-optimized for heterologous expression is provided, having a DNA sequence contained in SEQ NO:2, SEQ ID NO:81, SEQ ID NO:91 or SEQ ID NO:93, or a homolog thereof including variations due to the degeneracy of the genetic code. In some embodiments, the E11 desaturase gene is 50% to 100% identical, 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 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.

[0103] Z11 desaturase

[0104] In some embodiments, the disclosure provides a Z11 desaturase having at least 70% sequence identity to an amino acid sequence comprised in a Z11 desaturase of SEQ ID NO: 72, 74, 76, or 78. In some embodiments, the Z11 desaturase is expressed in a genetically engineered host cell of the disclosure. In some embodiments, a Z11 desaturase having at least 70% sequence identity to an amino acid sequence comprised in a Z11 desaturase of SEQ ID NO: 72, 74, 76, or 78 is used in a method disclosed herein. In some embodiments, the Z11 desaturase has 70% to 100% sequence identity, 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 to an amino acid sequence comprised in a Z11 desaturase of SEQ ID NO: 72, 74, 76, or 78.

[0105] Gene constructs

[0106] On the other hand, a polynucleotide construct comprising the E11 desaturase gene operably linked to one or more control sequences is provided. Such control sequences can be native sequences or heterologous sequences of the E11 desaturase gene. The polynucleotide construct can be further integrated into an expression vector to express the E11 desaturase gene in a host cell.

[0107] Genetically engineered host cells

[0108] In another aspect, a genetically engineered microbial cell producing (Z,E)-9,11-hexadecadienoyl-CoA is provided, the cell heterologously expressing an E11 desaturase of the present disclosure, which, in the presence of a (Z)-9-hexadecadienoyl-CoA substrate, introduces a double bond of E configuration at position 11 in the (Z)-9-hexadecadienoyl-CoA substrate, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA having a double bond of E configuration at position 11. In addition, the present disclosure provides a genetically engineered microbial cell as defined herein, which expresses a Δ9-desaturase capable of introducing a Z9 double bond in (E)-11-hexadecadienoyl-CoA to provide (Z,E)-9,11-hexadecadienoyl-CoA.

[0109] The cells provided herein can further comprise an efficient biosynthetic pathway for converting (Z,E)-9,11-hexadecadienoyl-CoA into a target compound selected from the group consisting of

[0110] a) (Z,E)-9,11-hexadecadien-1-ol;

[0111] b) (Z,E)-9,11-hexadecadienal; and / or

[0112] c) (Z,E)-9,11-hexadecadienyl acetate;

[0113] The pathway expresses one or more pathway polypeptides selected from the group consisting of:

[0114] a) Alcohol-forming fatty acyl-CoA reductase (FAR), which converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol;

[0115] b) acetyltransferase, converting (Z,E)-9,11-hexadecadien-1-ol into (Z,E)-9,11-hexadecadienyl acetate;

[0116] c) an alcohol dehydrogenase, which converts (Z,E)-9,11-hexadecadien-1-ol into (Z,E)-9,11-hexadecadienal; and / or

[0117] d) Fatty alcohol oxidase, which converts (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal.

[0118] In some embodiments, the cells provided herein further comprise an efficient biosynthetic pathway for converting (Z,E)-9,11-hexadecadienoyl-CoA into a target compound selected from a) (Z,E)-9,11-hexadecadien-1-ol;

[0119] b) (Z,E)-9,11-hexadecadienal; and / or

[0120] c) (Z,E)-9,11-hexadecadienyl acetate;

[0121] The pathway expresses one or more pathway polypeptides selected from the group consisting of:

[0122] d) alcohol-forming fatty acyl-CoA reductase (FAR), which converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol;

[0123] e) acetyltransferase converts (Z,E)-9,11-hexadecadien-1-ol into (Z,E)-9,11-hexadecadienyl acetate;

[0124] f) Fatty alcohol oxidase converts (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal.

[0125] In some embodiments, a cell is provided, wherein:

[0126] a) the 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;

[0127] b) an acetyltransferase that is at least 70% identical to the acetyltransferase contained in SEQ ID NO: 106;

[0128] c) the fatty alcohol oxidase is at least 70% identical to the fatty alcohol oxidase contained in SEQ ID NO:70.

[0129] When cells express enzymes of this pathway,

[0130] (I) the FAR is preferably at least 70% identical, such as at least 80%, such as at least 90%, such as at least 95%, such as 100% 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; (II) the acetyltransferase is preferably at least 70% identical, such as at least 80%, such as at least 90%, such as at least 95%, such as 100% identical to the acetyltransferase contained in SEQ ID NO:71; (III) the alcohol dehydrogenase is preferably at least 70% identical, such as at least 80% identical, such as at least 90% identical, such as at least 95% identical, such as 100% identical to the alcohol dehydrogenase contained in SEQ ID NO:68; and

[0131] The (IV) fatty alcohol oxidase is preferably at least 70% identical to the fatty alcohol oxidase comprised in SEQ ID NO: 69 or 70, such as at least 80%, such as at least 90%, such as at least 95%, such as 100% identical.

[0132] In some embodiments,

[0133] (I) a FAR that 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) an acetyltransferase that is at least 70% identical to the acetyltransferase contained in SEQ ID NO:71;

[0134] (III) the alcohol dehydrogenase is at least 70% identical to the alcohol dehydrogenase contained in SEQ ID NO:68; and

[0135] (IV) the fatty alcohol oxidase is at least 70% identical to the fatty alcohol oxidase contained in SEQ ID NO:69 or 70.

[0136] The genetically modified host cell may also comprise an efficient biosynthetic pathway for producing a (Z)-9-hexaenoyl-CoA substrate, in particular a pathway expressing one or more heterologous Δ9 desaturases that introduce a double bond into the hexaenoyl-CoA substrate in the Z configuration at position 9. Such Δ9 desaturases are suitably at least 70% identical, such as at least 80%, such as at least 90%, such as at least 95%, such as 100% identical to the Δ9 desaturase contained in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 82.

[0137] The genetically modified host cell may also comprise an efficient biosynthetic pathway for producing a (Z)-9-hexaenoyl-CoA substrate, in particular a pathway expressing one or more heterologous Δ9 desaturases that introduce a double bond into the hexaenoyl-CoA substrate in the Z configuration at position 9. Such Δ9 desaturases are suitably at least 70% identical, such as at least 80%, such as at least 90%, such as at least 95%, such as 100% identical to the Δ9 desaturase contained in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21 or 22.

[0138] In a further embodiment, the host cell is further modified so that one or more natural or endogenous genes are weakened, destroyed and / or deleted. Other modifications include overexpression of one or more pathway genes or providing an increased amount of substrate for modification of at least one enzyme of pathways described herein. Genetically modified host cells can also be further modified to show increased tolerance to one or more substrates, intermediates or product molecules of pathways described herein and / or host cells can include at least two copies of one or more genes in such pathways. In some embodiments, host cells can include at least two copies of one or more genes in (Z, E)-9,11-hexadecadiene-1-ol pathway.

[0139] Host cells provided herein are suitably fungal cells, such as yeast cells. Preferred yeasts include yeasts belonging to and selected from the following genus: Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipozyme, wherein the yeast cell optionally belongs to a species selected from Saccharomyces cerevisiae, Saccharomyces boulardii, Pichia, Kluyveromyces marxianus, Cryptococcus albus, oleaginous lipozyme, Starch lipozyme, Rhodosporidium toruloides, Rhodotorula glutinosus, budding Trichosporon and Yarrowia lipolytica.

[0140] However, filamentous fungal cells may also be useful, for example, selected from Aspergillus awamori, Aspergillus odoratus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus nicotianae, Pseudomonas carnegiei, Pseudomonas spp., Pseudomonas mycotoxins, White rot fungi, Pseudomonas flavoceae, Pseudomonas curvatum, Chrysosporium inops, Chrysosporium hornophilum, Chrysosporium rukvenii, Chrysosporium feces, Chrysosporium rukii, Chrysosporium quenslandii, Chrysosporium tropicalis, Chrysosporium zonulensis, Coprinus cinerea, Coriolus versicolor, Bacteroidetes, Fusarium cereus, Fusarium Crookwellii, Fusarium flavosum, Fusarium graminearum, Fusarium graminearum, Fusarium heterosporum, Fusarium negundii, Fusarium oxysporum, Fusarium reticularis, Fusarium roseum, Fusarium elder, Fusarium carnosus, Fusarium ramosissima, Fusarium sulphureus, Fusarium toro, Fusarium monoterminale, Fusarium venenatum, Humicola insolens, Humicola sparsely hairy, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium, Penicillium purpurogenum, Phanerochaete chrysosporium, Veins radiata, Pleurotus eryngii, Thielavia terrestris, Trametes velutipes, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei and Trichoderma viride.

[0141] In an alternative or additional aspect, provided herein is a genetically engineered yeast cell that produces (Z,E)-9,11-hexadecadienoyl-CoA and (Z,E)-9,11-hexadecadien-1-ol, the cell producing hexadecanoyl-CoA and expressing

[0142] a. Δ9 desaturase, catalyzing the formation of a Z-configuration (Z) double bond at position 9 in hexadecanoyl-CoA, thereby producing (Z)-9-hexadecenoyl-CoA;

[0143] b. E11 desaturase, catalyzing the formation of an E-configuration double bond at position 11 in the (Z)-9-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA; and

[0144] c. Alcohol-forming fatty acyl-CoA reductase (FAR) converts the (Z,E)-9,11-hexadecadienoyl-CoA into (Z,E)-9,11-hexadecadien-1-ol.

[0145] In further embodiments, the yeast cells may express one or more enzymes selected from:

[0146] a. Z11 desaturase, catalyzing the formation of a Z-configuration double bond at position 11 in hexadecenoyl-CoA, thereby producing (Z)-11-hexadecenoyl-CoA;

[0147] b. one or more alcohol-forming fatty acyl-CoA reductases (FARs), converting the hexadecanoyl-CoA into hexadecane-1-ol, converting the (Z)-9-hexadecenoyl-CoA into (Z)-9-hexadecene-1-ol, and converting the (Z)-11-hexadecenoyl-CoA into (Z)-11-hexadecene-1-ol, respectively.

[0148] In some embodiments, a genetically engineered yeast cell is provided that produces (Z,E)-9,11-hexadecadienoyl-CoA and (Z,E)-9,11-hexadecadien-1-ol, wherein the yeast cell produces hexadecanoyl-CoA and expresses (I) E11 desaturase, catalyzing the formation of an E-configuration double bond at position 11 in hexadecanoyl-CoA, thereby producing E-11-hexadecenoyl-CoA;

[0149] (II) Δ9 desaturase, which catalyzes the formation of a Z-configuration double bond at position 9 in (E)-11-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA;

[0150] (III) Alcohol-forming fatty acyl-CoA reductase (FAR) converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol.

[0151] As shown in the embodiments 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 desaturation in any order, i.e., first introducing an E-configuration double bond at position 11 and then introducing a Z-configuration double bond at position 9, or vice versa.

[0152] In a further embodiment,

[0153] a) an E11 desaturase having an amino acid sequence that is at least 50% identical to the E11 desaturase contained in SEQ ID NO: 1, e.g., 50% to 100%, 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 to the E11 desaturase contained in the E11 desaturase of SEQ ID NO: 1;

[0154] b) a Δ9 desaturase having an amino acid sequence that is at least 70% identical, such as at least 80% identical, such as at least 90% identical, such as at least 95% identical, such as 100% identical to the Δ9 desaturase contained in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 82;

[0155] c) a Z11 desaturase having an amino acid sequence that is at least 70% identical to the Z11 desaturase contained in SEQ ID NO:72, 74, 76 or 78, such as an amino acid sequence that is at least 80% identical, such as an amino acid sequence that is at least 90% identical, such as an amino acid sequence that is at least 95% identical, such as a 100% identical amino acid sequence; and

[0156] d) an alcohol forming fatty acyl-CoA reductase (FAR) having 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, such as an amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, such as a 100% identical amino acid sequence.

[0157] In this regard, the yeast cell is preferably Saccharomyces cerevisiae or Yarrowia lipolytica.

[0158] In some embodiments, a yeast cell is provided, wherein (I) the E11 desaturase has at least 70% sequence identity to the amino acid sequence contained in the E11 desaturase of SEQ ID NO: 1, 80, 90, 92, 96, 98, 100, 102, or 104;

[0159] (II) a Δ9 desaturase having at least 70% sequence identity to the amino acid sequence comprised in the Δ9 desaturase of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 82;

[0160] (III) the Z11 desaturase has at least 70% sequence identity to the amino acid sequence contained in the Z11 desaturase of SEQ ID NO: 72, 74, 76 or 78; and / or

[0161] (IV) an alcohol forming fatty acyl-CoA reductase (FAR) having at least 70% sequence identity to 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.

[0162] In some embodiments, a yeast cell is provided, wherein (I) the E11 desaturase has an amino acid sequence contained in the E11 desaturase of SEQ ID NO: 1, 80, 90, 92, 96, 98, 100, 102, or 104;

[0163] (II) a Δ9 desaturase having an amino acid sequence contained in the Δ9 desaturase of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 82;

[0164] (III) the Z11 desaturase has an amino acid sequence contained in the Z11 desaturase of SEQ ID NO: 72, 74, 76 or 78; and / or

[0165] (IV) 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, 59, 60, 61, 62, 63, 64, 65, 66, 88 or 95.

[0166] Culture

[0167] Another aspect provides a cell culture comprising a host cell as described herein and a growth medium. Suitable growth media for eukaryotic and prokaryotic cells are well known in the art.

[0168] Methods of producing compounds of the disclosure.

[0169] In another aspect, a method for producing a target compound is provided, wherein the target compound is selected from:

[0170] (I) (Z,E)-9,11-hexadecadienoyl-CoA;

[0171] (II) (Z,E)-9,11-hexadecadien-1-ol;

[0172] (III) (Z,E)-9,11-hexadecadienal; and / or

[0173] (IV) (Z,E)-9,11-hexadecadienyl acetate;

[0174] The method comprises culturing a cell culture described herein under conditions that allow the cell culture to produce a compound of interest; and optionally recovering and / or isolating the compound of interest.

[0175] Cell cultures can be cultured in a nutrient medium under conditions suitable for producing the target compound and / or its precursor and / or suitable for increasing cell counts using methods known in the art. For example, the culture can be cultured by shaking flasks or in a laboratory or industrial fermentor in a suitable medium under conditions that allow the host cells to grow and / or reproduce, either on a small scale or in large scale (including continuous fermentation, batch fermentation, fed-batch fermentation or solid-state fermentation), optionally recovered and / or separated.

[0176] Cultivation can be carried out using procedures known in the art in a suitable nutrient medium comprising a carbon source and a nitrogen source and an inorganic salt. Suitable medium can be obtained from a commercial supplier, or can be prepared according to a published formula (e.g., a catalog from the American Type Culture Collection). The selection of suitable medium can be based on the selection of host cells and / or based on the regulatory requirements of host cells. Such medium is available in the art. If desired, medium can include additional components that are conducive to transforming the expression host rather than other potential contaminating microorganisms. Therefore, in one embodiment, suitable nutrient medium includes a carbon source (e.g., glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellulosic biomass hydrolyzate, 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.).

[0177] The host cell can be cultured for a period of about 0.5 days to about 30 days. The culture process can be a batch process, a continuous process or a fed-batch process, suitably carried out at a temperature in the range of 0-100°C or 10-80°C, for example, about 20°C to about 50°C and / or 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. Appropriate conditions are generally selected based on the choice of the host cell. Therefore, in one embodiment, the method of the present disclosure further comprises one or more elements selected from the following:

[0178] (I) cultivating a cell culture in a nutrient medium;

[0179] (II) cultivating the cell culture under aerobic or anaerobic conditions;

[0180] (III) cultivating the cell culture under agitation;

[0181] (IV) cultivating the cell culture at a temperature between 25 and 50° C.;

[0182] (V) culturing the cell culture at a pH between 3 and 9; and

[0183] (VI) Cultivating the cell culture for 10 hours to 30 days.

[0184] The cell culture of the present disclosure may be recovered and / or separated using methods known in the art. For example, the target compound may be recovered from the nutrient medium by conventional procedures, including but not limited to centrifugation, filtration, spray drying or lyophilization. In certain embodiments, the method includes a recovery and / or separation step, including separating the liquid phase of the cells or cell culture from the solid phase of the cells or cell culture to obtain a supernatant containing the target compound and / or subjecting the supernatant to one or more steps selected from the following:

[0185] (I) disrupting cells of a cell culture to release a target compound within the cells into a supernatant;

[0186] (II) separating the supernatant from the solid phase of the cell culture, for example by filtration or gravity separation;

[0187] (III) contacting the supernatant with one or more adsorption resins to obtain at least a portion of the produced target compound;

[0188] (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;

[0189] (V) extracting the target compound; and / or

[0190] (VI) precipitating the E11 fatty acyl compound by crystallization or evaporation of the liquid phase solvent; and optionally isolating the target compound by filtration or gravity separation;

[0191] Thereby recovering and / or isolating the target compound.

[0192] The methods described herein may include one or more in vitro steps in the process of producing the target compound. Therefore, in one embodiment, the method also includes supplying one or more precursors or substrates in the target compound pathway to the cell culture. When the target compound is not the desired final product, further steps can be added to the method disclosed herein, and the target compound is modified chemically or biologically / enzymatically, such as oxidation and / or acetylation. Therefore, 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-hexadecadiene-1-ol (Z9, E11-16: OH), preferably using a reductase (FAR). In other attractive embodiments, the step performed in vitro includes chemically or enzymatically reducing (Z, E)-9,11-hexadecadienoic acid to (Z, E)-9,11-hexadecadiene-1-ol. In a further attractive embodiment, the step performed in vitro comprises 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 comprises chemically or enzymatically acetylating (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienyl acetate.

[0193] The method may also include recovering the target compound and mixing it with one or more carriers, agents, additives, adjuvants and / or excipients to produce a biopesticide composition.

[0194] In the method, one or more carriers, reagents, additives, adjuvants and / or excipients preferably include a protective agent, which preferably includes conjugated sulfur, which protects the target compound from being converted into acid. Such protective agents appropriately include compounds selected from pyrithione zinc, 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 pyrithione sodium, which have significant effects on stabilizing fatty aldehydes from oxidation. In some embodiments, the method also includes mixing at least 10 mg of protective agent per gram of aldehyde and / or alcohol. One or more carriers, medicaments, additives, adjuvants and / or excipients can also include a carrier that promotes the slow release of the target compound, optionally (i) a polymer matrix selected from plastics, wax emulsions, oil emulsions or microcapsules and / or (ii) zeolites.

[0195] 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 an alternative aspect, a method for producing the biopesticide composition described herein is provided, comprising (II) culturing a genetically engineered yeast cell that produces hexadecanoyl-CoA and expressing

[0196] a) a Δ9 desaturase, which catalyzes the formation of a Z-configuration double bond at position 9 in hexadecanoyl-CoA and thereby produces (Z)-9-hexadecenoyl-CoA;

[0197] b) an E11 desaturase, catalyzing the formation of an E-configuration double bond at position 11 in said (Z)-9-hexadecenoyl-CoA, and thereby producing (Z,E)-9,11-hexadecadienoyl-CoA;

[0198] c) an alcohol-forming fatty acyl-CoA reductase (FAR) that converts the (Z,E)-9,11-hexadecadienoyl-CoA into (Z,E)-9,11-hexadecadien-1-ol;

[0199] (IV) enzymatically or chemically converting (Z,E)-9,11-hexadecadien-1-ol into (Z,E)-9,11-hexadecadienal; and

[0200] (V) optionally recovering and / or isolating (Z,E)-9,11-hexadecadienal and optionally one or more precursors thereof.

[0201] In some embodiments of this alternative aspect, the genetically engineered yeast cell further expresses one or more enzymes selected from:

[0202] a) a Z11 desaturase, catalyzing the formation of a Z-configuration (Z) double bond at position 11 in hexadecenoyl-CoA and thereby producing (Z)-11-hexadecenoyl-CoA;

[0203] b) one or more alcohol-forming fatty acyl-CoA reductases (FARs) to convert the hexadecanoyl-CoA into hexadecane-1-ol, the (Z)-9-hexadecenoyl-CoA into (Z)-9-hexadecene-1-ol, and the (Z)-11-hexadecenoyl-CoA into (Z)-11-hexadecene-1-ol, respectively; and the method further comprises: enzymatically or chemically converting hexadecane-1-ol into hexadecanal, converting (Z)-9-hexadecene-1-ol into (Z)-9-hexadecene-1-ol, converting (Z)-11-hexadecene-1-ol and (Z)-11-hexadecene-1-ol into (Z)-11-hexadecene-1-ol; and optionally recovering and / or separating hexadecanal, (Z)-9-hexadecene-1-ol and (Z)-11-hexadecene-1-ol and optionally one or more precursors thereof.

[0204] In some embodiments, an alternative method of producing a biopesticide composition is provided, comprising the steps of:

[0205] (I) Cultivating genetically engineered yeast cells that produce hexadecanoyl-CoA and expressing

[0206] a. E11 desaturase, catalyzing the formation of an E-configuration double bond at position 11 in hexadecanoyl-CoA, thereby producing E-11-hexadecenoyl-CoA;

[0207] b. Δ9 desaturase, catalyzing the formation of a Z-configuration double bond at position 9 in (E)-11-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA;

[0208] c. Alcohol-forming fatty acyl-CoA reductase (FAR), which converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol;

[0209] (II) enzymatically or chemically converting (Z,E)-9,11-hexadecadien-1-ol into (Z,E)-9,11-hexadecadienal; and

[0210] (III) optionally recovering and / or isolating (Z,E)-9,11-hexadecadienal and optionally one or more precursors thereof.

[0211] Example 18 demonstrates the successful use of this alternative approach, showing the possibility of introducing the E11 double bond before the Z9 double bond in hexadecanoyl-CoA.

[0212] In some embodiments, the method is provided, wherein the genetically engineered yeast cell further expresses one or more enzymes selected from:

[0213] a. Z11 desaturase, which catalyzes the formation of a Z-configuration double bond at position 11 in hexadecanoyl-CoA, thereby producing (Z)-11-hexadecenoyl-CoA;

[0214] b. one or more alcohol-forming fatty acyl-CoA reductases (FARs) that convert hexadecanoyl-CoA to hexadecane-1-ol, (Z)-9-hexadecenoyl-CoA to (Z)-9-hexadecene-1-ol, and (Z)-11-hexadecenoyl-CoA to (Z)-11-hexadecene-1-ol, respectively;

[0215] The method also includes converting hexadecan-1-ol into hexadecanal, converting (Z)-9-hexadecen-1-ol into (Z)-9-hexadecenal, converting (Z)-11-hexadecen-1-ol and (Z)-11-hexadecen-1-ol into (Z)-11-hexadecenal by enzymatic or chemical methods; and / or optionally recovering and / or separating hexadecanal, (Z)-9-hexadecenal and (Z)-11-hexadecenal and optionally one or more precursors thereof.

[0216] In other embodiments of 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 precursors thereof with one or more carriers, reagents, additives, adjuvants and / or excipients to produce a biopesticide composition. These carriers, reagents, additives, adjuvants and / or excipients preferably comprise one or more compounds selected from the group consisting of

[0217] a) a protecting agent, the protecting agent comprising 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, for protecting the target compound from being converted into an acid; and / or

[0218] b) a carrier that promotes the slow release of (Z,E)-9,11-hexadecadienal, (Z)-9-hexadecenal, (Z)-11-hexadecenal and / or hexadecanal in the mixture, optionally (i) a polymer matrix selected from plastics, wax emulsions, oil emulsions or microcapsules and / or (ii) a zeolite.

[0219] Composition

[0220] Another aspect provides a biopesticide composition comprising a target compound selected from the group consisting of:

[0221] a)(Z,E)-9,11-hexadecadien-1-ol (Z9,E11-16:OH);

[0222] b) (Z,E)-9,11-hexadecadienal (Z9,E11-16:Ald); and / or

[0223] c) (Z,E)-9,11-hexadecadienyl acetate (Z9,E11-16:OAc); and

[0224] One or more carriers, agents, additives, adjuvants and / or excipients.

[0225] Another alternative 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-hexadecenal, (Z)-11-hexadecenal and / or hexadecanal, and in combination with one or more carriers, agents, additives, adjuvants and / or excipients. In certain embodiments, the biopesticide composition also 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, and other metabolites of the cell culture. In other embodiments, the biopesticide composition also 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 a genetically engineered host cell culture as described herein, and the composition optionally comprises one or more further compounds or metabolites from the cell culture. Such compounds and / or metabolites of the cell culture include precursors of the target compound and compounds selected from trace metals, vitamins, salts, yeast nitrogen base, carbon sources, YNB and / or fermented amino acids. In particular, the biopesticide composition comprises the target compound in a concentration 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, such as at least 500 mg / kg, such as at least 1.000 mg / kg, such as at least 5.000 mg / kg, such as at least 10.000 mg / kg, such as at least 50.000 mg / kg.

[0226] The composition may also advantageously include one or more protective agents containing conjugated sulfur, which protect the target compound from further conversion to acid. The protective agent includes 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 composition preferably contains at least 10 mg of the protective agent per gram of the target compound.

[0227] In some embodiments, the composition further comprises a carrier that promotes the slow release of the target compound, and the carrier is optionally (i) a polymer matrix selected from plastic, wax emulsion, oil emulsion or microcapsule and / or (ii) zeolite.

[0228] In some embodiments, the biopesticide composition comprises at least 50% biobased 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%.

[0229] In some embodiments, the biopesticide composition comprises at least 20% biobased carbon, such as at least 30% biobased carbon, such as at least 40% biobased carbon, such as at least 50% biobased carbon, such as at least 60% biobased carbon, such as at least 70% biobased carbon, such as at least 75% biobased carbon, such as at least 80% biobased carbon, such as at least 85% biobased carbon, such as at least 90% biobased carbon, such as at least 95% biobased carbon, such as 100% biobased carbon.

[0230] In some embodiments, the biopesticide composition comprises 20% to 100% biobased carbon, such as 30% to 100% biobased carbon, such as 40% to 100% biobased carbon, such as 50% to 100% biobased carbon, such as 60% to 100% biobased carbon, such as 70% to 100% biobased carbon, such as 75% to 100% biobased carbon, such as 80% to 100% biobased carbon, such as 85% to 100% biobased carbon, such as 90% to 100% biobased carbon, such as 95% to 100% biobased carbon, such as 100% biobased carbon.

[0231] In some embodiments, the biopesticide composition comprises no more than 50% fossil-based carbon, such as no more than 45%, such as no more than 40%, such as no more than 35%, such as no more than 30%, such as no more than 25%, such as no more than 20%, such as no more than 15%, such as no more than 10%, such as no more than 5%, such as no more than 1% fossil-based carbon.

[0232] In some embodiments, the biopesticide composition comprises 90% biobased carbon, 91% biobased carbon, 92% biobased carbon, 93% biobased carbon, 94% biobased carbon, 95% biobased carbon, 96% biobased carbon, 97% biobased carbon, 98% biobased carbon, 99% biobased carbon, or 100% biobased carbon, such as 94% biobased carbon.

[0233] In some embodiments, the biopesticide composition comprises Z9,E11-16:OH, which is at least 90% biobased, such as at least 92% biobased carbon, such as at least 94% biobased carbon, such as at least 96% biobased carbon, such as at least 98% biobased carbon, such as 100% biobased carbon, such as 94% biobased.

[0234] application

[0235] Another aspect provides a method for controlling or monitoring pests, comprising distributing the composition described herein in the habitat of the pests and allowing the target compound to control the pests. The target compound described herein is particularly effective against small sugarcane borers. According to a preferred embodiment, the habitat is a sugarcane field and the pest is a small sugarcane borer.

[0236] Sequence Listing

[0237] This application contains a sequence listing prepared in Patentl described below, but also submitted electronically in ST26 format, which sequence listing is incorporated herein by reference in its entirety.

[0238] Table A

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245] Example

[0246] Example 1 - Construction of BioBricks and Plasmids

[0247] All heterologous genes were synthesized by GeneArt (Life Technologies) and were codon optimized versions for Yarrowia lipolytica. Genes were either amplified by PCR using Phusion U Hot Start DNA polymerase (ThermoFisher) or obtained by restriction digestion to obtain fragments for cloning into yeast expression vectors. Primers are listed in Table 1 and the resulting DNA fragments (BioBricks) are listed in Table 2. PCR products or restriction digestion reactions were separated on 1% agarose gels containing Midori GreenAdvance (Nippon Genetics Europe GmbH). PCR / restriction digestion products of the correct size were cut from the gel and purified using Nucleospin Gel and PCR Clean-up kits (Macherey-Nagel).

[0248] The yeast vector with the USER box was linearized with FastDigest SfaAI (ThermoFisher) at 37°C for 2 hours and then cut with Nb.Bsml (New England Biolabs) at 65°C for 1 hour. The resulting vector containing 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 Escherichia coli DHα cells, and the cells were plated on Lysogeny Broth (LB) agar plates containing 100 mg / L ampicillin. The plates were incubated overnight at 37°C, and the resulting colonies were screened by colony PCR. Plasmids were purified from overnight E. coli liquid cultures, and the correct clones were confirmed by sequencing. The constructed vectors are listed in Table 3.

[0249] Table 1. Primers

[0250]

[0251]

[0252] *(Holkenbrink et al., 2018)

[0253] Table 2. DNA fragments obtained by PCR and restriction digestion using the indicated templates and primers (BioBricks)

[0254]

[0255]

[0256] *(Holkenbrink et al., 2018)

[0257] **(Holkenbrink et al., 2020)

[0258] Table 3. Vectors

[0259] Resistance Resistance Parent plasmid BioBricks pBP8662 BB1135,BB8679,BB8680,BB1631 pBP9002 Nourseothricin pCfB3405* BB8955 pBP9698 Nourseothricin pCfB3405* BB9506 pBP9700 Nourseothricin pBP9002 BB9454,BB9507 pBP10539 Nourseothricin pBP9698 BB9454,BB10122 pBP11063 Nourseothricin pBP10995 BB10398,BB10439

[0260] *(Holkenbrink et al., 2018)

[0261] Example 2 - Construction of yeast strains

[0262] Yeast strains were constructed by DNA vector transformation as described in (Holkenbrink et al., 2018) (Jensen et al., 2014). Strains were selected on yeast peptone dextrose (YPD) agar with appropriate antibiotics or on synthetic dropout medium (Sigma-Adrich) lacking specific amino acids. Correct genotypes were confirmed by colony PCR and, if necessary, by sequencing. The resulting strains are listed in Table 4.

[0263] Strains marked with "***" were constructed as follows. The indicated genes were amplified with gene-specific primers containing a 5'-overhang in the forward primer: "ACTTTTTGCAGTACUAACCGCAG" and a 3'-overhang "CACGCGAU" in the reverse primer. The first "ATG" of the target gene sequence was omitted. These PCR products were cloned into an integration vector or a free vector together with BB9454 as described in (Holkenbrink, et al. 2018).

[0264] Strains marked with "****" were constructed as follows. The indicated genes were cloned into an integration vector or free vector together with BB10398 and a synthetic minimal terminator (SEQ ID NO: 86) (ST13043) or a natural LIP2 terminator of Yarrowia lipolytica (ST13149, ST13247), and "Golden-Gate Assembly" was performed as described in (Pryor, JM, et al. 2020) or in accordance with the user manual of NEBridge Golden Gate Assembly Kit (New England BioLabs). The first "ATG" of the target gene sequence was omitted. The episomal vector pBP10995 was derived from pCfB3405 (Holkenbrink et al., 2018) for Golden-Gate assembly.

[0265] Table 4. Yeast strains

[0266]

[0267]

[0268] **(Holkenbrink, et al. 2020)

[0269] Example 3 - Strain cultivation and analysis of fatty alcohols and fatty acid methyl esters (FAME)

[0270] The Yarrowia 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) into 2.5 mL YPG medium (10 g / L yeast extract, 10 g / L peptone, 40 g / L glycerol) in a 24-well plate (EnzyScreen) with an initial OD600 of 0.2. The plate was incubated at 28°C with shaking at 300 rpm. After 24 hours, the plate was centrifuged at 4°C and 3,000 x g for 5 minutes. The supernatant was discarded, and the cells were suspended 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 N Na2MoO4.2H2O, 0.3 g / L CoCl2.6H2O, 0.1 g / L CuSO4.5H2O, 0.1 g / L KI, 15 g / L EDTA). If necessary, antibiotics were added to the medium. The plates were incubated at 28 °C for 26 h with shaking at 300 rpm.

[0271] To analyze fatty acids, centrifuge at 4°C and 3,000xg for 5 minutes and harvest 1mL per bottle. Extract each precipitate with 1000μL 1M HCl methanol solution (anhydrous). Vortex the sample for 20 seconds and then put it in a 70°C water bath for 2 hours. Vortex the sample for 10 seconds every 30 minutes. After the sample is cooled to room temperature, add 1000μL 1M NaOH methanol solution (anhydrous), 500μL NaCl saturated H2O, 990μL hexane and 10μL 19:Me (10mg / mL) as internal standards. Vortex the sample and centrifuge at 21°C and 3,000xg for 5 minutes. Analyze the upper organic phase by gas chromatography-mass spectrometry (GC-MS). For the analysis of fatty alcohols, centrifuge at 4°C and 3,000xg for 5 minutes and harvest 1mL per bottle. 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 samples were 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 samples were vortexed and centrifuged at 21°C and 3,000×g for 5 minutes. The upper organic phase was analyzed by gas chromatography-mass spectrometry (GC-MS). GC-MS analysis was performed on an Agilent 7820A GC coupled to 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. MS was operated in electron impact mode (70 eV), scanning between m / z 30 and 400, and the injector was configured in split mode 20:1 at 220°C. The oven temperature was set to 80°C for 1 minute, then increased to 210°C at a rate of 20°C / min, then held at 210°C for 7 minutes, and then increased to 230°C at a rate of 20°C / min. The compounds were identified by comparing the retention time and mass spectra of reference compounds. The data were analyzed by Agilent Masshunter software.

[0272] Example 4 - Production of Z9, E11-16:CoA in the yeast Yarrowia lipolytica

[0273] The newly identified desaturase Ds12389 from the small sugarcane borer was expressed in the Yarrowia lipolytica strain ST6629 (Holkenbrink et al., 2020) to generate strain ST12028. The empty expression vector (pBP9002) was transformed into the same parent strain to obtain ST10444 as a control strain. The cultivation of both strains and the extraction of fatty acids were carried out as described in Example 3.

[0274] FAME extract of strain ST12028 expressing Ds12389 ( Figure 2A, dotted line) contained diunsaturated 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). The elution retention time of this 16-2:Me (12.954 min) is the same as that of the authentic standard of Z9,E11-16:Me ( Figure 2A , solid line), and the mass spectrum matches that of the Z9,E11-16:Me standard ( Figure 3 In addition, strain ST12028 produces 16:Me and Z9-16:Me ( Figure 2C ). The titers of Z9,E11-16:Me are listed in Table 5.

[0275] Table 5. Production of Z9,E11-16:Me in the yeast Yarrowia lipolytica

[0276] strain ID Desaturase Z9,E11-16:Me(mg / L) ST10444 - 0 ST12028 Ds12389 0.43

[0277] Example 5 - Expression of Dendrolimus punctatus desaturase in Yarrowia lipolytica

[0278] The desaturase Dpu_APSQ gene from Dendrolimus punctatus was expressed in Yarrowia lipolytica strain ST6629 (Holkenbrink et al., 2020) to generate strain ST10746. Strain ST10444 carried only the empty expression vector and was used as a control strain.

[0279] The FAME extract of strain ST10746 expressing the desaturase Dpu_APSQ did not contain any compound eluting at 12.954 min, but a trace amount of an unknown diunsaturated C16-fatty acyl-CoA was detected with a retention time of 12.985 min ( Figure 2B ).

[0280] Example 6 - Production of Z9,E11-16:OH in Yarrowia lipolytica

[0281] The desaturase Ds12389 from C. canadensis was co-expressed with fatty acyl-CoA reductase in Yarrowia lipolytica. The strains were cultured and samples analyzed as described in Example 3, and fatty alcohol Z9,E11-16:OH was detected.

[0282] Example 7 - Production of (Z9,E11)-hexadecadienal

[0283] A primary aliphatic alcohol mixture containing 96 wt% (Z,E)-9,11-hexadecadien-1-ol (Z9,E11-16:OH) was used as a representative sample for conversion to aldehydes. Z9,E11-16:OH (560 mg) was dissolved in 1 mL of acetonitrile in a 10 mL round bottom flask with a magnetic stirrer. Then 39.0 mg of tetraacetonitrile copper (I) trifluoromethanesulfonate (5 mol%), 16.0 mg of 2,2'-bipyridine (Bipy) (5 mol%), 9.0 mg of 4-hydroxy TEMPO) (2.5 mol%), 8.5 mg of N-methylimidazole (5 mol%) were added to the reaction mixture and 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). The top heptane phase was then evaporated under reduced pressure until a clear residue was formed, yielding 493 mg of product containing 88.0 wt% (Z9,E11)-hexadecadienal (Z9,E11-16:Ald).

[0284] Example 8 - Production of Z9,E11-16:Acid and Z9,E11-16:OH in Saccharomyces cerevisiae

[0285] The Ds12389 desaturase (SEQ ID NO: 1) from C. cane borer was cloned alone and in combination with a fatty acyl reductase into a Saccharomyces cerevisiae gene expression vector and transformed into Saccharomyces cerevisiae as described by Jensen et al., 2014. Strain culture and sample extraction were performed as in Example 3. Strains expressing only the Ds12389 desaturase produced Z9,E11-16:Me, while strains expressing the fatty acyl reductase gene produced Z9,E11-16:OH.

[0286] Example 9 - Various fatty acyl reductases convert Z9,E11-16:Me to Z9,E11-16:OH in Yarrowia lipolytica

[0287] Fatty acyl-CoA reductases from various organisms were expressed in Yarrowia lipolytica. Strains were cultured and fatty alcohol samples extracted as described in Example 3, however the culture time in YPG medium was extended from 24 to 47 hours and 0.2 μl Z9,E11-16:Me was added to the production medium.

[0288] Extracts of strains ST12118-ST12123, ST12125, ST12126, ST12129, ST12131-ST12133, ST12138, ST12140, ST12141, ST12146, ST12151, ST12154, ST12156, ST12159 and ST12560 produce Z9,E11-16:OH. As an example, the GC-MS chromatograms and mass spectra of the extract of strain ST12118 and the pure standard of Z9,E11-16:OH are shown in Figures 1 and 2, respectively. Figure 4A 4B and 4C. The extract of strain ST12118 contained 5 mg / L Z9,E11-16:OH.

[0289] Example 10 - Bio-based Pheromone Precursor Mixture Production

[0290] The main pheromone of the small sugar cane borer is (Z,E)-9,11-hexadecadienal. The minor components are (Z)-11-hexadecenal, (Z)-9-hexadecenal and hexadecanal. The fatty alcohol precursors of the major and minor components can be produced in one yeast cell. To this end, the ΔE11 desaturase Ds12389 (SEQ ID NO: 1) is co-expressed with any ΔZ11-16 desaturase, such as Desat16 (SEQ ID NO: 72) from the navel orange borer, Desat51 (SEQ ID NO: 78) from the cotton bollworm, Desat37 (SEQ ID NO: 74) from the beet armyworm, and Desat38 (SEQ ID NO: 76) from the Spodoptera litura, as well as a suitable fatty acyl-CoA reductase known in the art, such as SEQ ID NO: 46. The strain is cultured and the fatty alcohol sample is extracted as described in Example 3.

[0291] 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.

[0292] The extract is chemically oxidized to obtain a composition containing (Z,E)-9,11-hexadecadienal, hexadecanal, (Z)-9-hexadecenal and (Z)-11-hexadecenal.

[0293] The biobased carbon content of the compositions was determined by C14 radiocarbon dating.

[0294] Example 11 - Co-expression of ΔZ9-16 desaturase and Ds12389 (SEQ ID NO: 1)

[0295] The ΔE11 desaturase Ds12389 is co-expressed with the ΔZ9-16 desaturase and optionally the fatty acyl-CoA reductase. The strains are cultured and the FAME and fatty alcohol samples are extracted as described in Example 3. The strain expressing Ds12389 and the ΔZ9-16 desaturase produces (Z,E)-9,11-hexadecadienoic acid methyl ester. If the fatty acyl-CoA reductase is expressed, (Z,E)-9,11-hexadecadien-1-ol is produced.

[0296] Example 12 - Production of Z9, E11-16:CoA in Yarrowia lipolytica by expression of DsaDes1 (SEQ ID NO:80)

[0297] Newly identified desaturases DsaDes1 and Ds12389 from C. serrata were expressed in Yarrowia lipolytica strain ST12834, generating strains ST13043 and ST13042, respectively. Strain ST12834 was derived from ST6629 (Holkenbrink et al., 2020) and additionally expressed a heterologous NAD(P)H cytochrome b5 oxidoreductase Ncb5or (SEQ ID NO: 84) from Cydia pomonella (WO2022 / 238404A1). The empty expression vector (pBP9002) was transformed into the same parent strain ST12834 to obtain ST13046 and used as a control strain. Both strains were cultured and fatty acid extraction was performed as described in Example 3.

[0298] FAME extracts of strains ST13043 and ST13042, expressing DsaDes1 and Ds12389, respectively (Figure 5), contained di-unsaturated C16 fatty acid methyl esters (16-2:Me) with the same elution time as the authentic standard of Z9, E11-16:Me (Figure 5). The mass spectrum of this di-unsaturated 16-2:Me matched 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 titers of Z9, E11-16:Me are listed in Table 6.

[0299] Table 6. Production of Z9,E11-16:Me in the yeast Yarrowia lipolytica

[0300] strain Z9,E11-16:Me(mg / L) ST13046 - ST13043 20±3 ST13042 3±3

[0301] Example 13 - Production of Z9,E11-16:OH in Yarrowia lipolytica by co-expression of DsaDes1 and fatty acyl-CoA reductase

[0302] The desaturase DsaDes1 from C. canadensis was co-expressed with fatty acyl-CoA reductase in Yarrowia lipolytica. The strains were grown and samples were analyzed as described in Example 3, and fatty alcohol Z9,E11-16:OH was detected.

[0303] Example 14 - Co-expression of ΔZ9-16 desaturase and DsaDes1 (SEQ ID NO: 80)

[0304] The ΔE11 desaturase DsaDes1 was co-expressed with a ΔZ9-16 desaturase and optionally a fatty acyl-CoA reductase. The strains were cultured and FAME and fatty alcohol samples were extracted as described in Example 3. The strains expressing DsaDes1 and ΔZ9-16 desaturase produced (Z,E)-9,11-hexadecadienoic acid methyl ester and, if a fatty acyl-CoA reductase was expressed, additionally produced (Z,E)-9,11-hexadecadien-1-ol.

[0305] Example 15 - Production of Z9,E11-16:Acid and Z9,E11-16:OH in Saccharomyces cerevisiae

[0306] Desaturases Ds12389 (SEQ ID NO: 1) and DsaDes1 (SEQ ID NO: 80) from C. cane borer were cloned into a Saccharomyces cerevisiae gene expression vector and transformed into the Saccharomyces cerevisiae CEN.PK strain as described by 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) with an initial OD600 of 0.1-0.2, supplemented with 2% glucose, and placed in a 24-well plate (EnzyScreen). Sample extraction was performed as in Example 3. The derived FAME samples of strains ST13093 and ST13150 expressing the desaturases Ds12389 and DsaDes1, respectively, both contained Z9, E11-16:Me (Table 7), whereas the derived samples of the control strain ST12515 only carried the empty expression vector and did not contain any Z9, E11-16:Me.

[0307] Table 7. Detection of Z9,E11-16:Me in derived FAME samples of S. cerevisiae strains.

[0308] strain ID Gene expression Z9,E11-16:Me(mg / L) ST12515 - 0 ST13093 Ds12389 1.6±0.6 ST13150 DsaDes1 1.3±0.6

[0309] Example 16 - Production of Z9,E11-16:OH in Y. lipolytica by co-expression of DsaDes1 and fatty acyl-CoA reductase

[0310] The desaturase DsaDes1 from the small sugar cane borer was co-expressed in the Yarrowia lipolytica strain ST6629 (Holkenbrink et al., 2020) in combination with fatty acyl reductases from different insect species. The empty expression vector (pBP9002) was transformed into the strain ST13146 expressing only DsaDes1 to obtain ST13151 as a control strain. The strains were cultured and the samples were analyzed according to the method of Example 3. The control strain ST13151 did not produce the expected fatty alcohols. Strains ST13147, ST13152, ST13162 and ST13251 express the newly identified fatty acyl reductase DsaFAR1 (SEQ ID NO: 88) from C. caneborer, FAR1 (SEQ ID NO: 46) from Helicoverpa armigera, FAR16 (SEQ ID NO: 56) from Spodoptera exigua and FAR25 (SEQ ID NO: 95) from Tyta alba, respectively, producing 16: OH, Z11-16: OH, Z9-16: OH and the target compound Z9, E11-16: OH (Table 8). The amount of 16: OH produced by strain ST13147 expressing DsaFAR1 is much lower than that of strains expressing FAR1 (ST13152), FAR16 (ST13162) or FAR25 (ST13251). This feature is conducive to achieving higher purity Z9, E11-16: OH production.

[0311] Table 8. Production of Z9,E11-16:OH in the yeast Yarrowia lipolytica.

[0312]

[0313] Example 17 - Co-expression of ΔZ9-16 desaturase and DsaDes1

[0314] In Yarrowia lipolytica strain ST6629 (Holkenbrink et al., 2020), the ΔE11 desaturase DsaDes1 (SEQ ID NO: 80) was co-expressed with the newly identified ΔZ9-16 desaturase DsaDes7 (SEQ ID NO: 82) from C. canephora to produce ST13149. The empty expression vector (pBP9002) was transformed into strain ST13146 expressing only DsaDes1 to obtain ST13151 as a control strain. The strains were cultured and FAME samples were extracted as described in Example 3. When DsaDes1 was expressed alone, the derivative sample of the control strain ST13151 contained 2.0% Z9,E11-16:Me and 10.1% Z9-16:Me (Table 9). The derived FAME samples of strain ST13149 co-expressing DsaDes1 and the ΔZ9-16 desaturase DsaDes7 contained 3.3% Z9,E11-16:Me and 15.2% Z9-16:Me. The presence of DsaDes7 increased the purity of Z9-16:Me by 50% and 65%, respectively. If a fatty acyl-CoA reductase is additionally expressed, (Z,E)-9,11-hexadecadien-1-ol can be produced. This property is conducive to achieving higher purity Z9,E11-16:OH production.

[0315] Table 9. Effect of ΔZ9-16 desaturase DsaDes7 on the purity of Z9-16:Me and Z9,E11-16:Me in Yarrowia lipolytica biomass strain-derived FAME samples.

[0316]

[0317] Example 18 - Conversion of E11-16:Me to Z9, E11-16:CoA by ΔZ9 desaturase in Yarrowia lipolytica

[0318] The ΔZ9 desaturase DsaDes7 (SEQ ID NO: 82) from C. saccharum was expressed in Yarrowia lipolytica strain ST6629 (Holkenbrink et al., 2020) to generate strain ST13247. The empty expression vector (pBP9002) was transformed into the same parent strain to obtain ST10444 as a control strain. Both strains were cultured as described in Example 3 with or without the addition of 0.2 g / L E11-16:Me to the culture. After cultivation, samples were extracted and converted into fatty acid methyl esters as described in Example 3. When E11-16:Me was not added to the culture, the derivatized FAME samples of strains ST10444 and ST13247 did not contain any Z9, E11-16:Me. However, in the derivatized samples of the two strains with the addition of E11-16:Me, Z9, E11-16:Me could be detected (Table 10). The purity of Z9,E11-16:Me in the samples of strain ST13247 was higher than that of the control strain ST10444. These results support that both the C. saccharum ΔZ9 desaturase DsaDes7 and the native Yarrowia lipolytica ΔZ9 desaturase OLE1 (SEQ ID NO: 17) can convert E11-16:CoA into Z9,E11-16:CoA.

[0319] Table 10. Detection of Z9, E11-16:Me in Yarrowia lipolytica strain-derived samples with and without added E11-16:Me.

[0320]

[0321]

[0322] Example 19 - Production of Z9, E11-16:CoA in Yarrowia lipolytica using an alternative E11 desaturase

[0323] A synthetic protein variant of the ΔE11 desaturase DsaDes1 (Holkenbrink et al., 2020) was designed and expressed in the Yarrowia lipolytica strain ST6629 to generate strains ST13284, ST13285, ST13286, ST13287, and ST13288. The empty expression vector (pBP9002) was transformed into the same parent strain to obtain ST10444 as a control strain. Both strains were cultured and fatty acid extraction was performed as described in Example 3. As expected, Z9,E11-16:Me was not detected in the FAME extract of the control strain ST10444. FAME extracts of strains ST13284, ST13285, ST13286, ST13287 and ST13288 expressed the designed 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 containing the target compound Z9, E11-16: Me (Table 11). The sequence identities of the synthetic protein variants can be seen in the table (Table 12). This example shows that a large number of E11 desaturase sequence variants can be used to provide the desired E-olefins.

[0324] Table 11. Detection of Z9,E11-16:Me in Y. lipolytica strain-derived samples.

[0325] strain ID Gene expression Z9,E11-16:Me(mg / L) ST10444 - 0 ST13284 Desat87 7.1±1.7 ST13285 Desat88 6.2±2.3 ST13286 Desat89 4.5±1.0 ST13287 Desat90 8.0±2.3 ST13288 Desat91 4.2±0.4

[0326] Table 12. Percent sequence identity between ΔE11 desaturases.

[0327]

[0328]

[0329] Example 20 - Measuring Biobased Carbon Content

[0330] The biobased carbon content of the fatty alcohol pheromone precursor co was measured using an analytical measurement method that can be called "modern carbon percentage (pMC)". This is the percentage of the isotope 14C measured in the sample relative to a modern reference standard (NIST4990C). The % biobased carbon content is calculated from the pMC by applying a small adjustment factor for the isotope 14C in the carbon dioxide in today's air. The strain STSCB designed to produce Z9,E11-16:OH was fermented and the product was recovered. The product sample consisted of a mixture of fatty alcohols including Z9,E11-16:OH. The "modern carbon percentage (pMC)" of the product sample was analyzed by the standard test method "ASTM D6866". The percentage of modern carbon was determined to be 93.80±0.33pMC, which is equivalent to a biobased carbon content of 94% (Table 13). This means that the pMC of Z9,E11-16:OH contained in the product sample is also 94%.

[0331] Table 13. Determination of percentage of modern carbon in product samples.

[0332]

[0333] Example 21 - Production of Z9,E11-16:OH in Saccharomyces cerevisiae

[0334] The E11 desaturase DsaDes1 (SEQ ID NO: 80) and fatty acyl-CoA reductase DsaFAR1 (SEQ ID NO: 88) from C. cane borer were cloned into a Saccharomyces cerevisiae gene expression vector and transformed into the Saccharomyces cerevisiae CEN.PK strain as described by Maury et al., 2016 and Jensen et al., 2014. The Saccharomyces cerevisiae strains were inoculated from synthetic dropout agar plates (lacking uracil, leucine and histidine) to an initial OD600 of 0.1-0.2 in 2.5 mL of synthetic dropout medium (lacking uracil, leucine and histidine) supplemented with 2% glucose in a 24-well plate (EnzyScreen). Sample extraction was performed as in Example 3. Strain ST13490 simultaneously expressed DsaDes1 and DsaFAR1 and produced Z9, E11-16: OH (Table 14). The control strain ST13491 only expressed DsaDes1 and did not produce any Z9,E11-16:OH.

[0335] Table 14. Production of Z9,E11-16:OH in Saccharomyces cerevisiae

[0336]

[0337] References

[0338] Holkenbrink,C.,Dam,M.I.,Kildegaard,K.,Beder,J.,Doménech,D.B.,&Borodina,I.(2018).EasyCloneYALI:CRISPR / Cas9-Based Synthetic Toolbox forEngineering of the Yeast Yarrowia lipolytica.Biotechnol J.

[0339] Holkenbrink,C.,Ding,B.-J.,Wang,H.-l.,Dam,M.I.,Petkevicius,K.,Kildegaard,K.R.,...Borodina,I.(2020).Production of moth sex pheromones forpest control by yeast fermentation.Metab Eng.,312-321.

[0340] Jensen,N.,Strucko,T.,Kildegaard,K.,David,F.,Maury,J.,Mortensen,U.,...Borodina,I.(2014).EasyClone:method for iterative chromosomal integrationof 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.,& C.(2010).Elucidation of the sex-pheromone biosynthesis producing5,7-dodecadienes in Dendrolimus punctatus reveals11-and9-desaturases with unusualcatalytic properties.Insect Biochemistry and Molecular Biology,440-452.

[0341] Zhao,C.-H.,Adolf,R.,& C.(2004).Sex pheromone biosynthesis inthe pine caterpillar moth,Dendrolimus punctatus:pathways leading to Z5-monoene and5,7-conjugated diene components.Insect Biochemistry and MolecularBiology,261-271.

[0342] Da Silva,M.,Cortes,A.,Svensson,G., C.,Lima,E.,&Zarbin,P.(2021).Identification of two additional behaviorally active glandconstituents of female Diatraea saccharalis(Fabricius)(LepidopteraCrambidae).Journal of the Brazilian Chemical Society.

[0343] Holkenbrink,C.,Dam,M.I.,Kildegaard,K.,Beder,J.,Doménech,D.B.,&Borodina,I.(2018).EasyCloneYALI:CRISPR / Cas9-Based Synthetic Toolbox forEngineering of the Yeast Yarrowia lipolytica.Biotechnol J.

[0344] Holkenbrink,C.,Ding,B.-J.,Wang,H.-l.,Dam,M.I.,Petkevicius,K.,Kildegaard,K.R.,...Borodina,I.(2020).Production of moth sex pheromones forpest control by yeast fermentation.Metab Eng.,312-321.

[0345] Jensen,N.,Strucko,T.,Kildegaard,K.,David,F.,Maury,J.,Mortensen,U.,...Borodina,I.(2014).EasyClone:method for iterative chromosomal integrationof 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 mainpheromone component in wind tunnel experiments.Journal of Applied Entomology.

[0346] Lienard,M.,Lassance,J.-M.,Wang,H.-L.,Zhao,C.-H.,Piskur,J.,Johansson,T.,& C.(2010).Elucidation of the sex-pheromone biosynthesisproducing5,7-dodecadienes in Dendrolimus punctatus reveals11-and9-desaturaseswith unusual catalytic properties.Insect Biochemistry and Molecular Biology,440-452.

[0347] Svatos,A.,Kalinova,B.,Kindl,J.,Kuldova,J.,Hovorka,O.,Rufino DoNascimento,R.,&Oldham,N.(2001).Chemical Characterization and Synthesis of theMajor Component of the Sex Pheromone of the Sugarcane Borer Diatraea saccharalis.Collect.Czech.Chem.Commun.,1682-1690.Zhao,C.-H.,Adolf,R.,& C.(2004).Sex pheromone biosynthesis in the pine caterpillar moth,Dendrolimus punctatus:pathways leading to Z5-monoene and5,7-conjugated dienecomponents.Insect Biochemistry and Molecular Biology,261-271.

[0348] Pryor, JM, Potapov, V., Kucera, RB, Bilotti, K., Cantor, EJ, Lohman, GJS (2020). Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design. PloS ONE 15(9): e0238592. Projects of this disclosure

[0349] The present disclosure also provides the following embodiments and projects:

[0350] Item 1. An E11 fatty acyl-CoA desaturase (E11 desaturase) comprising an amino acid sequence having at least 50% identity to the E11 desaturase contained in SEQ ID NO: 1 or SEQ ID NO: 80.

[0351] Item 2. The E11 desaturase according to Item 1, wherein the E11 desaturase introduces an E-configuration double bond at position 11 in the presence of a (Z)-9-hexadecenoyl-CoA substrate, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA.

[0352] Item 3. A polynucleotide sequence encoding the E11 desaturase as described in Item 1 and codon-optimized for heterologous expression, which has a DNA sequence contained in SEQ NO: 2 or SEQ ID NO: 81 or a homolog thereof, including variations due to the degeneracy of the genetic code.

[0353] Item 4. A polynucleotide construct comprising the polynucleotide sequence described in Item 3 operably linked to one or more control sequences.

[0354] Item 5. A polynucleotide construct according to Item 4, wherein the control sequence is heterologous to the polynucleotide.

[0355] Item 6. A vector comprising the polynucleotide construct of Item 4 or Item 5.

[0356] Item 7. A genetically engineered microbial cell that produces (Z,E)-9,11-hexadecadienoyl-CoA, wherein the cell heterologously expresses the E11 desaturase as described in Item 1 or 2, which introduces an E-configuration double bond at position 11 in the (Z)-9-hexadecadienoyl-CoA substrate in the presence of the (Z)-9-hexadecadienoyl-CoA substrate, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA having an E-configuration double bond at position 11.

[0357] Item 8. The cell according to Item 7, further comprising an effective biosynthetic pathway for converting the (Z,E)-9,11-hexadecadienoyl-CoA into a target compound selected from the following

[0358] a) (Z,E)-9,11-hexadecadien-1-ol;

[0359] b) (Z,E)-9,11-hexadecadienal; and / or

[0360] c) (Z,E)-9,11-hexadecadienyl acetate;

[0361] The pathway expresses one or more pathway polypeptides selected from the group consisting of:

[0362] a) Alcohol-forming fatty acyl-CoA reductase (FAR), which converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol;

[0363] b) acetyltransferase, converting (Z,E)-9,11-hexadecadien-1-ol into (Z,E)-9,11-hexadecadienyl acetate;

[0364] c) an alcohol dehydrogenase, which converts (Z,E)-9,11-hexadecadien-1-ol into (Z,E)-9,11-hexadecadienal; and / or

[0365] d) Fatty alcohol oxidase, which converts (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienal.

[0366] Item 9. A cell according to Item 8, wherein

[0367] a) the FAR 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;

[0368] b) an acetyltransferase that is at least 70% identical to the acetyltransferase contained in SEQ ID NO:71;

[0369] c) the alcohol dehydrogenase is at least 70% identical to the alcohol dehydrogenase contained in SEQ ID NO: 68; and

[0370] d) the fatty alcohol oxidase is at least 70% identical to the fatty alcohol oxidase contained in SEQ ID NO: 69 or 70.

[0371] Item 10. A cell according to items 7 to 9, which also comprises an efficient biosynthetic pathway for producing a (Z)-9-hexaenoyl-CoA substrate, wherein the pathway expresses one or more heterologous Δ9 desaturases, which introduce a double bond in the hexaenoyl-CoA substrate at position 9 in the Z configuration in the presence of the hexaenoyl-CoA substrate.

[0372] Item 11. A cell according to Item 10, wherein the Δ9 desaturase 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.

[0373] Item 12. The cell 11 according to Items 7 to 9, wherein one or more native or endogenous genes are attenuated, disrupted and / or deleted.

[0374] Item 13. A cell according to items 7 to 12, wherein one or more pathway genes are overexpressed.

[0375] Item 14. A cell according to items 7 to 13, which is further genetically modified to provide an increased amount of a substrate for at least one enzyme of the (Z,E)-9,11-hexadecadienal pathway.

[0376] Item 15. A cell according to items 7 to 14, which is further genetically modified to exhibit increased tolerance to one or more substrate, intermediate or product molecules of the (Z,E)-9,11-hexadecadienal pathway.

[0377] Item 16. A cell according to Items 7 to 15, comprising at least two copies of one or more genes in the (Z,E)-9,11-hexadecadienal pathway.

[0378] Item 17. A host cell according to Items 7 to 16, wherein the host cell is a fungal cell.

[0379] Item 18. The host cell according to Item 17, wherein the fungal cell is a yeast cell. Item 19. The host cell according to Item 18, wherein the yeast cell belongs to a genus selected from the group consisting of Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces, wherein the yeast cell optionally belongs to a species selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Pichia, Kluyveromyces marxianus, Cryptococcus albus, Lipomyces oleaginosus, Lipomyces stargensis, Rhodosporidium toruloides, Rhodotorula glutinosus, Trichosporon and Yarrowia lipolytica.

[0380] Item 20. A cell according to Item 17, wherein the fungal cell is selected from Aspergillus awamori, Aspergillus odoratus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus nicotianae, Pseudocercis carnegiei, Pseudocercis genus, Pseudocercis moldy, White rot fungus, Pseudocercis light yellow, Pseudocercis curvature, Chrysosporium narrow marginatum, Chrysosporium hornophilum, Chrysosporium rukvenica, Chrysosporium coprosporum, Chrysosporium rukvenica, Chrysosporium coprosporum, Chrysosporium quenslandica, Chrysosporium tropicalis, Chrysosporium banded, Coprinus cinereus, Coriolus versicolor, Bacteroides, Fusarium cerealis, Fusarium crookwellii, Fusarium yellow Filamentous fungal cells of species consisting of Fungi, Fusarium graminearum, Fusarium graminearum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticularis, Fusarium roseum, Fusarium elder, Fusarium carnosus, Fusarium ramosissima, Fusarium sulphureus, Fusarium toroides, Fusarium monoterme, Fusarium venenatum, Humicola insolens, Humicola sparsely hairy, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium, Penicillium purpurogenum, Phanerochaete chrysosporium, Veins radiata, Pleurotus eryngii, Thielavia terrestris, Trametes velutipes, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride. Project 21. Genetically engineered yeast cells producing (Z,E)-9,11-hexadecadienoyl-CoA and (Z,E)-9,11-hexadecadien-1-ol, wherein the cells produce hexadecanoyl-CoA and express

[0381] a. Δ9 desaturase, catalyzing the formation of a Z-configuration double bond at position 9 in hexadecanoyl-CoA, thereby producing (Z)-9-hexadecenoyl-CoA;

[0382] b. E11 desaturase, catalyzing the formation of an E-configuration double bond at position 11 in the (Z)-9-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA; and

[0383] c. Alcohol-forming fatty acyl-CoA reductase (FAR) converts the (Z,E)-9,11-hexadecadienoyl-CoA into (Z,E)-9,11-hexadecadien-1-ol.

[0384] Item 22. The yeast cell according to Item 21, which further expresses one or more enzymes selected from the following:

[0385] a. Z11 desaturase, catalyzing the formation of a Z-configuration double bond at position 11 in hexadecenoyl-CoA, thereby producing (Z)-11-hexadecenoyl-CoA;

[0386] b. one or more alcohol-forming fatty acyl-CoA reductases (FARs) that convert the hexadecanoyl-CoA into hexadecane-1-ol, the (Z)-9-hexadecenoyl-CoA into (Z)-9-hexadecene-1-ol, and the (Z)-11-hexadecenoyl-CoA into (Z)-11-hexadecene-1-ol, respectively. Item 23. The yeast cell according to Item 21 or 22, wherein

[0387] a) an E11 desaturase having an amino acid sequence contained in the E11 desaturase of SEQ ID NO: 1 or 80;

[0388] b) a Δ9 desaturase having an amino acid sequence contained in the Δ9 desaturase of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22 or 82;

[0389] c) a Z11 desaturase having an amino acid sequence contained in the Z11 desaturase of SEQ ID NO: 72, 74, 76 or 78; and

[0390] d) The alcohol forming fatty acyl-CoA reductase (FAR) has an amino acid sequence contained in the FAR of 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.

[0391] Item 24. The yeast cell according to Item 21 or 23, wherein the yeast cell belongs to the species Saccharomyces cerevisiae or Yarrowia lipolytica.

[0392] Item 25. A cell culture comprising the genetically engineered microbial cells of Items 27 to 24 and a growth medium.

[0393] Item 26. A method for producing a target compound selected from the following compounds:

[0394] a)(Z,E)-9,11-hexadecadienoyl-CoA

[0395] b) (Z,E)-9,11-hexadecadien-1-ol;

[0396] c) (Z,E)-9,11-hexadecadienal; and / or

[0397] d) (Z,E)-9,11-hexadecadienyl acetate;

[0398] The method comprises culturing the cell culture of item 27 under conditions that allow the cell culture to produce the target compound, and optionally recovering and / or isolating the target compound. Item 27. The method of item 26 comprises: feeding the cell culture exogenously with one or more substrates or precursors of the target compound pathway.

[0399] Item 28. The method according to Items 26 to 27, wherein one or more steps of producing the target compound are performed in vitro.

[0400] Item 29. The method according to Items 26 to 28, wherein the in vitro step includes: using a reductase (FAR) to reduce (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol.

[0401] Item 30. A method according to Items 26 to 28, wherein the in vitro step comprises: chemically or enzymatically reducing (Z,E)-9,11-hexadecadienoic acid to (Z,E)-9,11-hexadecadien-1-ol.

[0402] Item 31. The method according to items 26 to 29, wherein the step performed 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 performed in vitro comprises: chemically or enzymatically acetylating (Z,E)-9,11-hexadecadien-1-ol to (Z,E)-9,11-hexadecadienyl acetate.

[0403] Item 33. The method according to any one of Items 26 to 32 also includes: recovering the target compound and mixing it with one or more carriers, reagents, additives, adjuvants and / or excipients to produce a biopesticide composition.

[0404] Item 34. A method according to Item 33, wherein the one or more carriers, reagents, additives, adjuvants and / or excipients include a conjugated sulfur-containing protecting agent that protects the target compound from being converted into an acid.

[0405] Item 35. A 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.

[0406] Item 36. The method according to Items 34 to 35 comprises: mixing at least 10 mg of the protective agent per gram of aldehyde and / or alcohol.

[0407] Item 37. A method according to items 33 to 37, wherein the one or more carriers, agents, additives, adjuvants and / or excipients include a carrier that promotes the slow release of the target compound, optionally (i) a polymer matrix selected from plastics, wax emulsions, oil emulsions or microcapsules and / or (ii) zeolites. Item 38. A method for producing a biopesticide composition, comprising

[0408] (I) Cultivating genetically engineered yeast cells that produce hexadecanoyl-CoA and expressing

[0409] a. Δ9 desaturase, catalyzing the formation of a Z-configuration double bond at position 9 in hexadecanoyl-CoA, thereby producing (Z)-9-hexadecenoyl-CoA;

[0410] b. E11 desaturase, catalyzing the formation of an E-configuration double bond at position 11 in (Z)-9-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA;

[0411] c. Alcohol-forming fatty acyl-CoA reductase (FAR), which converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol;

[0412] (II) converting (Z,E)-9,11-hexadecadien-1-ol into (Z,E)-9,11-hexadecadienal by an enzymatic or chemical method; and

[0413] (III) optionally recovering and / or isolating the (Z,E)-9,11-hexadecadienal and optionally one or more precursors thereof.

[0414] Item 39. The method of Item 38, wherein the genetically engineered yeast cell further expresses one or more enzymes selected from the following:

[0415] a. Z11 desaturase, which catalyzes the formation of a Z-configuration double bond at position 11 in hexadecanoyl-CoA, thereby producing (Z)-11-hexadecenoyl-CoA;

[0416] b. one or more alcohol-forming fatty acyl-CoA reductases (FARs), respectively converting the hexadecanoyl-CoA into hexadecane-1-ol, converting the (Z)-9-hexadecenoyl-CoA into (Z)-9-hexadecene-1-ol, and converting the (Z)-11-hexadecenoyl-CoA into (Z)-11-hexadecene-1-ol; the method further comprising: enzymatically or chemically converting the hexadecane-1-ol into hexadecanal, converting the (Z)-9-hexadecene-1-ol into (Z)-9-hexadecene-1-ol, converting the (Z)-11-hexadecene-1-ol and (Z)-11-hexadecene-1-ol into (Z)-11-hexadecene-1-ol; and / or optionally recovering and / or separating the hexadecanal, the (Z)-9-hexadecene-1-ol and the (Z)-11-hexadecene-1-ol and optionally one or more precursors thereof.

[0417] Item 40. The method according to Items 38 to 39 also includes: 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, reagents, additives, adjuvants and / or excipients to produce a biopesticide composition.

[0418] Item 41. The method according to Item 40, wherein the carrier, reagent, additive, adjuvant and / or excipient comprises one or more compounds selected from the following

[0419] a) a protecting agent, the protecting agent comprising 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, for protecting the target compound from being converted into an acid; and / or

[0420] b) a carrier that promotes the slow release of (Z,E)-9,11-hexadecadienal, (Z)-9-hexadecenal, (Z)-11-hexadecenal and / or hexadecanal in the mixture, optionally (i) a polymer matrix selected from plastics, wax emulsions, oil emulsions or microcapsules and / or (ii) a zeolite.

[0421] 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, reagents, additives, adjuvants and / or excipients.

[0422] Item 43. A biopesticide composition according to Item 42, which also contains at least trace amounts of one or more compounds selected from hexadecene-1-ol, (Z)-9-hexadecene-1-ol, (Z)-11-hexadecene-1-ol and (Z,E)-9,11-hexadecadiene-1-ol, and optionally other metabolites of the cell culture.

[0423] Item 44. The biopesticide composition further comprises (Z,E)-9,11-hexadecadienyl acetate.

[0424] Item 45. A biopesticide composition according to items 42 to 44, wherein one or more of hexadecanoyl-CoA, (Z)-9-hexadecenoyl-CoA, (Z)-11-hexadecenoyl-CoA, (Z,E)-9,11-hexadecadienoyl-CoA, hexadecane-1-ol, (Z)-9-hexadecene-1-ol, (Z)-11-hexadecene-1-ol and (Z,E)-9,11-hexadecadiene-1-ol are obtained by the method according to items 26 to 41, and optionally, the composition contains one or more other compounds or metabolites from the cell culture as described in item 25.

[0425] Item 46. A biopesticide composition according to Items 42 to 45, wherein the concentration of the target compound is at least 1 mg / kg of the composition.

[0426] Item 47. The composition according to Items 42 to 46, further comprising a protecting agent, wherein the protecting agent comprises conjugated sulfur, and the protecting agent protects the target compound from being converted into an acid.

[0427] Item 48. A composition according to Item 47, 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.

[0428] Item 49. The composition according to items 47 to 48, comprising at least 10 mg of protective agent per gram of aldehyde and / or alcohol.

[0429] Item 50. The composition according to items 42 to 49, further comprising a carrier for promoting slow release of the target compound, the carrier being optionally (i) a polymer matrix selected from plastics, wax emulsions, oil emulsions or microcapsules and / or (ii) zeolite.

[0430] Item 51. A method for controlling or monitoring pests, comprising distributing the composition of items 42 to 50 in a habitat of the pests and allowing the target compound to control the pests.

[0431] Item 52. The method according to Item 51, wherein the habitat is a sugarcane field and the pest is sugarcane borer.

Claims

1. An E11 fatty acyl-CoA desaturase (E11 desaturase) comprising an amino acid sequence that is at least 50% identical to 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.

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

3. A polynucleotide sequence encoding the E11 desaturase of claim 1 that is codon-optimized for heterologous expression, having a DNA sequence contained in SEQ 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, SEQ ID NO:105 or a homologue thereof, including variations due to the degeneracy of the genetic code.

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

5. The polynucleotide construct of 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 engineered microbial cell for producing (Z,E)-9,11-hexadecadienoyl-CoA, wherein the cell heterologously expresses the E11 desaturase according to claim 1 or 2, which introduces an E-configuration double bond at position 11 in the (Z)-9-hexadecadienoyl-CoA substrate in the presence of the (Z)-9-hexadecadienoyl-CoA substrate, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA having an E-configuration double bond at position 11.

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

9. The cell according to claim 8, wherein a) the 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; 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.

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

11. The cell of claim 10, wherein the Δ9 desaturase 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.

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

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

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

15. The cell of claims 7 to 14, further genetically modified to exhibit increased tolerance to one or more substrate, intermediate or product molecules of the (Z,E)-9,11-hexadecadienal pathway.

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

17. The cell of claims 7 to 15, comprising at least two copies of one or more genes in 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 of claim 18, wherein the fungal cell is a yeast cell.

20. The host cell of claim 19, wherein the yeast cell belongs to a genus selected from the group consisting of Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces, wherein the yeast cell optionally belongs to a species selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Pichia, Kluyveromyces marxianus, Cryptococcus albicans, Lipomyces oleaginosus, Lipomyces stargensis, Rhodosporidium toruloides, Rhodotorula glutinosus, budding Trichosporon, and Yarrowia lipolytica.

21. The cell of claim 18, wherein the fungal cell is selected from the group consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus nicotianae, Pseudocercis carnegiei, Pseudocercis spp., Pseudocercis moldy, Pseudocercis white rot, Pseudocercis light yellow, Pseudocercis curvature, Chrysosporium stenospermum, Chrysosporium hornophilum, Chrysosporium rukvenii, Chrysosporium coprospermum, Chrysosporium crassifolium, Chrysosporium queenslandense, Chrysosporium tropicalis, Chrysosporium zonalis, Coprinus cinereus, Coriolus versicolor, Bacteroides, Fusarium cerealis, Fusarium crookwellii, Fusarium flavosum Filamentous fungal cells of species consisting of Fungi, Fusarium graminearum, Fusarium graminearum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticularis, Fusarium roseum, Fusarium elder, Fusarium carnosus, Fusarium ramosissima, Fusarium sulphureus, Fusarium toroides, Fusarium monoterme, Fusarium venenatum, Humicola insolens, Humicola sparsely hairy, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium, Penicillium purpurogenum, Phanerochaete chrysosporium, Veins radiata, Pleurotus eryngii, Thielavia terrestris, Trametes velutipes, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.

22. A genetically engineered yeast cell that produces (Z,E)-9,11-hexadecadienoyl-CoA and (Z,E)-9,11-hexadecadien-1-ol, wherein the cell produces hexadecanoyl-CoA and expresses a. Δ9 desaturase, catalyzing the formation of a Z-configuration double bond at position 9 in hexadecanoyl-CoA, thereby producing (Z)-9-hexadecenoyl-CoA; b. E11 desaturase, catalyzing the formation of an E-configuration double bond at position 11 in the (Z)-9-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA; and c. Alcohol-forming fatty acyl-CoA reductase (FAR) converts the (Z,E)-9,11-hexadecadienoyl-CoA into (Z,E)-9,11-hexadecadien-1-ol.

23. The yeast cell of claim 22, further expressing one or more enzymes selected from the group consisting of: a. Z11 desaturase, catalyzing the formation of a Z-configuration double bond at position 11 in hexadecenoyl-CoA, thereby producing (Z)-11-hexadecenoyl-CoA; b. one or more alcohol-forming fatty acyl-CoA reductases (FARs), converting the hexadecanoyl-CoA into hexadecane-1-ol, converting the (Z)-9-hexadecenoyl-CoA into (Z)-9-hexadecene-1-ol, and converting the (Z)-11-hexadecenoyl-CoA into (Z)-11-hexadecene-1-ol, respectively.

24. The yeast cell according to claim 22 or 23, wherein a) an E11 desaturase having at least 70% sequence identity to the amino acid sequence comprised in the E11 desaturase of SEQ ID NO: 1, 80, 90, 92, 96, 98, 100, 102 or 104; b) a Δ9 desaturase having at least 70% sequence identity to the amino acid sequence comprised 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 to the amino acid sequence comprised in the Z11 desaturase of SEQ ID NO: 72, 74, 76 or 78; and / or d) an alcohol forming fatty acyl-CoA reductase (FAR) having at least 70% sequence identity to 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.

25. The yeast cell according to claims 22 to 24, wherein a) an E11 desaturase having an amino acid sequence comprised in the E11 desaturase of SEQ ID NO: 1, 80, 90, 92, 96, 98, 100, 102 or 104; b) a Δ9 desaturase having 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.

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

27. A cell culture comprising the genetically engineered microbial cells of claims 7 to 26 and a growth medium.

28. A method for producing a target compound selected from the group consisting of: 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; The method comprises culturing the cell culture of claim 27 under conditions that allow the cell culture to produce the target compound, and optionally recovering and / or isolating the target compound.

29. The method according to claim 28, comprising: The cell culture is fed exogenously with one or more substrates or precursors of a pathway to the compound of interest.

30. The method of claims 28 to 29, wherein one or more steps of producing the target compound are performed in vitro.

31. The method of claims 28 to 30, wherein the step performed in vitro comprises: (Z,E)-9,11-hexadecadienoyl-CoA is reduced to (Z,E)-9,11-hexadecadien-1-ol using a reductase (FAR).

32. The method of claims 28 to 30, wherein the step performed in vitro comprises: (Z,E)-9,11-hexadecadienoic acid is reduced chemically or enzymatically to (Z,E)-9,11-hexadecadien-1-ol.

33. The method of claims 28 to 31, wherein the step performed in vitro comprises: (Z,E)-9,11-hexadecadien-1-ol is oxidized chemically or enzymatically to (Z,E)-9,11-hexadecadienal.

34. The method of claims 28 to 31, wherein the step performed in vitro comprises: (Z,E)-9,11-hexadecadien-1-ol is chemically or enzymatically acetylated to (Z,E)-9,11-hexadecadienyl acetate.

35. A method according to any one of claims 28 to 34, further comprising: The target compound is recovered and mixed with one or more carriers, agents, additives, adjuvants and / or excipients to produce a biopesticide composition.

36. The method of claim 35, wherein the one or more carriers, agents, additives, adjuvants and / or excipients include a conjugated sulfur-containing protecting agent that protects the target compound from conversion to an acid.

37. The method of claim 36, 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.

38. The method according to claims 36 to 37, comprising: At least 10 mg of the protective agent is mixed per gram of aldehyde and / or alcohol.

39. The method of claims 35 to 38, wherein the one or more carriers, agents, additives, adjuvants and / or excipients comprise a carrier that promotes slow release of the target compound, optionally (i) a polymer matrix selected from a plastic, a wax emulsion, an oil emulsion or a microcapsule and / or (ii) a zeolite.

40. A method for producing a biopesticide composition comprising the following steps: (I) Cultivating genetically engineered yeast cells that produce hexadecanoyl-CoA and expressing a. Δ9 desaturase, catalyzing the formation of a Z-configuration double bond at position 9 in hexadecanoyl-CoA, thereby producing (Z)-9-hexadecenoyl-CoA; b. E11 desaturase, catalyzing the formation of an E-configuration double bond at position 11 in (Z)-9-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA; c. Alcohol-forming fatty acyl-CoA reductase (FAR), which converts (Z,E)-9,11-hexadecadienoyl-CoA to (Z,E)-9,11-hexadecadien-1-ol; (II) converting (Z,E)-9,11-hexadecadien-1-ol into (Z,E)-9,11-hexadecadienal by an enzymatic or chemical method; as well as (III) optionally recovering and / or isolating the (Z,E)-9,11-hexadecadienal and optionally one or more precursors thereof.

41. The method of claim 40, wherein the genetically engineered yeast cell further expresses one or more enzymes selected from: a. Z11 desaturase, which catalyzes the formation of a Z-configuration double bond at position 11 in hexadecanoyl-CoA, thereby producing (Z)-11-hexadecenoyl-CoA; b. one or more alcohol-forming fatty acyl-CoA reductases (FARs) that convert the hexadecanoyl-CoA into hexadecane-1-ol, the (Z)-9-hexadecenoyl-CoA into (Z)-9-hexadecene-1-ol, and the (Z)-11-hexadecenoyl-CoA into (Z)-11-hexadecene-1-ol, respectively; The method further includes: The hexadecan-1-ol is converted into hexadecanal, the (Z)-9-hexadecen-1-ol is converted into (Z)-9-hexadecenal, the (Z)-11-hexadecen-1-ol and (Z)-11-hexadecen-1-ol are converted into (Z)-11-hexadecenal by enzymatic or chemical methods; and / or the hexadecanal, the (Z)-9-hexadecenal and the (Z)-11-hexadecenal and optionally one or more precursors thereof are recovered and / or separated.

42. A method for producing a biopesticide composition comprising the following steps: (I) Cultivating genetically engineered yeast cells that produce hexadecanoyl-CoA and expressing a. E11 desaturase, which catalyzes the formation of an E-configuration double bond at position 11 in hexadecanoyl-CoA, thereby producing (E)-11-hexadecenoyl-CoA; b. Δ9 desaturase, which catalyzes the formation of a Z-configuration double bond at position 9 in (E)-11-hexadecenoyl-CoA, thereby producing (Z,E)-9,11-hexadecadienoyl-CoA; c. an alcohol-forming fatty acyl-CoA reductase (FAR) that converts the (Z,E)-9,11-hexadecadienoyl-CoA into (Z,E)-9,11-hexadecadien-1-ol; (II) enzymatically or chemically converting the (Z,E)-9,11-hexadecadien-1-ol into (Z,E)-9,11-hexadecadienal; as well as (III) optionally recovering and / or isolating the (Z,E)-9,11-hexadecadienal and optionally one or more precursors thereof.

43. The method of claims 40 to 42, wherein the genetically engineered yeast cell further expresses one or more enzymes selected from: a. Z11 desaturase, catalyzing the formation of a Z-configuration double bond at position 11 in the hexadecanoyl-CoA, thereby producing (Z)-11-hexadecenoyl-CoA; b. one or more alcohol-forming fatty acyl-CoA reductases (FARs) that convert the hexadecanoyl-CoA into hexadecane-1-ol, the (Z)-9-hexadecenoyl-CoA into (Z)-9-hexadecene-1-ol, and the (Z)-11-hexadecenoyl-CoA into (Z)-11-hexadecene-1-ol, respectively; And the method also includes: The hexadecan-1-ol is converted into hexadecanal, the (Z)-9-hexadecen-1-ol is converted into (Z)-9-hexadecenal, the (Z)-11-hexadecen-1-ol and the (Z)-11-hexadecen-1-ol are converted into (Z)-11-hexadecenal by enzymatic or chemical methods; and / or the hexadecanal, the (Z)-9-hexadecenal and the (Z)-11-hexadecenal and optionally one or more precursors thereof are recovered and / or separated.

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

45. The method of claim 44, wherein the carrier, agent, additive, adjuvant and / or excipient comprises one or more compounds selected from the group consisting of a) a protecting agent, the protecting agent comprising 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, for protecting the target compound from being converted into an acid; and / or b) a carrier that promotes the slow release of (Z,E)-9,11-hexadecadienal, (Z)-9-hexadecenal, (Z)-11-hexadecenal and / or hexadecanal in the mixture, optionally (i) a polymer matrix selected from plastics, wax emulsions, oil emulsions or microcapsules and / or (ii) a zeolite.

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 of claim 46, further comprising 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, and optionally other metabolites of the cell culture.

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

49. The biopesticide composition according to claims 46 to 48, wherein 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-hexadecene-1-ol, (Z)-11-hexadecene-1-ol and (Z,E)-9,11-hexadecadiene-1-ol are obtained by the method according to claims 26 to 41, and optionally, the composition comprises one or more other compounds or metabolites from the cell culture according to claim 25.

50. The biopesticide composition of claims 46 to 49, wherein the biopesticide composition comprises at least 20% biobased carbon, such as at least 30% biobased carbon, such as at least 40% biobased carbon, such as at least 50% biobased carbon, such as at least 60% biobased carbon, such as at least 70% biobased carbon, such as at least 75% biobased carbon, such as at least 80% biobased carbon, such as at least 85% biobased carbon, such as at least 90% biobased carbon, such as at least 95% biobased carbon, such as 100% biobased carbon.

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

52. The biopesticide composition of claims 46 to 49, wherein the composition comprises 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%.

53. The biopesticide composition of claims 46 to 49, wherein the 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.

54. A biopesticide composition according to claims 46 to 52, wherein the composition comprises no more than 50% fossil based carbon, such as no more than 45%, such as no more than 40%, such as no more than 35%, such as no more than 30%, such as no more than 25%, such as no more than 20%, such as no more than 15%, such as no more than 10%, such as no more than 5%, such as no more than 1% fossil based carbon.

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

56. The biopesticide composition according to claims 46 to 55, further comprising a protectant comprising conjugated sulfur, which protects the target compound from degradation, for example by conversion into acid.

57. The biopesticide composition of claim 56, wherein the protectant comprises a compound selected from the group consisting of 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 biopesticide composition according to claims 56 to 57, comprising at least 10 mg of protective agent per gram of aldehyde and / or alcohol.

59. The biopesticide composition according to claims 46 to 58, further comprising a carrier that promotes the slow release of the target compound, the carrier being optionally (i) a polymer matrix selected from plastics, wax emulsions, oil emulsions or microcapsules and / or (ii) zeolites.

60. A method of controlling or monitoring pests, comprising distributing the biopesticide composition of claims 46 to 59 in a habitat of the pests and allowing the target compound to control the pests.

61. The method of claim 60, wherein the habitat is a sugar cane field and the pest is the sugar cane borer.

Images