Field

[0001] The present disclosure describes a biobased biopesticide compositions comprising sugarcane borer Diatraea saccharalis mating pheromone components and methods for the production of such biopesticide composition. Also described herein are engineered cells and enzymes they express as well as methods for the application of such biopesticide compositions for the control of pests.

Background

[0002] Integrated Pest Management (IPM) is playing an increasing role for both increasing the crop yield and for minimizing environmental impact and enabling organic food production. IPM employs alternative pest control methods, such as using pheromones for pest insect mating disruption or mass trapping, or attraction of beneficial insects etc.

[0003] Pheromones constitute a group of diverse chemical compounds that insects (like other organisms) use to communicate between individuals of the same species in various contexts, including mate attraction, alarm, trail marking and aggregation. Insect pheromones associated with long-range mate finding are already used in agriculture and forestry applications for monitoring and control of pests, as a safe and environmentally friendly alternative to pesticides. The biological production of pheromones for use in pest control is advantageous over chemical synthesis in respect to price, specificity, and environmental impact.

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

[0005] Known pheromones include fatty acyl alcohols, aldehydes and acetates having one or more double bonds at specific positions of the carbon backbone having specific Z or/and E orientation. The sugarcane borer D. saccharalis is a major pest of sugarcane in the Central and Southern America. Its major mating pheromone component is (Z,E)-9, 11- hexadecadienal (Svatos, et aL, 2001). The minor pheromone components are (Z)-ll-hexadecenal, (Z)-9-hexadecenal, and hexadecanal (Kalinova, Kindi, Hovorka, Hoskovec, & Svatos, 2005) (Da Silva, et al., 2021).

[0006] Zhao et al (2004) relates to sex pheromones (Z)-5-dodecenol and (Z,E)-5,7-dodecadienol of the moth Dendrolimus punctatus and studies the formation of compounds produced when topically applying labelled fatty acids to the moth gland. The paper further speculates that the pathway for D.punctatus sex pheromones includes formation of (Z,E)-9,ll-hexadecadienoyl-CoA intermediate compounds, but no such compound is in fact detected in any of the reported experiments, although searched for. Lienard et al (2010) described yeast cells expressing a desaturase from D. punctatus named Dpu_APSQ. The authors speculate that the desaturase Dpu_APSQ introduces a Ell- desaturation into (Z)-9-hexadecenoic acid natively produced in yeast producing the doubleunsaturated C16-fatty acid (Z,E)-9,ll-hexadecadienoic acid. However, the present inventors have found that the Dpu_APSQ does not produce (Z,E)-9,ll-hexadecadienoic acid, but another unknown double unsaturated hexadecadienoic acid eluting slightly later than the (Z,E)-9,ll-hexadecadienoic acid.

[0007] Accordingly, until now a bio-based method for producing D. saccharalis pheromones has not been available and accordingly, there is a need for identifying enzymes that can carry out biosynthesis of pheromone precursor and developing recombinant strains and methods for the production of such pheromones.

Summary

[0008] Until the present disclosure no enzyme has been available for expression in a genetically modified host cell for introducing Ell double bond in (Z)-9-hexadecenoyl-CoA (Z9-16:CoA) - a functionality which is essential to the production of the major mating pheromone component of D. saccharalis. However, disclosed herein is a novel enzyme which surprisingly catalyses the Ell desaturation in a (Z)-9-hexadecenoyl-CoA substrate and in the presence of such (Z)-9-hexadecenoyl- CoA substrate produces (Z,E)-9,ll-hexadecadienoyl-CoA, which is a precursor for D. saccharalis pheromone (Z,E)-9,ll-hexadecadienal (Z9, Ell-16 : Aid ). Further, the present disclosure provides a A9- desaturase capable of introducing a Z9 double bond in (E)-ll-hexadecenoyl-CoA to provide (Z,E)-9,11- hexadecadienoyl-CoA. Accordingly, the present inventors succeeded in preparing for the first time biobased biopesticide compositions comprising D. saccharalis pheromone components formulated for the use to control pests such as D. saccharalis. Accordingly, in a first aspect provided for herein is a biopesticide composition comprising (Z,E)-9,ll-hexadecandienal, and optionally one or more compounds selected from (Z)-9-hexadecenal, (Z)-ll-hexadecenal and/or hexadecanal, in combination with one or more carriers, agents, additives, adjuvants and/or excipients.

[0009] In a further aspect described herein is a method for controlling a pest comprising distributing the composition described herein in a habitat for the pest and allowing the target compound to control the pest.

[0010] In a still further aspect described herein is a method for producing the biopesticide composition of this disclosure comprising (I) culturing a genetically engineered yeast cell producing hexadecanoyl-CoA and expressing a) a A9 desaturase catalyzing the formation of a double bond in a Z configuration in position

9 in the hexadecanoyl-CoA and thereby producing a (Z)-9-hexadecenoyl-CoA; b) an Ell desaturase catalyzing the formation of a double bond in a E configuration in position 11 in the (Z)-9-hexadecenoyl-CoA and thereby producing a (Z,E)-9,11- hexadecandienoyl-CoA; c) an alcohol-forming fatty acyl-CoA reductase (FAR) converting the (Z,E)-9,11- hexadecandienoyl-CoA into (Z,E)-9,ll-hexadecandien-l-ol;

(II) enzymatically or chemically converting the (Z,E)-9,ll-hexadecandien-l-ol to (Z,E)-9,11- hexadecandienal; and

(III) optionally recovering and/or isolating the (Z,E)-9,ll-hexadecandienal and optionally one or more precursors thereof.

[0011] In a still further aspect described herein is a genetically engineered yeast cell producing (Z,E)- 9,11-hexadecadienoyl-CoA and (Z,E)-9,ll-hexadecadien-l-ol, said cell producing hexadecanoyl-CoA and expressing a. a A9 desaturase catalyzing the formation of a double bond in a Z configuration in position 9 in the hexadecanoyl-CoA thereby producing a (Z)-9-hexadecenoyl-CoA; b. an Ell desaturase catalyzing the formation of a double bond in a E configuration in position 11 in the (Z)-9-hexadecenoyl-CoA thereby producing a (Z,E)-9,ll-hexadecadienoyl-CoA; and c. an alcohol-forming fatty acyl-CoA reductase (FAR) converting the (Z,E)-9,ll-hexadecadienyl- CoA into (Z,E)-9,ll-hexadecadien-l-ol.

[0012] In a still further aspect described herein is a cell culture comprising the genetically engineered microbial cell described herein and a growth medium.

[0013] In a still further aspect described herein is an Ell desaturase having an amino acid sequence comprised in the Ell desaturase of SEQ. ID NO: 1.

Drawings and figures

[0014] Figure 1 shows the method for producing the major and minor pheromone components of D. saccharalis - (Z,E)-9,ll-hexadecadienal. The method comprises biosynthesis of (Z,E)-9,11- hexadecadien-l-ol and subsequent chemical oxidation into the corresponding aldehyde (Z,E)-9 ll- hexadecadienal. Due to the side biochemical reactions, the minor components of the D. saccharalis can be produced in the same process as shown.

[0015] Figure 2 shows GC-MS analyses of FAME extracts from Yarrowia lipolytica strains expressing D. saccharalis and D. punctatus desaturases. (A) Total ion chromatogram of FAME extracts from strains expressing Dsl2389 D. saccharalis desaturase (the dashed line) were compared with extracts from the control strain transformed with an empty vector (the dotted line) and authentic standard of (Z, E)-9, 11- hexadecadienoic acid methyl ester (Z9, Ell-16:Me) (the solid line). (B) Total ion chromatogram of FAME extracts from strains expressing Dpu_APSQ (the dotted dash line) were compared with extracts from the control strain transformed with an empty vector (the dot line) and authentic standard of Z9, Ell-16:Me (the solid line).

[0016] Figure 3 shows mass spectra of detected products from GC-MS chromatograms. (A) the product eluted at 12. 954 min from the FAME extracts of Y. lipolytica strains expressing Dsl2389. (B) authentic standard of Z9, Ell-16:Me.

[0017] Figure 4 shows representative GC chromatograms of (A) extracts of strains ST12118 (cultivated in medium supplemented with Z9, Ell-16:Me), expressing fatty acyl-CoA reductase HarFAR (also referred to as FAR1 herein), and Z9,E11-16:OH analytical standard. Mass spectra of ( B) extract of ST12118 and (C) Z9, E11-16:OH analytical standard at retention time 13.6-13.7 min.

[0018] Figure 5 shows representative GC chromatograms of (A) extracts of strains ST13046, ST13042, ST13043 and Z9,Ell-16:Me analytical standard. Mass spectra of (B) extract of ST13046, (C) extract of ST13042, (D) extract of ST13043 and (E) Z9,Ell-16:Me analytical standard at retention time 11.08 min.

Incorporation by reference

[0019] All publications, patents, and patent applications referred to 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 between a term herein and a term in an incorporated reference, the term herein prevails and controls.

Details

Definitions

[0020] Throughout this disclosure where reference is made to a pheromone component or a precursor for example (Z,E)-9,ll-hexandecadienal referring to the fatty aldehyde having a carbon chain of 16, having an aldehyde at Cl, having a Z configured double bond at C9 and an E configured double bond at Cll, alternative terms such as Z9,Ell-16:Ald or (Z9, Ell)-hexadecadienal may be used interchangeably. Similar nomenclature may be used about other pathway compounds such as corresponding fatty acids, CoA derivatives, alcohols, acids or acetates.

[0021] The term "saturated" refers to a compound which is devoid of double or triple carbon-carbon bonds.

[0022] The term "desaturated" as used herein interchangeably with the term "unsaturated" about compounds refers to the compound containing one or more double or triple carbon-carbon bonds, preferably a double carbon-carbon bond. The following nomenclature is used herein throughout: a A/ desaturated compound, where / is an integer, refers to a compound having a double or triple carboncarbon bond in position / of the carbon chain. The carbon chain length is thus at least equal to /. For example, a A12 desaturated compound refers to a compound having a double or triple carbon-carbon bond in position 12 and having a carbon chain length of 13 or more. The double or triple bond can be in an E configuration or in a Z configuration. Thus, an E/ or a Z/ desaturated compound will refer to a compound having a double carbon-carbon bond in an E configuration or in a Z configuration, respectively, in position / of the carbon chain, which has a total length at least equal to /. For example, an Ell fatty alcohol has a desaturation in position 11 in an E configuration and has a carbon chain length of 12 or more.

[0023] The term "All desaturase" as used herein refers to a desaturase enzyme which catalyzes the introduction of a double bond in position between Cll and C12 in 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.

[0024] The term "Ell desaturase" as used herein refers to a All desaturase enzyme which catalyzes the introduction of a double bond in a E configuration in position 11 between Cll and C12 in 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.

[0025] The term "A9 desaturase" as used herein refers to a desaturase enzyme which catalyzes the introduction of a double bond in position between C9 and CIO in 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.

[0026] The term "Z9 desaturase" as used herein refers to a A9 desaturase enzyme which catalyzes the introduction of a double bond in a Z configuration in position between C9 and CIO in 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.

[0027] The term "biopesticide" as used herein refers to a contraction of 'biological pesticide' and refers to several types of pest management interventions: through predatory, parasitic, or chemical relationships. In the EU, biopesticides have been defined as "a form of pesticide based on microorganisms or natural products". In the US, they are defined by the EPA as "including naturally occurring substances that control pests (biochemical pesticides), microorganisms that control pests (microbial pesticides), and pesticidal substances produced by plants containing added genetic material (plantincorporated protectants) or PIPs". The present disclosure relates more particularly to biopesticides comprising natural products or naturally occurring substances. In the present context, these are manufactured by cultivating and concentrating naturally occurring organisms and/or their metabolites including bacteria and other microbes, fungi, nematodes, proteins, etc. These compounds are considered to be important components of integrated pest management (IPM) programs and have received much practical attention as substitutes to synthetic chemical plant protection products (PPPs). The Manual of Biocontrol Agents (2009: formerly the Biopesticide Manual) gives a review of the available biological insecticide (and other biology-based control) products.

[0028] The term "biobased" as used herein is used to characterize biobased products wherein:

(I) the total carbon content of the product is at least 30%, and

(II) the carbon content of a renewable raw material (biobased) is at least 20%.

[0029] As recognized by the Circular Bio-based Europe Joint Undertaking (CBE Joint Undertaking) established in 2021, developing biobased materials is essential if the EU is to reach its climate targets as set out in the European Green Deal. The present disclosure provides a methodology for efficiently providing fatty alcohols and fatty aldehydes having a high content of biobased carbon (%).

[0030] Both fossil and renewable raw materials consist mainly of carbon (C). Carbon occurs in several isotopes. Isotope ¹⁴ C is radioactive and occurs naturally in all living organisms (plants, animals, etc) in a fixed relative concentration which is nearly identical to the relative ¹⁴ C concentration in the atmosphere. At this concentration, the radioactivity level of ¹⁴ C is 100%. Once an organism is no longer living, this concentration, and thus the radioactivity rate, decays with a half-life of approximately 5700 years. The radioactive ¹⁴ C level of an unknown substance can therefore help determine how old the carbon contained in the substance is.

[0031] "Young" carbon (0 to 10 years) derived from renewable raw materials, such as plants or animals, has a relative isotope ¹⁴ C concentration which is nearly identical to the relative ¹⁴ C concentration in the atmosphere and the radioactive ¹⁴ C level of such young carbon is thus about 100%.

[0032] "Old" carbon (millions of years) derived from synthetic or fossil (petrochemical) sources is greatly depleted from isotope ¹⁴ C as the age of such synthetic and fossil sources far exceeds the halflife of isotope ¹⁴ C which is approximately 5700 years. Hence, carbon derived from synthetic or fossil sources has a relative isotope ¹⁴ C concentration around 0% and the radioactive ¹⁴ C level of such old carbon is thus about 0%.

[0033] In one embodiment the term "radioactive ¹⁴ C level" refer to the total radioactive ¹⁴ C level of a given substance, product, or composition, as defined above.

[0034] The isotope ¹⁴ C method may be used to determine the concentration of young (renewable) materials in comparison with the concentration of old (fossil) resources. The carbon content of a renewable raw material is referred to as the "biobased carbon content". The carbon content of a renewable raw material or the "biobased carbon content" may be determined as described below.

[0035] When measuring the biobased carbon content, the result may be reported as "% biobased carbon". This indicates the percentage carbon from "natural" (plant or animal by-product) sources versus "synthetic" or "fossil" (petrochemical) sources. For reference, 100 % biobased carbon indicates that a material is entirely sourced from plants or animal by-products and 0 % biobased carbon indicates that a material did not contain any carbon from plants or animal by-products. A value in between represents a mixture of natural and fossil sources.

[0036] Example: If a product has a radioactive ¹⁴ C level of 80%, it means that the product consists of 80% renewable and 20% fossil carbon (C). In other words, the product is 80% biobased.

[0037] The analytical measurement may be cited as "percent modern carbon (pMC)". This is the percentage of ¹⁴ C measured in the sample relative to a modern reference standard (NIST 4990C). The % Biobased Carbon content is calculated from pMC by applying a small adjustment factor for ¹⁴ C in carbon dioxide in air today. It is important to note that all internationally recognized standards using ¹⁴ C assume that the plant or biomass feedstocks were obtained from natural environments. pMC may be analysed by a standard test method, such as "ASTM D6866".

[0038] The term "fatty acyl compounds" as used herein refers to fatty compounds having a long aliphatic chain, i.e. an aliphatic chain typically having between 12 and 28 carbon atoms, such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 1 or 28 carbon atoms. Most naturally occurring fatty acids are unbranched. They can be saturated, or desaturated. Fatty acyl compounds can include various functional end groups.

[0039] The term "Fatty acyl-CoA" as used herein interchangeably with "fatty acyl-CoA ester" refers to compounds of general formula R-CO-SCoA, where R is a fatty 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 fatty carbon chain is joined to the -SH group of CoA by a thioester bond. Fatty acyl- CoAs can be saturated or desaturated, depending on whether the fatty acid which it is derived from is saturated or desaturated.

[0040] The term "fatty alcohol" as used herein 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. Fatty alcohols can be saturated or desaturated.

[0041] The term "fatty alcohol acetate" as used herein refers to an acetate having a fatty carbon chain, i.e. an aliphatic chain 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. Fatty acyl acetates can be saturated or desaturated.

[0042] The term "fatty aldehyde" as used herein 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. Fatty aldehydes can be saturated or desaturated.

[0043] The term "functional variant" as used herein refers to functional variants of an enzyme, which retain at least some of the activity of the parent enzyme. Thus, functional variants of desaturases, or other pathway enzymes catalyze similar reactions as their parent enzymes, although the efficiency and specificity of reaction may be different, e.g. the efficiency is decreased or increased compared to the parent enzyme.

[0044] The term "heterologous" or "recombinant" or "genetically modified" and their grammatical equivalents as used herein interchangeably about nucleotides, polypeptides and cells refers to entities "derived from a different species or cell". For example, a heterologous or recombinant polynucleotide gene is a gene in a host cell not naturally containing that gene, i.e. the gene is from a different species or cell type than the host cell. A heterologous or recombinant polypeptide is a polypeptide produced in a host cell not naturally containing the polypeptide, i.e. the polypeptide is from a different species or cell type than the host cell. Where the terms as used herein about host cells, they refer to host cells comprising and expressing heterologous or recombinant polynucleotides

[0045] The term "% identity" is used herein about the relatedness between two amino acid sequences or between two nucleotide sequences usings standard alignment software known in the art, and applying settings as instructed for the software, including gaps, to achieve the maximum percent identity/similarity/homology, and if necessary considering any conservative substitutions according to the NCIUB rules (hftp://www. chem.qmul.ac.uk/iubmb/misc/naseq.html; NC-IUB, Eur J Biochem (1985)) as part of the sequence identity. 5' or 3' extensions nor insertions (for nucleic acids) or N' or C' extensions nor insertions (for polypeptides) usually result in a reduction of identity, similarity or homology using such standard software.

[0046] The term "degeneracy" as used herein about genetic code reflects that a number of different variant nucleotide sequences can encode the same polypeptide, since there is more than one nucleotide triplet that serves as codon for a given amino acid. Thus, codons in the coding sequence for a given polypeptide can be modified such that optimal expression in a particular host is obtained, using appropriate codon bias tables for a particular host cell.

[0047] The term "pest" as used herein refers to an organism, in particular an animal such as an insect considered harmful to humans or animals or to cultivated crops, in particular within agriculture or livestock production. A pest is any living organism which is invasive or prolific, harmful, troublesome, noxious, destructive, to either crops or animals, human or human concerns, livestock, human structures, wild ecosystems etc. Particularly used herein pest is used about the sugarcane borer moth D. saccharalis and any of its preceding life stages (larvae). [0048] The term "pheromone" as used herein is used about naturally occurring signaling compounds used in nature for chemical communication between indiviuals of a species. Lepidopteran pheromones for example are designated by an unbranched aliphatic chain (between 9 and 18 carbons, such as 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 carbon atoms) ending in an alcohol, aldehyde or acetate functional group and containing up to 3 double bonds in the aliphatic backbone. Thus, desaturated fatty alcohols, desaturated fatty aldehydes and desaturated fatty alcohol acetates are typically comprised in pheromones. Pheromone compositions may be produced chemically or biochemically, for example as described herein. Pheromones thus comprise desaturated fatty alcohols, desaturated fatty aldehydes and/or desaturated fatty alcohol acetates, such as can be obtained by the methods and cells described herein.

[0049] The titer of a compound as used herein refers herein to the produced concentration of a compound. When the compound is produced by a cell, the term refers to the total concentration produced by the cell, i.e. the total amount of the compound divided by the volume of the culture medium. This means that, particularly for volatile compounds, the titer includes the portion of the compound which may have evaporated from the culture medium, and it is thus determined by collecting the produced compound from the fermentation broth and from potential off-gas from the fermenter.

[0050] The term "pathway" or "biosynthetic pathway" or "metabolic pathway" as used herein interchangeably refers to one or more enzymes acting in concert in a live cell to convert one or more substrate precursors into a chemical product. A pathway may include one enzyme or multiple enzymes acting in sequence or in combination. A pathway including only one enzyme may also herein be referred to as "bioconversion" in particular relevant for embodiments where a host cell is fed with a precursor or substrate exogenously to be converted by the enzyme into a desired end product. Enzymes are characterized by having catalytic activity, which can change the chemical structure of the substrate(s). An enzyme may have more than one substrate and produce more than one product. The enzyme may also depend on cofactors, which can be inorganic chemical compounds or organic compounds (co-factor and/or co-enzymes) which may or may not be considered part of the pathway. [0051] The term "in vivo", as used herein refers to within a living cell or organism, including, for example animal, a plant or a microorganism.

[0052] The term "in vitro" as used herein refers to outside a living cell or organism, including, without limitation, for example, in a microwell plate, a tube, a flask, a beaker, a tank, a reactor and the like.

[0053] 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 both compounds generated in situ by an enzymatic reaction in a cell or exogenously provided compounds, such as exogenously provided organic molecules which the host cell can metabolize into a desired compound.

[0054] The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.

[0055] The term "expression vector" refers to a DNA molecule, either single- or double stranded, either linear or circular, which comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression. Expression vectors include expression cassettes for the integration of genes into a host cell as well as plasmids and/or chromosomes comprising such genes.

[0056] The term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide to be expressed in the host cell. Host cell encompasses any progeny of a parent cell including those that are not identical to the parent cell due to mutations that occur during replication.

[0057] The term "polynucleotide construct" refers to a polynucleotide, either single- or double stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises a polynucleotide encoding a polypeptide and one or more control sequences.

[0058] The term "operably linked" refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding polynucleotide such that the control sequence directs expression of the coding polynucleotide.

[0059] The terms "nucleotide sequence" and "polynucleotide" are used herein interchangeably.

[0060] The term "comprise" and "include" as used throughout the specification and the accompanying items as well as variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. These words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[0061] The articles "a" and "an" are used herein refers to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

[0062] Terms like "preferably", "commonly", "particularly", and "typically" are not utilized herein to limit the scope of the itemed invention or to imply that certain features are critical, essential, or even important to the structure or function of the itemed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention. [0063] The term "cell culture" as used herein refers to a culture medium comprising a plurality of the host cells described herein. A cell culture may comprise a single strain of host cells or may comprise two or more distinct host cell strains. The culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., a carbon source; a nitrogen source; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases; and the like.

[0064] Term "endogenous" or "native" as used herein refers to a gene or a polypeptide in a host cell which originates from the same host cell.

[0065] The term "deletion" as used herein refers to manipulation of a gene so that it is no longer expressed in a host cell.

[0066] The term "disruption" as used herein refers to manipulation of a gene or any of the machinery participating in the expression the gene, so that it is no longer expressed in a host cell.

[0067] The term "attenuation" as used herein refers to manipulation of a gene or any of the machinery participating in the expression the gene, so that it the expression of the gene is reduced as compared to expression without the manipulation.

[0068] All methods described herein can be performed in any suitable order of steps unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0069] All percentages, ratios and proportions herein are by weight, unless otherwise specified. A weight percent (weight %, also as wt. %} of a component, unless specifically stated to the contrary, is based on the total weight of the composition in which the component is included (e.g., on the total amount of the reaction mixture).

[0070] The terms "substantially" or "approximately" or "about", as used herein refers to a reasonable deviation around a value or parameter such that the value or parameter is not significantly changed. These terms of deviation from a value should be construed as including a deviation of the value where the deviation would not negate the meaning of the value deviated from. For example, in relation to a reference numerical value the terms of degree can include a range of values plus or minus 10% from that value. For example, deviation from a value can include a specified value plus or minus a certain percentage from that value, such as plus or minus 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the specified value.

[0071] The term "and/or" as used herein is intended to represent an inclusive "or". The wording X and/or Y is meant to mean both X or Y and X and Y. Further the wording X, Y and/or Z is intended to mean X, Y and Z alone or any combination of X, Y, and Z.

[0072] The term "isolated" as used herein about a compound, refers to any compound, which by means of human intervention, has been put in a form or environment that differs from the form or environment in which it is found in nature. Isolated compounds include but are not limited to compounds of the disclosure for which the ratio of the compounds relative to other constituents with which they are associated in nature is increased or decreased. In an important embodiment the amount of compound is increased relative to other constituents with which the compound is associated in nature. In an embodiment the compound of the disclosure may be isolated into a pure or substantially pure form. In this context a substantially pure compound means that the compound is separated from other extraneous or unwanted material present from the onset of producing the compound or generated in the manufacturing process. Such a substantially pure compound preparation contains less than 10%, such as less than 8%, such as less than 6%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, such as less than 1 %, such as less than 0.5% by weight of other extraneous or unwanted material usually associated with the compound when expressed natively or recombinantly. In an 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. [0073] 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 appearing as mature spliced mRNA.

[0074] The term "coding sequence" refers to a nucleotide sequence, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, orTGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0075] The term "control sequence" as used herein refers to a nucleotide sequence necessary for expression of a polynucleotide encoding a polypeptide. A control sequence may be native (i.e., from the same gene) or heterologous or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide. Control sequences include, but are not limited to leader sequences, polyadenylation sequence, pro-peptide coding sequence, promoter sequences, signal peptide coding sequence, translation terminator (stop) sequences and transcription terminator (stop) sequences. To be operational control sequences usually must include promoter sequences, transcriptional and translational stop signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with a coding region of a polynucleotide encoding a polypeptide.

A9 desaturases

[0024] The present disclosure further provides a A9 desaturase comprising an amino acid sequence which is the desaturase comprised in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82 or is a functional variant at least 50% identical to the desaturase comprised in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82. In some embodiments the A9 desaturase is from 50% to 100% identical to the A9 desaturase comprised in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82, such as from 50% to 60%, , such as from 60% to 70%, such as from 70% to 80%, such as from 80% to 90%, such as from 90% to 92%, such as from 92% to 94%, such as from 94% to 96%, such as from 96% to 98%, such as from 98% to 99%, such 100% identical. The A9 desaturase of the disclosure refers to a desaturase enzyme which catalyzes the introduction of a double bond in position between C9 and CIO in 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 carbon atoms.

A9 genes

[0076] A further aspect provides for a polynucleotide sequence (A9 desaturase gene) encoding a A9 desaturase said polynucleotide sequence being at least 50% identical to the A9 desaturase coding sequence comprised in SEQ NO:83. In some embodiments the A9 desaturase gene is from 50% to 100% identical to the A9 desaturase gene comprised in SEQ ID NO: 83, such as from 50% to 60%, such as from 60% to 70%, such as from 70% to 80%, such as from 80% to 90%, such as from 90% to 92%, such as from 92% to 94%, such as from 94% to 96%, such as from 96% to 98%, such as from 98% to 99%, such 100% identical. In preferred embodiments, the A9 desaturase gene encodes the A9 desaturase of SEQ IN NO: 82 or the described functional variants. On other embodiments the A9 desaturase gene is codon optimized for heterologous expression in a microorganism, in particular a yeast.

Ell desaturases

[0077] One aspect provides for an Ell fatty acyl-CoA desaturase (Ell desaturase) comprising an amino acid sequence which is the Ell desaturase comprised in SEQ ID NO:1 or is a functional variant at least 50% identical to the Ell desaturase comprised in SEQ ID NO: 1. In some embodiments the Ell desaturase is from 50% to 100% identical to the Ell desaturase comprised in SEQ ID NO: 1, such as from 50% to 60%, , such as from 60% to 70%, such as from 70% to 80%, such as from 80% to 90%, such as from 90% to 92%, such as from 92% to 94%, such as from 94% to 96%, such as from 96% to 98%, such as from 98% to 99%, such 100% identical. In more specific embodiments the Ell desaturase catalyzes the introduction of a double bond in a E configuration in position 11 in a (Z9)-9- hexadecenoyl-CoA substrate a (Z,E)-9,ll-hexadecadienoyl-CoA having a double bond in position 9 in Z configuration and in position 11 in E configuration.

[0078] A further aspect provides for an Ell fatty acyl-CoA desaturase (Ell desaturase) comprising an amino acid sequence which is the Ell desaturase comprised in SEQ ID NO:1 or 80 or is a functional variant at least 50% identical to the Ell desaturase comprised in SEQ ID NO: 1 or 80. In some embodiments the Ell desaturase is from 50% to 100% identical to the Ell desaturase comprised in SEQ I D NO: 1 or 80, such as from 50% to 60%, , such as from 60% to 70%, such as from 70% to 80%, such as from 80% to 90%, such as from 90% to 92%, such as from 92% to 94%, such as from 94% to 96%, such as from 96% to 98%, such as from 98% to 99%, such 100% identical. In more specific embodiments the Ell desaturase catalyzes the introduction of a double bond in a E configuration in position 11 in a (Z9)-9-hexadecenoyl-CoA substrate to provide (Z,E)-9,ll-hexadecadienoyl-CoA having a double bond in position 9 in Z configuration and in position 11 in E configuration.

[0079] A further aspect provides for an Ell fatty acyl-CoA desaturase (Ell desaturase) comprising an amino acid sequence which is the Ell desaturase comprised in SEQ ID NQ:80 or is a functional variant at least 50% identical to the Ell desaturase comprised in SEQ ID NO: 80. In some embodiments the Ell desaturase is from 50% to 100% identical to the Ell desaturase comprised in SEQ ID NO: 80, such as from 50% to 60%, , such as from 60% to 70%, such as from 70% to 80%, such as from 80% to 90%, such as from 90% to 92%, such as from 92% to 94%, such as from 94% to 96%, such as from 96% to 98%, such as from 98% to 99%, such 100% identical. In more specific embodiments the Ell desaturase catalyzes the introduction of a double bond in a E configuration in position 11 in a (Z9)-9- hexadecenoyl-CoA substrate to provide (Z,E)-9,ll-hexadecadienoyl-CoA having a double bond in position 9 in Z configuration and in position 11 in E configuration.

[0080] In some embodiments, an Ell fatty acyl-CoA desaturase (Ell desaturase) is provided comprising an amino acid sequence having at least 50% identity to the Ell desaturase comprised in SEQ ID NO: 1, SEQ ID NO: 80, SEQ ID NO: 90, or SEQ ID NO: 92, such as from 50% to 60%, such as from 60% to 70%, such as from 70% to 80%, such as from 80% to 90%, such as from 90% to 92%, such as from 92% to 94%, such as from 94% to 96%, such as from 96% to 98%, such as from 98% to 99%, such 100% identical. [0081] In some embodiments, an Ell fatty acyl-CoA desaturase (Ell desaturase) is provided comprising an amino acid sequence having at least 50% identity to the Ell desaturase comprised 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, such as from 50% to 60%, such as from 60% to 70%, such as from 70% to 80%, such as from 80% to 90%, such as from 90% to 92%, such as from 92% to 94%, such as from 94% to 96%, such as from 96% to 98%, such as from 98% to 99%, such 100% identical.

Ell genes

[0082] A further aspect provides for a polynucleotide sequence (Ell desaturase gene) encoding an Ell desaturase said polynucleotide sequence being at least 50% identical to the Ell desaturase coding sequence comprised in SEQ NO:2. In some embodiments the Ell desaturase gene is from 50% to 100% identical to the Ell desaturase gene comprised in SEQ ID NO: 2 or 93, such as from 50% to 60%, such as from 60% to 70%, such as from 70% to 80%, such as from 80% to 90%, such as from 90% to 92%, such as from 92% to 94%, such as from 94% to 96%, such as from 96% to 98%, such as from 98% to 99%, such 100% identical. In preferred embodiments the Ell desaturase gene encodes the Ell desaturase of SEQ IN NO: 1 or the described functional variants. On other embodiments the Ell desaturase gene is codon optimized for heterologous expression in a microorganism, in particular a yeast.

[0083] A further aspect provides for a polynucleotide sequence (Ell desaturase gene) encoding an Ell desaturase said polynucleotide sequence being at least 50% identical to the Ell desaturase coding sequence comprised in SEQ NO:2 or 81. In some embodiments the Ell desaturase gene is from 50% to 100% identical to the Ell desaturase gene comprised in SEQ ID NO: 2, 81 or 93, such as from 50% to 60%, such as from 60% to 70%, such as from 70% to 80%, such as from 80% to 90%, such as from 90% to 92%, such as from 92% to 94%, such as from 94% to 96%, such as from 96% to 98%, such as from 98% to 99%, such 100% identical. In preferred embodiments the Ell desaturase gene encodes the Ell desaturase of SEQ IN NO: 1 or 80 or the described functional variants. On other embodiments the Ell desaturase gene is codon optimized for heterologous expression in a microorganism, in particular a yeast.

[0084] A further aspect provides for a polynucleotide sequence (Ell desaturase gene) encoding an Ell desaturase said polynucleotide sequence being at least 50% identical to the Ell desaturase coding sequence comprised in SEQ NO:81. In some embodiments the Ell desaturase gene is from 50% to 100% identical to the Ell desaturase gene comprised in SEQ ID NO: 81, such as from 50% to 60%, such as from 60% to 70%, such as from 70% to 80%, such as from 80% to 90%, such as from 90% to 92%, such as from 92% to 94%, such as from 94% to 96%, such as from 96% to 98%, such as from 98% to 99%, such 100% identical. In preferred embodiments the Ell desaturase gene encodes the Ell desaturase of SEQ IN NO: 80 or the described functional variants. On other embodiments the Ell desaturase gene is codon optimized for heterologous expression in a microorganism, in particular a yeast.

[0085] In some embodiments, a polynucleotide sequence codon optimized for heterologous expression encoding the Ell desaturase disclosed herein is provided having a DNA sequence comprised in SEQ NO:2, SEQ ID NO: 81, SEQ ID NO: 91, or SEQ ID NO: 93 or a homologue thereof including variations due to the degeneracy of the genetic code. In some embodiments, the Ell desaturase gene is from 50% to 100% identical to the Ell desaturase gene comprised in SEQ NO:2, SEQ ID NO: 81, SEQ ID NO: 91, or SEQ I D NO: 93, such as from 50% to 60%, such as from 60% to 70%, such as from 70% to 80%, such as from 80% to 90%, such as from 90% to 92%, such as from 92% to 94%, such as from 94% to 96%, such as from 96% to 98%, such as from 98% to 99%, such 100% identical.

[0086] In some embodiments, a polynucleotide sequence codon optimized for heterologous expression encoding the Ell desaturase disclosed herein is provided having a DNA sequence comprised in SEQ NO:2, SEQ ID NO: 81, SEQ ID NO: 91, or SEQ ID NO: 93 or a homologue thereof including variations due to the degeneracy of the genetic code. In some embodiments, the Ell desaturase gene is from 50% to 100% identical to the Ell desaturase gene comprised 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, or SEQ I D NO: 105, such as from 50% to 60%, such as from 60% to 70%, such as from 70% to 80%, such as from 80% to 90%, such as from 90% to 92%, such as from 92% to 94%, such as from 94% to 96%, such as from 96% to 98%, such as from 98% to 99%, such 100% identical.

Zll desaturases

[0087] In some embodiments, the present disclosure provides a Zll desaturase having at least 70% sequence identity to an amino acid sequence comprised in the Zll desaturase of SEQ ID NO: 72, 74, 76, or 78. In some embodiments, the Zll desaturase is expressed in the genetically engineered host cell of the present disclosure. In some embodiments, the Zll desaturase having at least 70% sequence identity to an amino acid sequence comprised in the Zll desaturase of SEQ ID NO: 72, 74, 76, or 78 is employed in a method disclosed herein. In some embodiments, the Zll desaturase has from 70% to 100% sequence identity to an amino acid sequence comprised in the Zll desaturase of SEQ ID NO: 72, 74, 76, or 78, such as from 70% to 80%, such as from 80% to 90%, such as from 90% to 92%, such as from 92% to 94%, such as from 94% to 96%, such as from 96% to 98%, such as from 98% to 99%, such 100% sequence identity. Gene constructs

[0088] A further aspect provides for a polynucleotide construct comprising an Ell desaturase gene as described operably linked to one or more control sequences. Such control sequence may be native or heterologous to the Ell desaturase gene. The polynucleotide construct can further be integrated in an expression vector for expression of the Ell desaturase gene in a host cell.

Genetically engineered host cells

[0089] A further aspect provides for a genetically engineered microbial cell producing a (Z,E)-9,11- hexadecadienoyl-CoA said cell heterologously expressing the Ell desaturase of this disclosure, which in the presence of a (Z)-9-hexadecenoyl-CoA substrate introduces a double bond in a E configuration in position 11 in the (Z)-9-hexadecenoyl-CoA substrate and thereby produces the (Z,E)-9,11- hexadecadienoyl-CoA having a double bond in position 11 in E configuration. Further, the present disclosure provides a genetically engineered microbial cell as defined herein expressing a A9- desaturase capable of introducing a Z9 double bond in (E)-ll-hexadecenoyl-CoA to provide (Z,E)-9,11- hexadecadienoyl-CoA.

[0090] The cell provided herein can further comprise an operative biosynthetic pathway converting the (Z,E)-9,ll-hexadecadienoyl-CoA into a target compound selected from a) (Z,E)-9,ll-hexadecadien-l-ol; b) (Z,E)-9,ll-hexadecadienal; and/or c) (Z,E)-9,ll-hexadecadienyl acetate; said pathway expressing one or more pathway polypeptides selected from: a) an alcohol-forming fatty acyl-CoA reductase (FAR) converting (Z,E)-9,ll-hexadecadienoyl-CoA into (Z,E)-9,ll-hexadecadien-l-ol; b) an acetyltransferase converting (Z,E)-9,ll-hexadecadien-l-ol into (Z,E)-9,ll-hexadecadienyl acetate; c) an alcohol dehydrogenase converting (Z,E)-9,ll-hexadecadien-l-ol into (Z,E)-9,11- hexadecadienal; and/or d) a fatty alcohol oxidase converting (Z,E)-9,ll-hexadecadien-l-ol into (Z,E)-9,ll-hexadecadienal. [0091] In some embodiments, the cell provided herein further comprises an operative biosynthetic pathway converting the (Z,E)-9,ll-hexadecadienoyl-CoA into a target compound selected from a) (Z,E)-9,ll-hexadecadien-l-ol; b) (Z,E)-9,ll-hexadecadienal; and/or c) (Z,E)-9,ll-hexadecadienyl acetate; said pathway expressing one or more pathway polypeptides selected from: d) an alcohol-forming fatty acyl-CoA reductase (FAR) converting (Z,E)-9,ll-hexadecadienoyl-CoA into (Z,E)-9,ll-hexadecadien-l-ol; e) an acetyltransferase converting (Z,E)-9,ll-hexadecadien-l-ol into (Z,E)-9,ll-hexadecadienyl acetate; f) a fatty alcohol oxidase converting (Z,E)-9,ll-hexadecadien-l-ol into (Z,E)-9,11- hexadecadienal.

[0092] In some embodiments, the cell is provided wherein: a) the FAR is at least 70% identical to the FAR comprised 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 comprised in SEQ ID NO: 106; c) the fatty alcohol oxidase is at least 70% identical to the fatty alcohol oxidase comprised in SEQ ID NO: 70.

[0093] Where the cell expresses such pathway enzymes the

(I) 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 comprised 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 comprised in SEQ ID NO: 71;

(III) the alcohol dehydrogenase 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 alcohol dehydrogenase comprised in SEQ ID NO: 68; and

(IV) the fatty alcohol oxidase 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 fatty alcohol oxidase comprised in SEQ ID NO: 69 or 70.

[0094] In some embodiments,

(I) the FAR is at least 70% identical to the FAR comprised 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) the acetyltransferase is at least 70% identical to the acetyltransferase comprised in SEQ ID NO: 71;

(III) the alcohol dehydrogenase is at least 70% identical to the alcohol dehydrogenase comprised in SEQ ID NO: 68; and (IV) the fatty alcohol oxidase is at least 70% identical to the fatty alcohol oxidase comprised in SEQ ID NO: 69 or 70.

[0095] The genetically modified host cell can further comprise an operative biosynthetic pathway producing the (Z)-9-hexadecenoyl-CoA substrate, in particular a pathway expressing one or more heterologous A9 desaturases introducing a double bond in the hexadecenoyl-CoA substrate in a Z configuration in position 9. Such A9 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 A9 desaturase comprised in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82.

[0096] The genetically modified host cell can further comprise an operative biosynthetic pathway producing the (Z)-9-hexadecenoyl-CoA substrate, in particular a pathway expressing one or more heterologous A9 desaturases introducing a double bond in the hexadecenoyl-CoA substrate in a Z configuration in position 9. Such A9 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 A9 desaturase comprised in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, or 22.

[0097] In further embodiments the host cell is further modified so that one or more native or endogenous genes are attenuated, disrupted and/or deleted. Other modifications include overexpressing one or more pathway genes or modifications providing an increased amount of a substrate for at least one enzyme of the pathways described herein. The genetically modified host cell can also be further modified to exhibit increased tolerance towards one or more substrates, intermediates, or product molecules from the pathways described herein and/or the host cell can comprise at least two copies of one or more genes in such pathways. In some embodiments, the host cell can comprise at least two copies of one or more genes in the (Z,E)-9,ll-hexadecadien-l-ol pathway.

[0098] The host cells provided for herein are suitably fungal cells such as yeast cells. Preferred yeasts include those belonging to a genus selected from Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces, optionally wherein the yeast cell belongs to a species selected from Saccharomyces cerevisiae, Saccharomyces boulardi, Pichia pastoris, Kluyveromyces marxianus, Cryptococcus albidus, Lipomyces lipofera, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulan and Yarrowia lipolytica.

[0099] However, also filamentous fungal cell can be useful for example those selected from the species consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporiuminops, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.

[0100] In alternative or additional aspects provided for herein is a genetically engineered yeast cell producing (Z,E)-9,ll-hexadecadienoyl-CoA and (Z,E)-9,ll-hexadecadien-l-ol, said cell producing hexadecanoyl-CoA and expressing a) a A9 desaturase catalyzing the formation of a double bond in a Z configuration (Z) in position 9 in the hexadecanoyl-CoA thereby producing a (Z)-9-hexadecenoyl-CoA; b) an Ell desaturase catalyzing the formation of a double bond in a E configuration in position 11 in the (Z)-9-hexadecenoyl-CoA thereby producing a (Z,E)-9,ll-hexadecadienoyl-CoA; and c) an alcohol-forming fatty acyl-CoA reductase (FAR) converting the (Z,E)-9,ll-hexadecadienoyl- CoA into (Z,E)-9,ll-hexadecadien-l-ol.

[0101] The yeast cell may in further embodiments express one or more enzymes selected from: a) a Zll desaturase catalyzing the formation of a double bond in a Z configuration in position 11 in the hexadecanoyl-CoA thereby producing a (Z)-ll-hexadecenoyl-CoA; b) one or more alcohol-forming fatty acyl-CoA reductases (FAR) converting respectively the hexadecanoyl-CoA into hexadecane-l-ol, the (Z)-9-hexadecenoyl-CoA into (Z)-9-hexadecen-l- ol, and the (Z)-ll-hexadecenoyl-CoA into (Z)-ll-hexadecen-l-ol.

[0102] In some embodiments, a genetically engineered yeast cell is provided producing (Z,E)-9,11- hexadecadienoyl-CoA and (Z,E)-9,ll-hexadecadien-l-ol, said yeast cell producing hexadecanoyl-CoA and expressing

(I) an Ell desaturase catalyzing the formation of a double bond in a E configuration in position 11 in the hexadecanoyl-CoA and thereby producing an E-ll-hexadecenoyl-CoA;

(II) a A9 desaturase catalyzing the formation of a double bond in a Z configuration in position 9 in the (E)-ll-hexadecenoyl-CoA and thereby producing a (Z,E)-9,ll-hexadecadienoyl-CoA; (Ill) an alcohol-forming fatty acyl-CoA reductase (FAR) converting the (Z,E)-9,ll-hexadecadienoyl- CoA into (Z,E)-9,ll-hexadecadien-l-ol.

[0103] As demonstrated in the examples herein, including Example 18, the cells of the present disclosure can produce (Z,E)-9,ll-hexadecadienoyl-CoA and (Z,E)-9,ll-hexadecadien-l-ol through any order of desaturation i.e. by either first introducing the E-configured double bond at position 11 and then the Z-configured double bond at position 9, or vice versa.

[0104] In still further embodiments the a) Ell desaturase has an amino acid sequence which is at least 50% identical to the Ell desaturase comprised in SEQ ID NO: 1, such as from 50% to 100%, such as from 50% to 60%, such as from 60% to 70%, such as from 70% to 80%, such as from 80% to 90%, such as from 90% to 92%, such as from 92% to 94%, such as from 94% to 96%, such as from 96% to 98%, such as from 98% to 99%, such 100% identical to the Ell desaturase comprised in the Ell desaturase of SEQ ID NO: 1; b) A9 desaturase has an amino acid sequence which is 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 A9 desaturase comprised in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82; c) Zll desaturase has an amino acid sequence which is 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 Zll desaturase comprised in SEQ ID NO: 72, 74, 76 or 78; and d) alcohol-forming fatty acyl-CoA reductases (FAR) has an amino acid sequence which is 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 comprised 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.

[0105] In this aspect the yeast cell is preferably of the species Saccharomyces cerevisiae, or Yarrowia lipolytica.

[0106] In some embodiments, the yeast cell is provided wherein the

(I) Ell desaturase has at least 70% sequence identity to an amino acid sequence comprised in the Ell desaturase of SEQ ID NO: 1, 80, 90, 92, 96, 98, 100, 102, or 104;

(II) A9 desaturase has at least 70% sequence identity to an amino acid sequence comprised in the A9 desaturase of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82;

(III) Zll desaturase has at least 70% sequence identity to an amino acid sequence comprised in the Zll desaturase of SEQ ID NO: 72, 74, 76, or 78; and/or

(IV) alcohol-forming fatty acyl-CoA reductases (FAR) has at least 70% sequence identity to an amino acid sequence comprised 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.

[0107] In some embodiments, the yeast cell is provided wherein the

(I) Ell desaturase has an amino acid sequence comprised in the Ell desaturase of SEQ ID NO: 1, 80, 90, 92, 96, 98, 100, 102, or 104;

(II) A9 desaturase has an amino acid sequence comprised in the A9 desaturase of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82;

(III) Zll desaturase has an amino acid sequence comprised in the Zll desaturase of SEQ ID NO: 72, 74, 76, or 78; and/or

(IV) alcohol-forming fatty acyl-CoA reductases (FAR) has an amino acid sequence comprised 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.

Cultures

[0108] A further aspect provides for a cell culture, comprising host cells described herein and a growth medium. Suitable growth mediums for eukaryotic and prokaryotic cell are well known in the art.

Methods of producing compounds of the disclosure.

[0109] A further aspect provides for a method for producing a target compound selected from:

(I) (Z,E)-9,ll-hexadecadienoyl-CoA;

(II) (Z,E)-9,ll-hexadecadien-l-ol;

(III) (Z,E)-9,ll-hexadecadienal; and/or

(IV) (Z,E)-9,ll-hexadecadienyl acetate; said method comprising culturing the cell culture described herein at conditions allowing the cell culture to produce the target compound; and optionally recovering and/or isolating the target compound.

[0110] The cell culture can be cultivated in a nutrient medium using methods known in the art at conditions suitable for production of the target compound and/or its precursors and/or for propagating cell count. For example, the culture may be cultivated by shake flask cultivation, or small- scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentations) in laboratory or industrial fermenters in a suitable medium and under conditions allowing the host cells to grow and/or propagate, optionally to be recovered and/or isolated.

[0111] The cultivation can take place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. from catalogues of the American Type Culture Collection). The selection of the appropriate medium may be based on the choice of host cell and/or based on the regulatory requirements for the host cell. Such media are available in the art. The medium may, if desired, contain additional components favoring the transformed expression hosts over other potentially contaminating microorganisms. Accordingly, in an embodiment a suitable nutrient medium comprises a carbon source (e.g. glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellolytic biomass hydrolysate, etc.), a nitrogen source (e. g. ammonium sulphate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (e.g. yeast extract, malt extract, peptone, etc.) and inorganic nutrient sources (e.g. phosphate, magnesium, potassium, zinc, iron, etc.).

[0112] Culturing of the host cell may be performed over a period of about 0.5 to about 30 days. The cultivation process may be a batch process, continuous or fed-batch process, suitably performed at a temperature in the range of 0-100 °C or 10-80 °C, for example, from about 20°C to about 50 °C and/or at a pH, for example, from about 2 to about 10. Preferred fermentation conditions for yeast and filamentous fungi are a temperature in the range of from about 25 °C to about 55 °C and at a pH of from about 3 to about 9. The appropriate conditions are usually selected based on the choice of host cell. Accordingly, in an embodiment the method of the disclosure further comprises one or more elements selected from:

(I) culturing the cell culture in a nutrient medium;

(II) culturing the cell culture under aerobic or anaerobic conditions

(III) culturing the cell culture under agitation;

(IV) culturing the cell culture at a temperature of between 25 to 50 °C;

(V) culturing the cell culture at a pH of between 3-9; and

(VI) culturing the cell culture for between 10 hours to 30 days.

[0113] The cell culture of the disclosure may be recovered and or isolated 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 a particular embodiment the method includes a recovery and/or isolation step comprising separating a liquid phase of the cell or cell culture from a solid phase of the cell or cell culture to obtain a supernatant comprising the target compound and/or subjecting the supernatant to one or more steps selected from:

(I) disrupting the cells of the cell culture to release intracellular target compound into the supernatant;

(II) separating the supernatant from the solid phase of the cell culture, such as by filtration or gravity separation;

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

(IV) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of the target compound;

(V) extracting the target compound; and/or

(VI) precipitating the Ell fatty acyl compound by crystallization or evaporating the solvent of the liquid phase; and optionally isolating the target compound by filtration or gravity separation; thereby recovering and/or isolating the target compound.

[0114] The method described herein may comprise one or more in vitro steps in the process of producing the target compound. Accordingly, in one embodiment the method further comprises feeding the cell culture with one or more precursor or substrates in the pathway of the target compound. Where the target compound is not the desired end products further steps may be added to the method of the disclosure either chemically or biologically/enzymatically modifying the target compound, such as oxidation and/or acetylation. Accordingly in attractive embodiments the in vitro performed step comprises chemically or enzymatically reducing (Z,E)-9,ll-hexadecadienoyl-CoA into (Z,E)-9,ll-hexadecadien-l-ol (Z9, E11-16:OH), preferably using a reductase enzyme (FAR). In other attractive embodiments the in vitro performed step comprises chemically or enzymatically reducing a (Z,E)-9,ll-hexadecadienoic acid into (Z,E)-9,ll-hexadecadien-l-ol. In further attractive embodiments the in vitro performed step comprises chemical or enzymatic oxidation of (Z,E)-9,ll-hexadecadien-l- ol into (Z,E)-9,ll-hexadecadienal. In still further attractive embodiments the in vitro performed step comprises chemical or enzymatic acetylation of (Z,E)-9,ll-hexadecadien-l-ol into (Z,E)-9,11- hexadecadienyl acetate.

[0115] The method can further comprise recovering the target compound and mixing it with one or more carriers, agents, additives, adjuvants and/or excipients to produce a biopesticide composition.

[0116] In the method the one or more carriers, agents, additives, adjuvants and/or excipients preferably comprises a protective agent preferably comprises a conjugated sulfur, which protects the target compound from being converted into an acid. Such protective agents suitably include compounds selected from zinc pyrithione, 5-amino-l,3,4-thiadiazole-2-thiol, 2-thiazoline-2-thiol, 5- methyl-l,3,4-thiadiazole-2-thiol, 2-mercapto-benzimidazole, 2-mercapto-l-methylimidazole and sodium pyrithione shown to have a remarkably high effect in stabilizing fatty aldehydes from oxidation. In some embodiments the method further comprises mixing in at least 10 mg of the protective agent per gram aldehyde and/or alcohol. The one or more carriers, agents, additives, adjuvants and/or excipients may also comprise a carrier facilitating slow release of the target compound, optionally being (i) a polymeric substrate selected from plastic, wax emulsion, oil emulsion, or microcapsules and/or (ii) zeolite.

[0117] In preferred embodiments target compound is a (Z,E)-9,ll-hexadecadien-l-ol, a (Z,E)-9,11- hexadecadienal or a (Z,E)-9,ll-hexadecadienyl acetate (Z9, Ell-16:OAc).ln an alternative aspect a method is provided for producing a biopesticide composition described herein comprising

(II) culturing a genetically engineered yeast cell producing hexadecanoyl-CoA and expressing a) a A9 desaturase catalyzing the formation of a double bond in a Z configuration in position

9 in the hexadecanoyl-CoA and thereby producing a (Z)-9-hexadecenoyl-CoA; b) an Ell desaturase catalyzing the formation of a double bond in a E configuration in position 11 in the (Z)-9-hexadecenoyl-CoA and thereby producing a (Z,E)-9,11- hexadecadienoyl-CoA; c) an alcohol-forming fatty acyl-CoA reductase (FAR) converting the (Z,E)-9,11- hexadecadienoyl-CoA into (Z,E)-9,ll-hexadecadien-l-ol;

(IV) enzymatically or chemically converting the (Z,E)-9,ll-hexadecadien-l-ol to (Z,E)-9,11- hexadecadienal; and

(V) optionally recovering and/or isolating the (Z,E)-9,ll-hexadecadienal and optionally one or more precursors thereof.

[0118] In some embodiments in this alternative aspect the genetically engineered yeast cell further expresses one or more enzymes selected from: a) a Zll desaturase catalyzing the formation of a double bond in a Z configuration (Z) in position 11 in the hexadecanoyl-CoA and thereby producing a (Z)-ll-hexadecenoyl-CoA; b) one or more alcohol-forming fatty acyl-CoA reductases (FAR) converting respectively the hexadecanoyl-CoA into hexadecan-l-ol, the (Z)-9-hexadecenoyl-CoA into (Z)-9-hexadecen-l-ol, and the (Z)-ll-hexadecenoyl-CoA into (Z)-ll-hexadecen-l-ol; and the method further comprises enzymatically or chemically converting the hexadecane-l-ol into hexadecanal, the (Z)-9-hexadecen-l-ol into (Z)-9-hexadecenal and the (Z)-ll-hexadecen-l-ol and the (Z)-ll-hexadecen-l-ol into (Z)-ll-hexadecenal; and optionally recovering and/or isolating the hexadecanal, the (Z)-9-hexadecenal and the (Z)-ll-hexadecenal and optionally one or more precursors thereof.

[0119] In some embodiments, an alternative method for producing a biopesticide composition is provided comprising the steps:

(I) culturing a genetically engineered yeast cell producing hexadecanoyl-CoA and expressing a. an Ell desaturase catalyzing the formation of a double bond in a E configuration in position 11 in the hexadecanoyl-CoA and thereby producing an E-ll-hexadecenoyl-CoA; b. a A9 desaturase catalyzing the formation of a double bond in a Z configuration in position 9 in the (E)-ll-hexadecenoyl-CoA and thereby producing a (Z,E)-9,ll-hexadecadienoyl- CoA; c. an alcohol-forming fatty acyl-CoA reductase (FAR) converting the (Z,E)-9,11- hexadecadienoyl-CoA into (Z,E)-9,ll-hexadecadien-l-ol;

(II) enzymatically or chemically converting the (Z,E)-9,ll-hexadecadien-l-ol to (Z E)-9,ll- hexadecadienal; and

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

[0120] The successful use of this alternative method is demonstrated in Example 18, showcasing the possibility of introducing the Ell double bond prior to the Z9 double bond in the hexadecanoyl-CoA.

[0121] In some embodiments, the method is provided wherein the genetically engineered yeast cell further expresses one or more enzymes selected from: a. a Zll desaturase catalyzing the formation of a double bond in a Z configuration in position 11 in the hexadecanoyl-CoA and thereby producing a (Z)-ll-hexadecenoyl-CoA; b. one or more alcohol-forming fatty acyl-CoA reductases (FAR) converting respectively the hexadecanoyl-CoA into hexadecan-l-ol, the (Z)-9-hexadecenoyl-CoA into (Z)-9-hexadecen-l-ol, and the (Z)-ll-hexadecenoyl-CoA into (Z)-ll-hexadecen-l-ol; and the method further comprises enzymatically or chemically converting the hexadecan-l-ol into hexadecanal, the (Z)-9-hexadecen-l-ol into (Z)-9-hexadecenal and the (Z)-ll-hexadecen-l-ol and the (Z)-ll-hexadecen-l-ol into (Z)-ll-hexadecenal; and/or optionally recovering and/or isolating the hexadecanal, the (Z)-9-hexadecenal and the (Z)-ll-hexadecenal and optionally one or more precursors thereof.

[0122] In other embodiments in this alternative aspect the method further comprises mixing the recovered (Z,E)-9,ll-hexadecadienal, (Z)-9-hexadecenal, (Z)-ll-hexadecenal, hexadecanal and optionally one or more precursors thereof with one or more carriers, agents, additives, adjuvants and/or excipients to produce the biopesticide composition. These carriers, agents, additives, adjuvants and/or excipients preferably comprise one or more compounds selected from a) a protective agent comprising a conjugated sulfur compound selected from zinc pyrithione, 5- amino-l,3,4-thiadiazole-2-thiol, 2-thiazoline-2-thiol, 5-methyl-l,3,4-thiadiazole-2-thiol, 2- mercapto-benzimidazole, 2-mercapto-l-methylimidazole and sodium pyrithione, which protects the target compound from being converted into an acid; and/or b) a carrier facilitating slow release of the (Z,E)-9,ll-hexadecadienal, (Z)-9-hexadecenal, (Z)-ll- hexadecenal and/or hexadecanal from the mixture, optionally being (i) a polymeric substrate selected from plastic, wax emulsion, oil emulsion, or microcapsules and/or (ii) a zeolite.

Composition

[0123] A further aspect provides for a biopesticide composition comprising a target compound selected from: a) (Z,E)-9,ll-hexadecadien-l-ol (Z9, E11-16:OH); b) (Z,E)-9,ll-hexadecadienal (Z9, Ell-16: Aid); and/or c) (Z,E)-9,ll-hexadecadienyl acetate (Z9, Ell-16:OAc); and one or more carriers, agents, additives, adjuvants and/or excipients.

[0124] A further alternative aspect provides for a biopesticide composition comprising a target compound selected from (Z,E)-9,ll-hexadecadienal, and optionally one or more compounds selected from (Z)-9-hexadecenal, (Z)-ll-hexadecenal and/or hexadecanal, in combination with one or more carriers, agents, additives, adjuvants and/or excipients. The biopesticide composition further comprises in certain embodiments at least trace amounts of one or more compounds selected from hexadecan-l-ol, (Z)-9-hexadecen-l-ol, (Z)-ll-hexadecen-l-ol, and (Z,E)-9,ll-hexadecadien-l-ol, other metabolites of the cell culture. In other embodiments he biopesticide composition further comprises (Z,E)-9,ll-hexadecandienyl acetate. In preferred embodiments, in the biopesticide composition at least the one or more of the hexadecan-l-ol, (Z)-9-hexadecen-l-ol, (Z)-ll-hexadecen-l-ol, and (Z,E)- 9,11-hexadecadien-l-ol are obtained from culturering genetically engineered host cell described herein and optionally the composition comprises one or more further compounds or metabolites from the cell culture. Such compounds and/or metabolites of the cell culture includes precursors for the target compound as well as compounds selected from trace metals, vitamins, salts, yeast nitrogen base, carbon source, YNB, and/or amino acids of the fermentation. In particular the biopesticide composition comprises a concentration of the target compound of at least 1 mg/kg 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.

[0125] The composition can also advantageously include one or more a protective agents comprising a conjugated sulfur, which protects the target compound from from being further converted into an acid. Such protective agents comprise compounds selected from zinc pyrithione, 5-amino-l,3,4- th iad iazole-2-thiol, 2-thiazoline-2-thiol, 5-methyl-l,3,4-thiadiazole-2-thiol, 2-mercapto- benzimidazole, 2-mercapto-l-methylimidazole and sodium pyrithione. The composition preferably comprises at least 10 mg of the protective agent per gram of target compound.

[0126] The composition further comprises in some embodiments a carrier facilitating slow release of the target compound, optionally being (i) a polymeric substrate selected from plastic, wax emulsion, oil emulsion, or microcapsules and/or (ii) zeolite.

[0127] 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%.

[0128] 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.

[0129] In some embodiments, the biopesticide composition comprises from 20% to 100% biobased carbon, such as from 30% to 100% biobased carbon, such as from 40% to 100% biobased carbon, such as from 50% to 100% biobased carbon, such as from 60% to 100% biobased carbon, such as from 70% to 100% biobased carbon, such as from 75% to 100% biobased carbon, such as from 80% to 100% biobased carbon, such as from 85% to 100% biobased carbon, such as from 90% to 100% biobased carbon, such as from 95% to 100% biobased carbon, such as 100% biobased carbon.

[0130] In some embodiments, the biopesticide composition comprises no more than 50% fossilbased 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.

[0131] 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, for example 94% biobased carbon.

[0132] 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, for example 94% biobased.

Applications

[0133] A further aspect provides for a method of controlling or monitoring a pest comprising distributing the composition described herein in a habitat for the pest and allowing the target compound to control the pest. The target compounds described herein are particularly effective again D. saccharalis and according the in preferred embodiments the habitat is a sugar cane field, and the pest is D. saccharalis.

Sequence listings

[0134] The present application contains a Sequence Listing prepared in Patentin included below but also submitted electronically in ST26 format which is hereby incorporated by reference in its entirety. Table A SEQIDNO: DNA/RNA sequence of Primer from Artificial

23 SEQIDNO: protein sequence of FAR from Yponomeuta rorellus

50 SEQIDNO: DNA encoding Zll desaturase from Spodoptera litura

77

Examples

Example 1 - Construction of BioBricks and plasmids

[0135] All heterologous genes were synthesized by GeneArt (Life Technologies) in codon-optimized versions for Y. lipolytica. Genes were either amplified by PCR using Phusion U Hot Start DNA Polymerase (ThermoFisher), or obtained by restriction digestions, to obtain the fragments for cloning into yeast expression vectors. The primers are listed in Table 1 and the resulting DNA fragments (BioBricks) are listed in Table 2. The PCR products or restriction digest reaction were separated on a 1%-agarose gel containing Midori Green Advance (Nippon Genetics Europe GmbH). PCR/restriction digest products of the correct size were excised from the gel and purified using the Nucleospin Gel and PCR Clean-up kit (Macherey-Nagel).

[0136] Yeast vectors with a USER cassette were linearized with FastDigest SfaAl (ThermoFisher) for 2 hours at 37°C and then nicked with Nb.Bsml (New England Biolabs) for 1 hour at 65°C. The resulting vectors containing sticky ends were separated by gel electrophoresis, excised from the gel, and gel- purified using the Nucleospin Gel and PCR Clean-up kit (Macherey-Nagel). The DNA fragments were cloned into vectors by USER-cloning as described in (Holkenbrink, et al., 2018) (Jensen, et al., 2014). The USER reaction was transformed into chemically competent Escherichia coli DHa cells and the cells were plated on Lysogeny Broth (LB) agar plates with 100 mg/L ampicillin. The plates were incubated overnight at 37°C and the resulting colonies were screened by colony PCR. The plasmids were purified from overnight E. coli liquid cultures and the correct cloning was confirmed by sequencing. The constructed vectors are listed in Table 3.

Table 1. Primers

* (Holke nbri nk, et al., 2018)

Table 2. DNA fragments (BioBricks) obtained by PCR and restriction digestion using the indicated template and primers

* (Holken brin k, et al., 2018)

** (Holkenbrink, et al., 2020)

Table 3. Vectors * (Holkenbrink, et al., 2018)

Example 2 - Construction of yeast strains

[0137] Yeast strains were constructed by transformation of DNA vectors as described in (Holkenbrink, et al., 2018) (Jensen, et al., 2014). Strains were selected on yeast peptone dextrose (YPD) agar with appropriate antibiotics selection or on synthetic drop-out medium (Sigma-Adrich) lacking a specific amino acid. Correct genotype was confirmed by colony PCR and when needed by sequencing. The resulting strains are listed in Table 4.

[0138] Strains marked with "***" were constructed as follows. The indicated genes were amplified with gene-specific primers containing a 5'-overhang of: "AC I I I I I GCAGTACUAACCGCAG" in the forward primer and a 3'-overhang of "CACGCGAU" in the reverse primer. The first "ATG" of the target gene sequence was omitted. These PCR products were cloned together with BB9454 either into integrative vectors or episomal vectors as described in (Holkenbrink, et al., 2018)

[0139] Strains marked with "****" were constructed as follows. The indicated genes were cloned together with BB10398 and a synthetic minimal terminator (SEQ ID NO: 86) (ST13043) or Y. lipolytica native LIP2 terminator (ST13149, ST13247) either into integrative vectors or episomal vectors by "Golden-Gate Assembly" as described in (Pryor, J.M., et al., 2020) or by following the User's Manual of NEBridge Golden Gate Assembly Kit (New England BioLabs, Inc.). The first "ATG" of the target gene sequence was omitted. The episomal vector pBP10995 is derived from pCfB3405 (Holkenbrink et al. 2018), adopted for Golden-Gate assembly.

Table 4. Yeast strains

** (Holkenbrink, et al., 2020)

Example 3 - Cultivation of strains and analysis of fatty alcohols and fatty acid methyl esters (FAME) [0140] Y. lipolytica strains were inoculated from a YPD agar plate (10 g/L yeast extract, 10 g/L peptone, 20 g/L glucose, 15 g/L agar agar) to an initial OD600 of 0.2 into 2.5 mL YPG medium (10 g/L yeast extract, 10 g/L peptone, 40 g/L glycerol) in 24 well-plates (EnzyScreen). The plates were incubated at 28°C, shaken at 300 rpm. After 24 h, the plates were centrifuged for 5 min at 4°C and 3,000 xg. The supernatant was discarded and the cells were resuspended in 1.25 mL production medium per well (50 g/L glycerol, 5 g/L yeast extract, 4 g/L KH2PO4, 1.5 g/L MgSC , 0.2 g/L NaCI, 0.265 g/L CaCl2.2H2O, 2 mL/L trace elements solution: 4.5 g/L CaCl2.2H2O, 4.5 g/L ZnSO4.7H2O, 3 g/L FeSO ₄ .7H ₂ O, 1 g/L H3BO3, 1 g/L MnCI ₂ .4H ₂ O, 0.4 g/L N Na ₂ MoO ₄ .2H ₂ O, 0.3 g/L CoCI ₂ .6H ₂ O, 0.1 g/L CUSO4.5H2O, 0.1 g/L KI, 15 g/L EDTA). The media was supplemented with antibiotics if necessary. The plate was incubated for 26 hours at 28°C, shaken at 300 rpm.

[0141] For analysis of the fatty acids, 1 mL of each vial was harvested by centrifugation for 5 min at 4°C and 3,000 xg. Each pellet was extracted with 1000 pL IM HCI in Methanol (anhydrous). The samples were vortexed for 20 sec and placed in the 70°C water bath for 2 h. The samples were vortexed every 30 min for 10 sec. After cooling down of the samples to room temperature, 1000 pL of IM NaOH in Methanol (anhydrous), 500 pL of NaCI saturated H2O, 990 pL of hexane and 10 pL of 19:Me (10 mg/mL) as internal standard were added. The samples were vortexed and centrifuged for 5 min at 21°C and 3,000 x g. The upper organic phase was analyzed via gas chromatography-mass spectrometry (GC-MS). For analysis of fatty alcohols, 1 mL of each vial was harvested by centrifugation for 5 min at 4°C and 3,000 xg. Each cell pellet was extracted with 1 ml of ethyl acetate:ethanol (84:15) and 10 pL of 19:Me (10 mg/mL) was added as internal standard. The samples were vortexed for 20 sec and incubated for 1 hour at room temperature, followed by 5 min of vortexing. 300 pL of H2O was added to each sample. The samples were vortexed and centrifuged for 5 min at 21°C and 3,000 x g. The upper organic phase was analyzed via gas chromatography-mass spectrometry (GC-MS). GC-MS analyses were performed on an Agilent 7820A GC coupled to a mass selective detector Agilent 5977B. The GC was equipped with an DB Fatwax column (30 mx0.25mm x0.25 pm), and helium was used as carrier gas. The MS was operated in electron impact mode (70eV), scanning between m/z 30 and 400, and the injector was configured in split mode 20:1 at 220°C. Oven temperature was set to 80°C for 1 min, then increased at a rate of 20°C /min to 210°C, followed by a hold at 210°C for 7 min, and then increased at a rate of 20 C/min to 230°C. Compounds were identified by comparison of retention times and mass spectra of the reference compounds. Data were analyzed by the Agilent Masshunter software.

Example 4 - Production of Z9, Ell-16:CoA in the yeast Y. lipolytica

[0142] The newly identified desaturase Dsl2389 from D. saccharalis was expressed in Y. lipolytica strain ST6629 (Holkenbrink et al., 2020) to generate strain ST12028. The empty expression vector (pBP9002) was transformed into the same parent strain to give ST10444 as a control strain. Cultivation of both strains and extraction of fatty acids were performed as described in Example 3.

[0143] FAME extracts of strain ST12028, expressing Dsl2389 (Figure 2A, the dash line), contained a double unsaturated C16-fatty acid methyl ester (16-2:Me), which was not observed in the FAME extracts of the control strain ST10444 (Figure 2A, the dot line). This 16-2:Me eluted at the same retention time (12.954 min) as the authentic standard of Z9, Ell-16:Me (Figure 2A, the solid line) and the mass spectrum matches the one of the Z9, Ell-16:Me standard (Figure 3). Additionally, strain ST12028 produced 16:Me and Z9-16:Me (Figure 2C). The titers of Z9, Ell-16:Me are listed in Table 5.

Table 5. Production of Z9, Ell-16:Me in the yeast Y. lipolytica

Example 5 - Expression of D. punctatus desaturases in Y. lipolytica

[0144] Desaturase Dpu_APSQ gene from D. punctatus, was expressed in Y. lipolytica strain ST6629 (Holkenbrink et al., 2020) to generate strain ST10746. Strain ST10444, carrying the empty expression vector only, serves as a control strain.

[0145] FAME extracts of strain ST10746, expressing desaturase Dpu_APSQ, did not contain any compound eluting at 12.954 min but instead trace amounts of an unknown double unsaturated CIS- fatty acyl CoA with a retention time of 12.985 min was detected (Figure 2B).

Example 6 - Production of Z9,E11-16:OH in Y. lipolytica

[0146] Desaturase Dsl2389 from D. saccharalis is co-expressed with a fatty-acyl CoA reductase in Y. lipolytica. The strain is cultivated and samples analyzed as described in Example 3 and the fatty alcohol Z9,E11-16:OH is detected.

Example 7 - Production of (Z9, Ell)-hexadecadienal

[0147] A mixture of primary fatty alcohols, containing 96 wt% (Z,E)-9,ll-hexadecadien-l-ol (Z9, El 1- 16:01-1), was used as a representative sample for conversion to aldehydes. Z9,E11-16:OH (560 mg) was dissoved in 1 mL of acetonitrile in a 10 mL round bottom flask with magnetic stirrer. Then 39.0 mg of Tetrakisacetonitrile copper(l) triflate (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-methyl imidazole (5 mol%) were added to the reaction mixture and stirred at 30 °C for 2 hours. The reaction mixture was extracted with 10 mL heptane and the acetonitrile layer was discarded. The heptane phase was washed with 5 ml of citric acid solution (0.15 wt% in water). Then the top heptane phase was evaporated at reduced pressure until a clear residue is formed, yielding 493 mg product containing 88.0 wt% (Z9, Ell)-hexadecadienal (Z9,E11- 16:Ald).

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

[0148] The Dsl2389 desaturase (SEQ ID NO:1) from D. saccharalis is cloned alone and in combination with a fatty acyl reductase into a S. cerevisiae gene expression vectors and transformed into S. cerevisiae as described in Jensen, et al., 2014. Strain cultivation and sample extraction is performed as in Example 3. A strain expressing only Dsl2389 desaturase produces Z9, Ell-16:Me, while the strain additionally expressing a fatty acyl reductase gene produce Z9, E11-16:OH.

Example 9 - Conversion of Z9,Ell-16:Me to Z9,E11-16:OH in Y. lipolytica b various fatty acyl reductases

[0149] Fatty acyl-CoA reductases from various organisms were expressed in Y. lipolytica. The strains were cultivated and fatty alcohol samples extracted as described in Example 3 however the cultivation in YPG medium was extended from 24 to 47 hours and that 0.2 pl of Z9,Ell-16:Me was added to the production medium.

[0150] Extracts of strains ST12118-ST12123, ST12125, ST12126, ST12129, ST12131-ST12133, ST12138, ST12140, ST12141, ST12146, ST12151, ST12154, ST12156, ST12159 and ST12560 produced Z9,E11-16:OH. As an example, the GC-MS chromatogram and mass spectrum of the extract of strain ST12118 and a pure standard of Z9,E11-16:OH is shown in Figure 4A and Figure 4B,C, respectively. The extracts of strain ST12118 contained 5 mg/L Z9,E11-16:OH.

Example 10 - Bio-based pheromone precursor blend production

[0151] The D. saccharalis major pheromone is (Z,E)-9,ll-hexdecadienal. Minor components are (Z)- 11-hexadecenal, (Z)-9-hexadecenal, and hexadecanal. The fatty alcohol precursor of both, the major and minor components, can be produced in one yeast cells. To do so, AE11 desaturase Dsl2389 (SEQ ID NO:1) is co-expressed with any AZ11-16 desaturase, such as Desatl6 from Amelyois transitella (SEQ ID NO: 72), Desat51 from Helicoverpa zea (SEQ ID NO:78, Desat37 from Spodoptera exigua (SEQ ID NO:74), and Desat38 from Spodoptera litura (SEQ ID NO: 76), and a suitable fatty acyl-CoA reductase known in the art for example SEQ ID NO: 46. The strains are cultivated, and fatty alcohol samples are extracted as described in Example 3.

[0152] The extract of the strain contains (Z,E)-9,ll-hexadecadien-l-ol, hexadecane-l-ol, (Z)-9- hexadecen-l-ol and (Z)-ll-hexadecen-l-ol.

[0153] The extract is chemically oxidized to give a composition that contains (Z,E)-9,11- hexadecadienal, hexadecanal, (Z)-9-hexadecenal and (Z)-ll-hexadecenal.

[0154] The bio-based carbon content of the composition is determined by C14 radiocarbon dating.

Example 11 - Co-expression of AZ9-16 desaturases and DS12389 (SEQ ID NO:1)

[0155] The AE11 desaturase Dsl2389 is co-expressed with a AZ9-16 desaturase and optionally with a fatty acyl-CoA reductase. The strains are cultivated, and FAME and fatty alcohol samples are extracted as described in Example 3. The strain expressing Dsl2389, and a AZ9-16 desaturase produces (Z,E)-

9.11-hexadecadienoic acid methyl ester. If a fatty acyl-CoA reductase is expressed additionally (Z,E)-

9.11-hexadecadien-l-ol is produced.

Example 12 - Production of Z9, Ell-16:CoA in Y. lipolytica by the expression of DsaDesl (SEQ ID N0:80)

[0156] A newly identified desaturase DsaDesl and Dsl2389 from D. saccharalis were expressed in Y. lipolytica strain ST12834 to generate strain ST13043 and ST13042, respectively. Strain ST12834 is derived from ST6629 (Holkenbrink et al., 2020) and expressed additionally a heterologous NAD(P)H cytochrome b5 oxidoreductase Ncb5or (SEQ ID NO:84) from Cydia pomonella (WO 2022/238404 Al). The empty expression vector (pBP9002) was transformed into the same parent strain ST12834 to give ST13046 and served as a control strain. Cultivation of both strains and extraction of fatty acids were performed as described in Example 3.

FAME extracts of strain ST13043 and ST13042, expressing DsaDesl and Dsl2389, respectively, (Figure 5), contained a double unsaturated C16-fatty acid methyl ester (16-2:Me), which eluted at the same retention time as the authentic standard of Z9, Ell-16:Me (Figure 5). Mass spectrum of this double unsaturated 16-2:Me matches the Z9, Ell-16:Me standard (Figure 5). No Z9, Ell-16:Me was detected in the FAME extracts of the control strain ST13046 (Figure 5). The titers of Z9, Ell-16:Me are listed in Table 6.

Table 6. Production of Z9, Ell-16:Me in the yeast Y. lipolytica

Example 13 - Production of Z9,E11-16:OH in Y. lipolytica by the co-expression of DsaDesl and fatty acyl-CoA reductases

[0157] Desaturase DsaDesl from D. saccharalis is co-expressed with a fatty-acyl CoA reductase in Y. lipolytica. The strain is cultivated and samples analyzed as described in Example 3 and the fatty alcohol Z9,E11-16:OH is detected.

Example 14 - Co-expression of AZ9-16 desaturases and DsaDesl (SEQ ID N0:80)

[0158] The AE11 desaturase DsaDesl is co-expressed with a AZ9-16 desaturase and optionally with a fatty acyl-CoA reductase. The strains are cultivated, and FAME and fatty alcohol samples are extracted as described in Example 3. The strain expressing DsaDesl, and a AZ9-16 desaturase produces (Z,E)-

9.11-hexadecadienoic acid methyl ester If a fatty acyl-CoA reductase is expressed additionally (Z,E)-

9.11-hexadecadien-l-ol is produced.

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

[0159] The desaturase Dsl2389 (SEQ ID NO:1) and DsaDesl (SEQ ID NQ:80) from D. saccharalis were cloned into S. cerevisiae gene expression vectors and transformed into S. cerevisiae CEN.PK strains as described in Jensen, et al., 2014. Saccharomyces cerevisiae strains were inoculated from synthetic drop-out agar plates (lacking uracil, leucine and histidine) to an initial QD600 of 0.1-0.2 into 2.5 mL synthetic drop-out medium (lacking uracil, leucine and histidine) supplemented with 2% glucose in 24 well-plates (EnzyScreen). Sample extraction is performed as in Example 3. Derivatized FAME samples of Strain ST13093 and ST13150, expressing the desaturase Dsl2389 and DsaDesl, respectively, both contained Z9, Ell-16:Me (Table 7), while derivatized samples of control strain ST12515, carrying an empty expression vectors only, did not contain any Z9,Ell-16:Me.

Table 7. Detection of Z9, Ell-16:Me in derivatized FAME samples of Saccharomyces cerevisiae strains.

Example 16 - Production of Z9,E11-16:OH in Y. lipolytica by the co-expression of DsaDesl and fatty acyl-CoA reductases

[0160] Desaturase DsaDesl from D. saccharalis was co-expressed in Y. lipolytica strain ST6629 (Holkenbrink et al., 2020) in combination with fatty acyl reductases from different insect species. An empty expression vector (pBP9002) was transformed into strain ST13146, which expressing DsaDesl only, to generate ST13151 as a control strain. Strains are cultivated and samples were analyzed as described in Example 3. The control strain ST13151 did not produce any fatty alcohols as expected. Strains ST13147, ST13152, ST13162 and ST13251, expressing the newly identified fatty acyl reductases DsaFARl from D. saccharalis (SEQ ID NO:88), FAR1 from Helicoverpa armigera (SEQ ID NO:46), FAR16 from Spodoptera exigua (SEQ ID NO:56) and FAR25 from Tyta alba (SEQ ID NO: 95), respectively, produced 16:OH, Z11-16:OH, Z9-16:OH and the target compound Z9,E11-16:OH (Table 8). Strain ST13147, expressing DsaFARl, produced much lower amount of 16:OH than strains expressing FAR1 (ST13152), FAR16 (ST13162) or FAR25 (ST13251). This property is advantageous for achieving Z9,E11- 16:01-1 production with higher purity.

Table 8. Production of Z9,E11-16:OH in the yeast Y. lipolytica. Example 17 - Co-expression of AZ9-16 desaturases and DsaDesl

[0161] The AE11 desaturase DsaDesl (SEQ ID NO:80) was co-expressed with a newly identified AZ9- 16 desaturase DsaDes7 from Diatraea saccharalis (SEQ. ID NO:82) in Y. lipolytica strain ST6629 (Holkenbrink et al., 2020) to generate ST13149. The empty expression vector (pBP9002) was transformed into strain ST13146, which expresses DsaDesl only, to give ST13151 as a control strain. The strains were cultivated, and FAME samples were extracted as described in Example 3. When DsaDesl was expressed alone, derivatized samples of the control strain ST13151 contained 2.0% of Z9,Ell-16:Me and 10.1 % of Z9-16:Me (Table 9). While derivatized FAME samples of strain ST13149, co-expressing DsaDesl and the AZ9-16 desaturase DsaDes7, contained 3.3% of Z9,Ell-16:Me and 15.2% of Z9-16:Me. The presence of DsaDes7 improved the purity of Z9-16:Me by 50% and Z9, Ell- 16:Me by 65%, respectively. If a fatty acyl-CoA reductase is expressed additionally, (Z,E)-9,11- hexadecadien-l-ol can be produced. This property is advantageous for achieving Z9,E11-16:OH production with higher purity.

Table 9. Effect of the AZ9-16 desaturase DsaDes7 on Z9-16:Me and Z9, Ell-16:Me purity in derivatized FAME samples of Yarrowia lipolytica strains.

Example 18 - Conversion of Ell-16:Me to Z9,Ell-16:CoA in Y. lipolytica by AZ9 desaturases

[0162] The AZ9 desaturase DsaDes7 (SEQ ID NO:82) from D. saccharalis was expressed in Y. lipolytica strain ST6629 (Holkenbrink et al., 2020) to generate strain ST13247. The empty expression vector (pBP9002) was transformed into the same parent strain to give ST10444 as a control strain. Both strains were cultivated as described in Example 3 either with or without additional supplementation of 0.2 g/L of Ell-16:Me to the cultures. After cultivation, samples were extracted and converted into fatty acid methyl ester as described in example 3. When the cultivation cultures were not supplied with Ell-16:Me, derivatized FAME samples of Strain ST10444 and ST13247 did not contain any Z9,E11- 16:Me. However, in derivatized samples of both strains with supplementation of Ell-16:Me, Z9,E11- 16:Me could be detected (Table 10). Samples of strain ST13247 had a higher purity of Z9,Ell-16:Me than the control strain ST10444. These results support that both D. saccharalis AZ9 desaturase DsaDes7 and the native Y. lipolytica AZ9 desaturase OLE1 (SEQ ID NO:17) can convert Ell-16:CoA into Z9,Ell-16:CoA.

Table 10. Detection of Z9,Ell-16:Me in derivatized samples of Y. lipolytica strains with and without supplementation of Ell-16:Me.

Example 19 - Production of Z9,Ell-16:CoA in Yarrowia lipolytica using alternative Ell desaturases [0163] Synthetic protein variants of the AE11 desaturase DsaDesl were designed and expressed in Y. lipolytica strain ST6629 (Holkenbrink et al., 2020) to generate strain ST13284, ST13285, ST13286, ST13287 and ST13288. The empty expression vector (pB P9002) was transformed into the same parent strain to give ST10444 as a control strain. Cultivation of both strains and extraction of fatty acids were performed as described in Example 3. No Z9,Ell-16:Me was detected from FAME extract of the control strain ST10444 as expected. FAME extracts of strains ST13284, ST13285, ST13286, ST13287 and ST13288, expressing the designed variants Desat87 (SEQ ID NO:96), Desat88 (SEQ ID NO:98), Desat89 (SEQ ID NQ:100), Desat90 (SEQ ID NQ:102) and Desat91 (SEQ ID NQ:102), respectively, all contained the target compound Z9,Ell-16:Me (Table 11). The sequence identities of synthetic protein variants can be seen in the table (Table 12). The present example demonstrates that a broad number of Ell desaturase sequence variants can be used to provide the desired E-alkene.

Table 11. Detection of Z9, Ell-16: Me in derivatized samples of Y. lipolytica strains.

Table 12. Sequence identity percentage between AE11 desaturases.

Example 20 - Measurement of biobased carbon content

[0164] The biobased carbon content of a fatty alcohol pheromone precursor cowas measured by using the analytical measurementthat may be cited as "percent modern carbon (pMC)". This is the percentage of isotope 14C measured in the sample relative to a modern reference standard (NIST 4990C). The % biobased carbon content is calculated from pMC by applying a small adjustment factor for isotope 14C in carbon dioxide in air today. Strain STSCB, engineered for production of Z9,E11- 16:01-1, was fermented and the product was recovered. The product sample was composed of a mix of fatty alcohols including Z9,E11-16:OH. The "percent modern carbon (pMC)" of the product sample was analyzed by the standard test method "ASTM D6866". The percent of modern carbon was determined to be 93.80 ± 0.33 pMC, corresponding to 94% of biobased carbon content (Table 13). This implies that the pMC of Z9,E11-16:OH contained in the product sample also was 94%.

Table 13. Determination of percent of modern carbon in product sample.

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

[0165] The Ell desaturase DsaDesl (SEQ. ID NO:80) and the fatty acyl-CoA reductase DsaFARl (SEQ ID NO:88) from D. saccharalis were cloned into S. cerevisiae gene expression vectors and transformed into S. cerevisiae CEN.PK strains as described in Maury, et al., 2016 and Jensen, et al., 2014. Saccharomyces cerevisiae strains were inoculated from synthetic drop-out agar plates (lacking uracil, leucine and histidine) to an initial OD600 of 0.1-0.2 into 2.5 mL synthetic drop-out medium (lacking uracil, leucine and histidine) supplemented with 2% glucose in 24 well-plates (EnzyScreen). Sample extraction is performed as in Example 3. Strain ST13490, expressing both the DsaDesl and DsaFARl, produced Z9, E11-16:OH (Table 14). While the control strain ST13491, expressing DsaDesl only, did not produce any Z9,E11-16:OH.

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

References

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

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

Jensen, N., Strucko, T., Kildegaard, K., David, F., Maury, J., Mortensen, U., . . . Borodina, I. (2014). EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae. FEMS Yeast Research, 238-48.

Lienard, M., Lassance, J.-M., Wang, H.-L., Zhao, C.-H., Piskur, J., Johansson, T., & Lbfstedt, C. (2010). Elucidation of the sex-pheromone biosynthesis producing 5,7-dodecadienes in Dendrolimus punctatus reveals 11- and 9-desaturases with unusual catalytic properties. Insect Biochemistry and Molecular Biology, 440-452.

Zhao, C.-H., Adolf, R., & Lofstedt, C. (2004). Sex pheromone biosynthesis in the pine caterpillar moth, Dendrolimus punctatus: pathways leading to Z5-monoene and 5,7-conjugated diene components. Insect Biochemistry and Molecular Biology, 261-271.

Da Silva, M., Cortes, A., Svensson, G., Lofstedt, C., Lima, E., & Zarbin, P. (2021). Identification of two additional behaviorally active gland constituents of female Diatraea saccharalis (Fabricius) (Lepidoptera Crambidae). Journal of the Brazilian Chemical Society.

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

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

Jensen, N., Strucko, T., Kildegaard, K., David, F., Maury, J., Mortensen, U., . . . Borodina, I. (2014). EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae. FEMS Yeast Research, 238-48.

Kalinova, B., Kindi, J., Hovorka, O., Hoskovec, M., & Svatos, A. (2005). (llZ)-hexadec-ll-enal enhances the attractiveness of Diatraea saccharalis main pheromone component in wind tunnel experiments. Journal of Applied Entomology.

Lienard, M., Lassance, J.-M., Wang, H.-L., Zhao, C.-H., Piskur, J., Johansson, T., & Lofstedt, C. (2010). Elucidation of the sex-pheromone biosynthesis producing 5,7-dodecadienes in Dendrolimus punctatus reveals 11- and 9-desaturases with unusual catalytic properties. Insect Biochemistry and Molecular Biology, 440-452.

Svatos, A., Kalinova, B., Kindi, J., Kuldova, J., Hovorka, O., Rufino Do Nascimento, R., & Oldham, N. (2001). Chemical Characterization and Synthesis of the Major Component of the Sex Pheromone of the Sugarcane Borer Diatraea saccharalis. Collect. Czech. Chem. Commun., 1682-1690. Zhao, C.-H., Adolf, R., & Lbfstedt, C. (2004). Sex pheromone biosynthesis in the pine caterpillar moth, Dendrolimus punctatus: pathways leading to Z5-monoene and 5,7-conjugated diene components. Insect Biochemistry and Molecular Biology, 261-271.

Pryor, J. M., Potapov, V., Kucera, R. B., Bilotti, K., Cantor, E. J., Lohman, G. J. S. (2020). Enabling one- pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design. PloS ONE 15(9): e0238592.

Items of the disclosure

[0166] The present disclosure further provides the following embodiments and items:

Item 1. An Ell fatty acyl-CoA desaturase (Ell desaturase) comprising an amino acid sequence having at least 50% identity to the of the Ell desaturase comprised in SEQ ID NO: 1 or SEQ ID NO: 80.

Item 2. The Ell desaturase of item 1 wherein the Ell desaturase in the presence of a (Z)-9- hexadecenoyl-CoA substrate introduces a double bond in a E configuration in position 11 thereby producing a (Z,E)-9,ll-hexadecadienoyl-CoA.

Item 3. A polynucleotide sequence codon optimized for heterologous expression encoding the Ell desaturase of item 1 having a DNA sequence comprised in SEQ NO:2 or SEQ ID NO: 81, or a homologue thereof including variations due to the degeneracy of the genetic code.

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

Item 5. The polynucleotide construct of item 4 wherein the control sequence is heterologous to the polynucleotide.

Item 6. A vector comprising the polynucleotide construct of item 4 or 5.

Item 7. A genetically engineered microbial cell producing a (Z,E)-9,ll-hexadecadienoyl-CoA said cell heterologously expressing the Ell desaturase of item 1 or 2 which in the presence of a (Z)-9- hexadecenoyl-CoA substrate introduces a double bond in a E configuration in position 11 in the (Z)-9- hexadecenoyl-CoA substrate and thereby produces the (Z,E)-9,ll-hexadecadienoyl-CoA having a double bond in position 11 in E configuration.

Item 8. The cell of item 7 further comprising an operative biosynthetic pathway converting the (Z,E)- 9,11-hexadecadienoyl-CoA into a target compound selected from a) (Z,E)-9,ll-hexadecadien-l-ol; b) (Z,E)-9,ll-hexadecadienal; and/or c) (Z,E)-9,ll-hexadecadienyl acetate; said pathway expressing one or more pathway polypeptides selected from: a) an alcohol-forming fatty acyl-CoA reductase (FAR) converting (Z,E)-9,ll-hexadecadienoyl-CoA into (Z,E)-9,ll-hexadecadien-l-ol; b) an acetyltransferase converting (Z,E)-9,ll-hexadecadien-l-ol into (Z,E)-9,ll-hexadecadienyl acetate; c) an alcohol dehydrogenase converting (Z,E)-9,ll-hexadecadien-l-ol into (Z,E)-9,11- hexadecadienal; and/or d) a fatty alcohol oxidase converting (Z,E)-9,ll-hexadecadien-l-ol into (Z,E)-9,ll-hexadecadienal.

Item 9. The cell of item 8 wherein a) the FAR is at least 70% identical to the FAR comprised 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; b) the acetyltransferase is at least 70% identical to the acetyltransferase comprised in SEQ ID NO: 71; c) the alcohol dehydrogenase is at least 70% identical to the alcohol dehydrogenase comprised in SEQ ID NO: 68; and d) the fatty alcohol oxidase is at least 70% identical to the fatty alcohol oxidase comprised in SEQ ID NO: 69 or 70.

Item 10. The cell of item 7 to 9 further comprising an operative biosynthetic pathway producing (Z)-9- hexadecenoyl-CoA substrate, said pathway expressing one or more heterologous A9 desaturases which in the presence of a hexadecenoyl-CoA substrate introduces a double bond in the hexadecenoyl-CoA substrate in a Z configuration in position 9.

Item 11. The cell of item 10 wherein the A9 desaturase is the at least 70% identical to the A9 desaturase comprised in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82. Item 12. The cell of item 7 to 11, wherein one or more native or endogenous genes are attenuated, disrupted and/or deleted.

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

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

Item 15. The cell of item 7 to 14, further genetically modified to exhibit increased tolerance towards one or more substrates, intermediates, or product molecules from the (Z,E)-9,ll-hexadecadienal pathway.

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

Item 17. The host cell of item 7 to 16, wherein the host cell is a fungal cell.

Item 18. The host cell of item 17, wherein the fungal cell is a yeast cell.

Item 19. The host cell of item 18, wherein the yeast cell belongs to a genus selected from Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces, optionally wherein the yeast cell belongs to a species selected from Saccharomyces cerevisiae, Saccharomyces boulardi, Pichia pastoris, Kluyveromyces marxianus, Cryptococcus albidus, Lipomyces lipofera, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulan and Yarrowia lipolytica.

Item 20. The cell of item 17 wherein the fungal cell is a filamentous fungal cell selected from the species consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporiuminops, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium gueenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.

Item 21. A genetically engineered yeast cell producing (Z,E)-9,ll-hexadecadienoyl-CoA and (Z, E )-9, 11- hexadecadien-l-ol, said cell producing hexadecanoyl-CoA and expressing a. a A9 desaturase catalyzing the formation of a double bond in a Z configuration in position 9 in the hexadecanoyl-CoA thereby producing a (Z)-9-hexadecenoyl-CoA; b. an Ell desaturase catalyzing the formation of a double bond in a E configuration in position 11 in the (Z)-9-hexadecenoyl-CoA thereby producing a (Z,E)-9,ll-hexadecadienoyl-CoA; and c. an alcohol-forming fatty acyl-CoA reductase (FAR) converting the (Z,E)-9,ll-hexadecadienoyl- CoA into (Z,E)-9,ll-hexadecadien-l-ol.

Item 22. The yeast cell of item 21 further expressing one or more enzymes selected from: a. a Zll desaturase catalyzing the formation of a double bond in a Z configuration in position 11 in the hexadecanoyl-CoA thereby producing a (Z)-ll-hexadecenoyl-CoA; b. one or more alcohol-forming fatty acyl-CoA reductases (FAR) converting respectively the hexadecanoyl-CoA into hexadecan-l-ol, the (Z)-9-hexadecenoyl-CoA into (Z)-9-hexadecen-l-ol, and the (Z)-ll-hexadecenoyl-CoA into (Z)-ll-hexadecen-l-ol.

Item 23. The yeast cell of item 21 or 22 wherein the a) Ell desaturase has an amino acid sequence comprised in the Ell desaturase of SEQ ID NO: 1 or 80; b) A9 desaturase has an amino acid sequence comprised in the A9 desaturase of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, or 82; c) Zll desaturase has an amino acid sequence comprised in the Zll desaturase of SEQ ID NO: 72, 74, 76, or 78; and d) alcohol-forming fatty acyl-CoA reductases (FAR) has an amino acid sequence comprised 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.

Item 24. The yeast cell of item 21 or 23, wherein the yeast cell is of the species Saccharomyces cerevisiae, or Yarrowia lipolytica.

Item 25. A cell culture comprising the genetically engineered microbial cell of item 7 to 24 and a growth medium.

Item 26. A method for producing a target compound selected from: a) (Z,E)-9,ll-hexadecadienoyl-CoA b) (Z,E)-9,ll-hexadecadien-l-ol; c) (Z,E)-9,ll-hexadecadienal; and/or d) (Z,E)-9,ll-hexadecadienyl acetate; said method comprising culturing the cell culture of item 25 at conditions allowing the cell culture to produce the target compound, and optionally recovering and/or isolating the target compound.

Item 27. The method of item 26, comprising feeding the cell culture exogenously with one or more substrates or precursors of the target compound pathway.

Item 28. The method of item 26 to 27, wherein one or more steps of producing the target compound is performed in vitro.

Item 29. The method of item 26 to 28, wherein the in vitro performed step comprises reducing (Z,E)- 9,11-hexadecadienoyl-CoA into (Z,E)-9,ll-hexadecadien-l-ol using a reductase enzyme (FAR).

Item 30. The method of item 26 to 28, wherein the in vitro performed step comprises chemically or enzymatically reducing a (Z,E)-9,ll-hexadecadienic acid into (Z,E)-9,ll-hexadecadien-l-ol.

Item 31. The method of item 26 to 29, wherein the in vitro performed step comprises chemical or enzymatic oxidization of (Z,E)-9,ll-hexadecadien-l-ol into (Z,E)-9,ll-hexadecadienal.

Item 32. The method of item 26 to 29, wherein the in vitro performed step comprises chemical or enzymatic acetylation of (Z,E)-9,ll-hexadecadien-l-ol into (Z,E)-9,ll-hexadecadienyl acetate. Item 33. The method of any one of item 26 to 32, further comprising recovering the target compound and mixing it with one or more carriers, agents, additives, adjuvants and/or excipients to produce a biopesticide composition.

Item 34. The method of item 33 wherein the one or more carriers, agents, additives, adjuvants and/or excipients comprises a protective agent comprising a conjugated sulfur, which protects the target compound from being converted into an acid.

Item 35. The method of item 34 wherein the protective agent comprises a compound selected from zinc pyrithione, 5-amino-l,3,4-thiadiazole-2-thiol, 2-th iazoline-2-thiol, 5-methyl-l,3,4-thiadiazole-2- thiol, 2-mercapto-benzimidazole, 2-mercapto-l-methylimidazole and sodium pyrithione.

Item 36. The method of item 34 to 35 comprising mixing in at least 10 mg of the protective agent per gram aldehyde and/or alcohol.

Item 37. The method of item 33 to 37, wherein the one or more carriers, agents, additives, adjuvants and/or excipients comprises a carrier facilitating slow release of the target compound, optionally being

(i) a polymeric substrate selected from plastic, wax emulsion, oil emulsion, or microcapsules and/or

(ii) zeolite.

Item 38. A method for producing a biopesticide composition comprising

(I) culturing a genetically engineered yeast cell producing hexadecanoyl-CoA and expressing a. a A9 desaturase catalyzing the formation of a double bond in a Z configuration in position

9 in the hexadecanoyl-CoA and thereby producing a (Z)-9-hexadecenoyl-CoA; b. an Ell desaturase catalyzing the formation of a double bond in a E configuration in position 11 in the (Z)-9-hexadecenoyl-CoA and thereby producing a (Z,E)-9,11- hexadecadienoyl-CoA; c. an alcohol-forming fatty acyl-CoA reductase (FAR) converting the (Z,E)-9,11- hexadecadienoyl-CoA into (Z,E)-9,ll-hexadecadien-l-ol;

(II) enzymatically or chemically converting the (Z,E)-9,ll-hexadecadien-l-ol to (Z,E)-9,11- hexadecadienal; and

(III) optionally recovering and/or isolating the (Z,E)-9,ll-hexadecadienal and optionally one or more precursors thereof. Item 39. The method of item 38, wherein the genetically engineered yeast cell further expresses one or more enzymes selected from: a. a Zll desaturase catalyzing the formation of a double bond in a Z configuration in position 11 in the hexadecanoyl-CoA and thereby producing a (Z)-ll-hexadecenoyl-CoA; b. one or more alcohol-forming fatty acyl-CoA reductases (FAR) converting respectively the hexadecanoyl-CoA into hexadecan-l-ol, the (Z)-9-hexadecenoyl-CoA into (Z)-9-hexadecen-l-ol, and the (Z)-ll-hexadecenoyl-CoA into (Z)-ll-hexadecen-l-ol; and the method further comprises enzymatically or chemically converting the hexadecan-l-ol into hexadecanal, the (Z)-9-hexadecen-l-ol into (Z)-9-hexadecenal and the (Z)-ll-hexadecen-l-ol and the (Z)-ll-hexadecen-l-ol into (Z)-ll-hexadecenal; and/or optionally recovering and/or isolating the hexadecanal, the (Z)-9-hexadecenal and the (Z)-ll-hexadecenal and optionally one or more precursors thereof.

Item 40. The method of item 38 to 39, further comprising mixing the recovered (Z,E)-9,11- hexadecadienal, (Z)-9-hexadecenal, (Z)-ll-hexadecenal, hexadecanal and optionally one or more precursors thereof with one or more carriers, agents, additives, adjuvants and/or excipients to produce the biopesticide composition.

Item 41. The method of item 40 wherein the carriers, agents, additives, adjuvants and/or excipients comprise one or more compounds selected from a) a protective agent comprising a conjugated sulfur compound selected from zinc pyrithione, 5- amino-l,3,4-thiadiazole-2-thiol, 2-thiazoline-2-thiol, 5-methyl-l,3,4-thiadiazole-2-thiol, 2- mercapto-benzimidazole, 2-mercapto-l-methylimidazole and sodium pyrithione, which protects the target compound from being converted into an acid; and/or b) a carrier facilitating slow release of the (Z,E)-9,ll-hexadecadienal, (Z)-9-hexadecenal, (Z)-ll- hexadecenal and/or hexadecanal from the mixture, optionally being (i) a polymeric substrate selected from plastic, wax emulsion, oil emulsion, or microcapsules and/or (ii) a zeolite.

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)-ll- hexadecenal and/or hexadecanal, in combination with one or more carriers, agents, additives, adjuvants and/or excipients.

Item 43. The biopesticide composition of item 42, further comprising at least trace amounts of one or more compounds selected from hexadecan-l-ol, (Z)-9-hexadecen-l-ol, (Z)-ll-hexadecen-l-ol, and (Z,E)-9,ll-hexadecadien-l-ol, and optionally other metabolites of the cell culture.

Item 44. The biopesticide composition further comprising (Z,E)-9,ll-hexadecandienyl acetate.

Item 45. The biopesticide composition of item 42 to 44 wherein one or more of the hexadecanoyl- CoA, (Z)-9-hexadecenoyl-CoA, (Z)-ll-hexadecenoyl-CoA, (Z,E)-9,ll-hexadecadienoyl-CoA, hexadecan- l-ol, (Z)-9-hexadecen-l-ol, (Z)-ll-hexadecen-l-ol, and (Z,E)-9,ll-hexadecadien-l-ol are obtained from the method of item 26 to 41 and optionally the composition comprises one or more further compounds or metabolites from the cell culture of item 25.

Item 46. The biopesticide composition of item 42 to 45 wherein the concentration of the target compound is at least 1 mg/kg composition.

Item 47. The composition of item 42 to 46 further comprising a protective agent comprising a conjugated sulfur, which protects the target compound frombeing converted into an acid.

Item 48. The composition of item 47 wherein the protective agent comprises a compound selected from zinc pyrithione, 5-amino-l,3,4-thiadiazole-2-thiol, 2-thiazoline-2-thiol, 5-methyl-l,3,4- th iad iazole-2-thiol, 2-mercapto-benzimidazole, 2-mercapto-l-methylimidazole and sodium pyrithione.

Item 49. The composition of item 47 to 48 comprising at least 10 mg of the protective agent per gram aldehyde and/or alcohol.

Item 50. The composition of item 42 to 49, further comprising a carrier facilitating slow release of the target compound, optionally being (i) a polymeric substrate selected from plastic, wax emulsion, oil emulsion, or microcapsules and/or (ii) zeolite.

Item 51. A method of controlling or monitoring a pest comprising distributing the composition of item 42 to 50 in a habitat for the pest and allowing the target compound to control the pest.

Item 52. The method of item 51, wherein the habitat is a sugar cane field, and the pest is Diatraea saccharalis.