WO2021262906A1 - Synthesis of complex insect pheromone blends and reaction apparatuses for the same - Google Patents

Synthesis of complex insect pheromone blends and reaction apparatuses for the same Download PDF

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WO2021262906A1
WO2021262906A1 PCT/US2021/038775 US2021038775W WO2021262906A1 WO 2021262906 A1 WO2021262906 A1 WO 2021262906A1 US 2021038775 W US2021038775 W US 2021038775W WO 2021262906 A1 WO2021262906 A1 WO 2021262906A1
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16ald
16acid
18ald
desaturase
strains
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PCT/US2021/038775
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French (fr)
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Weslee GLENN
Micah SHEPPARD
Kim Nguyen
Kati WU
Toni Lee
Thomas HEEL
David Rozzell
Effendi Leonard
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Provivi, Inc.
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Publication of WO2021262906A1 publication Critical patent/WO2021262906A1/en

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/013—NADP-retinol dehydrogenase (1.1.1.300)
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    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
    • C12Y103/01093—Very-long-chain enoyl-CoA reductase (1.3.1.93)
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    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
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    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
    • C12Y114/19001—Stearoyl-CoA 9-desaturase (1.14.19.1), i.e. DELTA9-desaturase
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    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01051—1-Acylglycerol-3-phosphate O-acyltransferase (2.3.1.51)
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    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01076—Retinol O-fatty-acyltransferase (2.3.1.76)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01199—Very-long-chain 3-oxoacyl-CoA synthase (2.3.1.199)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01M—CATCHING, TRAPPING OR SCARING OF ANIMALS; APPARATUS FOR THE DESTRUCTION OF NOXIOUS ANIMALS OR NOXIOUS PLANTS
    • A01M29/00—Scaring or repelling devices, e.g. bird-scaring apparatus
    • A01M29/12—Scaring or repelling devices, e.g. bird-scaring apparatus using odoriferous substances, e.g. aromas, pheromones or chemical agents

Definitions

  • the disclosure relates to the production of complex blends of regiospecific and stereospecific unsaturated fatty aldehydes that are biologically active as insect pheromone compositions, and/or complex blends of precursors thereof. More specifically, the disclosure relates to the coordination of multiple catalytic activities in a single reaction volume to produce a plurality of particular fatty acids and/or derivatives thereof (e.g., fatty alcohols, fatty acetates, and fatty aldehydes) in a ratio that elicits a behavior response an insect; for example, to disrupt mating and confer crop protection in a host plant of the insect, or to produce a plurality of precursors of the active forms in relative amounts corresponding to the amounts of the active forms in the biologically active blend compositions.
  • fatty acids and/or derivatives thereof e.g., fatty alcohols, fatty acetates, and fatty aldehydes
  • the latter method represents a non-toxic means of pest control and utilizes synthetic pheromones to mask naturally occurring pheromones, thereby confusing insects and disrupting their mating.
  • pheromones are biodegradable and are non-toxic to humans. These properties make pheromones ideal candidates for modern eco-friendly crop protection.
  • females emit an airborne trail of a specific chemical blend constituting that species’ sex pheromone. This aerial trail is referred to as a pheromone plume. Males of that species use the information contained in the pheromone plume to locate the emitting female.
  • Mating disruption exploits the male insects’ natural response to follow the plume by introducing a synthetic pheromone blend into the insects’ habitat.
  • the blend is designed to mimic the sex pheromone produced by the female insect.
  • Insect sex pheromones contain a diverse group of chemical compounds including regiospecific and stereospecific unsaturated fatty aldehyde isomers.
  • the sex pheromones of different insect species are specific blends of the constituent compounds in characteristic ratios, which in the current state of the art requires for effective synthetic pheromone blends to be assembled from isolated and purified compounds according to the requirements of a particular target insect species.
  • the constituent compounds of biologically active synthetic insect pheromones are regiospecific and stereospecific unsaturated fatty aldehyde isomers comprising lipid moieties with specific chain lengths, so the independent synthesis and work-up of the constituent compounds typically requires the expenditure of considerable resources. Therefore, although pheromones have significant potential in agricultural insect control, the cost of synthesizing pheromones using currently available techniques is very high, and the widespread use of this effective, eco-friendly, and sustainable technology in other than high-value crops is effectively prohibited.
  • Described herein are apparatuses and methods for producing complex compositions in a single reaction volume that comprise particular amounts of specific fatty acids, fatty alcohols, fatty aldehydes, and/or fatty acetates.
  • Such complex compositions may be biologically active in an insect pest (for example, by mimicking the natural pheromone of the insect), or may contain corresponding amounts of chemical precursors of the biologically active composition compounds.
  • the fatty acids and/or derivatives thereof e.g., fatty alcohols, fatty acetates, and fatty aldehydes
  • produced according to methods and compositions described herein may be fragrances, flavoring agents, and/or polymer intermediates.
  • a complex blend comprising particular amounts of regiospecific and stereospecific product isomers (i.e., fatty acids and/or derivatives thereof comprising lipid moieties with specific chain lengths) is produced from organic substrate(s) (e.g., molecules comprising saturated or unsaturated lipid moieties) by combining activities representing steps in the synthesis of each of the products.
  • the activities are comprised in a biocatalyst (a naturally occurring cell or organism, or genetically modified cell or organism), with or without conversion by non-biological means of reaction intermediates (e.g., regiospecific and/or stereospecific isomers comprising saturated or unsaturated lipid moieties) as one or more of the successive reaction steps.
  • the substrate(s) are subjected to successive chemical reactions in a single reaction volume, for example, without separation or purification of intermediates or products during the reaction process.
  • reagents or other reaction components e.g., catalysts, biocatalysts, and substrates
  • an apparatus for producing a complex composition in a single reaction volume is assembled for the one-pot synthesis of a complex blend comprising particular amounts of regiospecific and stereospecific product isomers comprising lipid moieties with specific chain lengths.
  • one or more unsaturated lipid moieties utilized as a reactant in one or more step(s) of the synthesis may be introduced into the reaction volume, or produced therein by a biocatalyst from a saturated lipid moiety or organic substrate (e.g., glucose).
  • a biocatalyst from a saturated lipid moiety or organic substrate (e.g., glucose).
  • unsaturated lipid moiety-containing reactants may be reduced in the reaction volume directly to produce one or more fatty alcohols, one or more fatty aldehydes, and/or one or more fatty acetates.
  • such unsaturated lipid moieties may be chemically converted in the reaction volume to one or more free fatty acids (FFAs), fatty acid alkyl esters (FAAEs) (e.g., fatty acid methyl esters (FAMEs)), fatty alcohols, fatty acetates, and/or fatty aldehydes.
  • FFAs free fatty acids
  • FAAEs fatty acid alkyl esters
  • FAMEs fatty acid methyl esters
  • an apparatus for producing a complex composition in a single reaction volume comprises the reaction volume within a single compartment in the apparatus.
  • an apparatus for producing a complex composition in a single reaction volume may comprise more than one compartment in the apparatus, disposed therein such that they are integrally connected to comprise the reaction volume.
  • one or more compartment of the apparatus may comprise one or more points of access for the addition and/or removal of fluids, gases, chemicals, reactants, catalysts, biocatalysts, etc.
  • one or more compartment of the apparatus may comprise a working station, for example, adapted to facilitate observation or monitoring of the reaction process.
  • a biocatalyst may be used in embodiments herein to catalyze, for example, any combination of the following reactions, in one or more steps: synthesis of a saturated lipid moiety or unsaturated lipid moiety isomer (e.g., from an organic substrate); production of an unsaturated lipid moiety isomer (e.g., from a saturated lipid moiety); production of a fatty alcohol (e.g., from an unsaturated lipid moiety isomer); production of a fatty aldehyde (e.g., from an unsaturated lipid moiety isomer); production of a FFA (e.g., from an unsaturated lipid moiety isomer); production of a FAE (e.g., from an unsaturated lipid moiety isomer, and from a FFA); production of a FAME (e.g., from an unsaturated lipid moiety isomer, and from a FFA); production of a saturated lipid moiety or uns
  • a biocatalyst according to embodiments herein may be an organism (e.g., a genetically modified organism).
  • the organism is a fungus or bacteria.
  • the organism is a yeast; for example, an oleaginous yeast.
  • the organism may an oleaginous yeast from the genus Yarrowia or Candida; e.g., Yarrowia lipolytica, Candida viswanathii, and Candida tropicalis.
  • the biocatalyst may be a genetically modified strain of Y. lipolytica.
  • the organism is a bacteria, for example, a gram-negative bacteria or cyanobacteria.
  • the organism may be a gram-negative bacteria from the genus Myxococcus, e.g., M. xanthus.
  • the organism is a cyanobacteria species from the genus Synechococcus, e.g., S. elongatus.
  • the organism is an insect, such as Amyelois transitella or Pyralis farinalis in one specific example.
  • the organism is a plant, or algae, or bacteria.
  • the organism is a plant from the family Proteaceae, or the family Rhamnaceae.
  • the organism is a plant species from the genus Gevuina, Calendula, Limnanthes, Lunaria, Carum, Daucus, Conandrum, Macadamia, Myristica, Licania, Aleurites, Ricinus, Corylus, Kermadecia, Asclepias, Cardwellia, Grevillea, Orites, Ziziphus, Hicksbeachia, Hippophae, Ephedra, Placospermum, Xylomelum, or Simmondsia.
  • the organism may be a plant species selected from the group consisting of Gevuina avellana, Corylus avellana, Kermadecia sinuata, Asclepias syriaca, Cardwellia sublimis, Grevillea exul var. rubiginosa, Orites diversifolius, Orites revoluta, Ziziphus jujube, Hicksbeachia pinnatifolia, and Grevillea decora.
  • the organism is an algae, for example, a green algae.
  • the organism may be a green algae species from the genus Pediastrum, e.g., Pediastrum simplex.
  • a complex composition is created as a synthetic pheromone in an insect selected from the group consisting of the Asiatic rice borer or striped rice stemborer (i.e, Chilo suppressalis), corn earworm (i.e, Helicoverpa zea), cotton bollworm (i.e, Helicoverpa armigera), rice leafroller (i.e, Cnaphalocrocis medinalis), yellow stem borer or rice yellow stem borer (i.e, Scirpophaga incertulas, fall armyworm (i.e, Spodoptera frugiperda), soybean looper moth (i.e, Pseudoplusia includens), and jasmine bud borer (i.e, Trichophysetis cretacea).
  • the Asiatic rice borer or striped rice stemborer i.e, Chilo suppressalis
  • corn earworm i.e, Helicoverpa zea
  • cotton bollworm i.e, Helicoverpa
  • Apparatuses and methods according to the embodiments herein provide significant advantages over the prior art by enabling the one-pot synthesis of complex compositions comprising the constituent compounds in relative amounts that are within a biologically relevant range, or compositions comprising precursors of the constituent compounds in relative amounts that are immediately able to be purified and processed without separation into a biologically active complex composition comprising the constituent compounds in relative amounts within the biologically active range.
  • a complex composition is produced as a synthetic pheromone, wherein the composition comprises a combination of constituent compounds selected from the group consisting of Z9-14Acid, Z9-14OH, Z9-14Ald, Z9-14Ac, Z9-16Acid, Z9-16OH, Z9-16Ald, Z9-16Ac, Z11-16Acid, Z11-16OH, Z11-16Ald, Z11-16Ac, Z9-18Acid, Z9-18OH, Z9-18Ald, Z9-18Ac, Z11-18Acid, Z11-18OH, Z11-18Ald, Z11-18Ac, Z13-18Acid, Z13- 18OH, Z13-18Ald, and Z13-18Ac.
  • a complex composition is produced as a synthetic pheromone, wherein the composition comprises Z9-16Ald.
  • a complex composition is biologically active as a synthetic pheromone in Chilo suppressalis, Scirpophaga incertulas, Helicoverpa zea, or Helicoverpa armigera, wherein the composition comprises 0.5- 40% (e.g., 0.5-10%, 5-40%, 3-15%, 1-20%, 1-4%, 5-12%, 5-15%, 15-25%, 15-33%, about 3%, about 7%, about 9%, about 19%, and about 25%) Z9-16Ald.
  • 0.5- 40% e.g., 0.5-10%, 5-40%, 3-15%, 1-20%, 1-4%, 5-12%, 5-15%, 15-25%, 15-33%, about 3%, about 7%, about 9%, about 19%, and about 25%
  • a complex composition that is biologically active as a synthetic pheromone in C. suppressalis may comprise 7-9% (e.g, 7%, 8%, and 9%) Z9-16Ald.
  • a complex composition is produced that is biologically active as a synthetic pheromone, wherein the composition comprises Z11-16Ald.
  • such a complex composition is biologically active as a synthetic pheromone in Chilo suppressalis, Scirpophaga incertulas, Trichophysetis cretacea, Helicoverpa zea, or Helicoverpa armigera, wherein the composition comprises at least 40% Z11-16Ald.
  • a complex composition that is biologically active as a synthetic pheromone in Chilo suppressalis, Scirpophaga incertulas, Trichophysetis cretacea, Helicoverpa zea, or H. armigera may comprise 40-99.5% (e.g., 40-90%, 40-97%, 60-80%, 60-90%, 66-85%, 90-99.5%, 47-97%, 70-80%, 70-94%, 75-90%, 96-99%, about 74%, about 75%, about 81%, and about 97%) Z11- 16Ald.
  • the complex composition is biologically active as a synthetic pheromone in T.
  • the complex composition is biologically active as a synthetic pheromone in C. suppressalis, wherein the composition comprises 74-85% (e.g., 74-81%, about 74%, about 75%, and about 81%) Z11-16Ald.
  • the complex composition is biologically active as a synthetic pheromone in H. zea, or H. armigera, wherein the composition comprises about 97% Z11-16Ald.
  • the complex composition is biologically active as a synthetic pheromone in S. incertulas, wherein the composition comprises about 75% Z11-16Ald.
  • a complex composition is produced that is biologically active as a synthetic pheromone, wherein the composition comprises Z9-16Ald and at least 40% Z11-16Ald.
  • a complex composition is biologically active as a synthetic pheromone in Chilo suppressalis, Scirpophaga incertulas, Helicoverpa zea, or Helicoverpa armigera, wherein the composition comprises 3%-25% Z9-16Ald and 40%-97% Z11-16Ald.
  • the complex composition is biologically active as a synthetic pheromone in C.
  • the composition comprises 7-9% (e.g, 7%, 8%, and 9%) Z9-16Ald and 74-85% (e.g., 74-81%, about 74%, about 75%, and about 81%) Z11-16Ald.
  • the complex composition is biologically active as a synthetic pheromone in H. zea, or H. armigera, wherein the composition comprises about 3% Z9-16Ald and about 97% Z11-16Ald.
  • the complex composition is biologically active as a synthetic pheromone in S. incertulas, wherein the composition comprises about 25% Z9-16Ald and about 75% Z11-16Ald.
  • a complex composition is produced that is biologically active as a synthetic pheromone, wherein the composition comprises Z13-18Ald.
  • such a complex composition is biologically active as a synthetic pheromone in Cnaphalocrocis medinalis or Chilo suppressalis, wherein the composition comprises 8%-90% Z13-18Ald.
  • the complex composition is a synthetic pheromone in C. medinalis, wherein the composition comprises about 90% Z13-18Ald.
  • the complex composition is a synthetic pheromone in C. suppressalis, wherein the composition comprises 8-11% (e.g., 8.5%, 9.5%, and 10%) Z13-18Ald.
  • a complex composition may be produced that comprises at least one additional constituent compound selected from the group consisting of Z9-14Acid, Z9-14OH, Z9-14Ald, Z9-14Ac, Z9-16Acid, Z9-16OH, Z9-16Ald, Z9- 16Ac, Z11-16Acid, Z11-16OH, Z11-16Ald, Z11-16Ac, Z9-18Acid, Z9-18OH, Z9-18Ald, Z9- 18Ac, Z11-18Acid, Z11-18OH, Z11-18Ald, Z11-18Ac, Z13-18Acid, Z13-18OH, Z13-18Ald, and Z13-18Ac.
  • the complex composition may be biologically active as a synthetic pheromone in Chilo suppressalis, wherein the composition comprises 7-9% (e.g, 7%, 8%, and 9%) Z9-16Ald, 74-85% (e.g., 74-81%, about 74%, about 75%, and about 81%) Z11-16Ald, and 8-11% (e.g., 8.5%, 9.5%, and 10%) Z13-18Ald.
  • such a composition may comprise about 81% Z11-16Ald, about 9% Z9-16Ald, and about 10% Z13-18Ald.
  • the complex composition may be biologically active as a synthetic pheromone in Cnaphalocrocis medinalis, wherein the composition comprises 90% Z13-18Ald and 10% Z11-18Ald.
  • the complex composition may be biologically active as a synthetic pheromone in Trichophysetis cretacea, wherein the composition comprises 47% Z11-16Ald, 47% Z11-16Ac, and 5% Z11-16OH.
  • a complex composition may be biologically active as a synthetic pheromone in an insect, wherein the composition comprises compounds that are not active ingredients in the pheromone of that insect.
  • a complex composition may be biologically active as a synthetic pheromone in Chilo suppressalis, wherein the composition comprises the active pheromone ingredients Z11-16Ald, Z9-16Ald, and Z13- 18Ald, and further comprises Z9-18Ald.
  • the composition comprises 7-9% (e.g, 7%, 8%, and 9%) Z9-16Ald, 74-85% (e.g., 74-81%, about 74%, about 75%, and about 81%) Z11-16Ald, 8-11% (e.g., 8.5%, 9.5%, and 10%) Z13-18Ald, and 7-11% (e.g., about 7.5% and about 10.5%) Z9-18Ald.
  • a complex composition may be biologically active as a synthetic pheromone in C.
  • the composition comprises the active pheromone ingredients Z11-16Ald, Z9-16Ald, and Z13-18Ald, and further comprises Z9-18Ald and Z11-18Ald.
  • Particular embodiments include a complex composition that is biologically active as a synthetic pheromone in a plurality of insects (e.g., 2, at least 2, and 3), wherein constituent compounds of the composition are active ingredients in the pheromones of the plurality of insects, but at least one of the constituent compounds is not an active ingredient in the pheromone of at least one of the plurality of insects.
  • a composition comprising Z11-16Ald, Z9-16Ald, Z13-18Ald, Z9-18Ald, and Z11-18Ald may be biologically active as a synthetic pheromone in C. suppressalis (Z11-16Ald, Z9-16Ald, Z13-Ald) and Cnaphalocrocis medinalis (Z13-18Ald, Z11-18Ald).
  • a complex composition may be produced that comprises a combination of constituent compounds selected from the group consisting of Z9- 14Acid, Z9-14OH, Z9-14Ald, Z9-14Ac, Z9-16Acid, Z9-16OH, Z9-16Ac, Z11-16Acid, Z11- 16OH, Z11-16Ac, Z9-18Acid, Z9-18OH, Z9-18Ald, Z9-18Ac, Z11-18Acid, Z11-18OH, Z11- 18Ac, Z13-18Acid, Z13-18OH, and Z13-18Ac, wherein the composition does not comprise Z9-16Ald, Z11-16Ald, Z11-18Ald, or Z13-18Ald.
  • the complex composition may be biologically active as a synthetic pheromone in Spodoptera frugiperda, wherein the composition comprises 82.8% Z9-14Ac and 12.9% Z11-16Ac.
  • a complex composition may be produced that comprises at least one additional constituent compound that is saturated lipid moiety, unsaturated lipid moiety isomer, fatty alcohol, fatty aldehyde, FFA, FAE, or fatty aldehyde.
  • the complex composition may be biologically active as a synthetic pheromone in Spodoptera frugiperda, wherein the composition comprises 82% Z9-14Ac, 12% Z11-16Ac, and 1% 14Ac.
  • the apparatuses and methods herein may also and alternatively specifically be utilized to produce complex compositions consisting essentially of chemical precursors of the biologically active synthetic pheromone constituent compounds in corresponding amounts to that of the biologically active synthetic pheromone constituent compounds. It is immediately understood by those in the art that a complex composition according to embodiments herein may comprise precursors of the constituent compounds in corresponding amounts to that of the synthetic pheromone constituent compounds that are nonetheless not precisely those of the synthetic pheromone, for example, because a process of purifying the biologically active complex composition from the reaction volume may be expected to result in a change in the relative amounts of the constituent compounds. By way of representative example, a complex composition of synthetic pheromone in C.
  • suppressalis may comprise 81% Z11-16Ald, 9% Z9- 16Ald, and 10% Z13-18Ald, but a complex composition comprising precursors of these compounds in corresponding amounts may comprise Z11-16Acid, Z9-16Acid, and Z13-18Acid in the relative amounts of 1 : 0.09 : 0.14-0.2 (Z11-16Acid : Z9-16Acid : Z13-18Acid), because a relative loss of Z13-18Acid would be anticipated and expected during a distillation of these compounds.
  • FIG. 2 includes the lineage of genetically engineered strains of the production platform.
  • FIG.2A shows the abridged SPV1994 and SPV1995 strain lineage.
  • OLE1 knockout strains are descendants of SPV1994, which is the 4-copy descendant of SPV1629.
  • FIG.2B shows the abridged SPV2072, SPV2073, SPV2074, SPV2075, and SPV2076 strain lineage.
  • FIG.2C shows the lineage of strains that are descendants of strain SPV1629 through the 4-copy strain SPV1994 and OLE replacement strain SPV2076.
  • FIG.2D shows the lineage of strains that are descendants of strain SPV1629.
  • FIG.2A shows the abridged SPV1994 and SPV1995 strain lineage.
  • OLE1 knockout strains are descendants of SPV1994, which is the 4-copy descendant of SPV1629.
  • FIG.2B shows the abridged SPV2072, SPV2073, SPV2074, SP
  • FIG. 3 includes a representation of Z9, Z9Z12, and Z13 fatty acid profiles of OLE1 replacement strains.
  • FIG. 3A shows Z9 and Z9 derived fatty acid titers for each OLE1 replacement strain.
  • FIG. 3B shows Z11 and Z11 derived fatty acids produced by each OLE1 replacement strain.
  • Z13-18Acid is derived from elongation of Z11-16Acid.
  • SPV1994 is the parental control.
  • FIG.4 includes the full fatty acid profiles for OLE1 replacement strains.
  • FIG.5 includes a representation of the final cell densities obtained in a 24-well plate bioconversion assay.
  • FIG.6 includes growth rates of OLE1 replacement strains. Strains were grown at 32 °C and 1500 rpm in a 48-well flower plate. Growth rates are shown in both Solulys ® and FERM1 media.
  • FIG.7 includes fatty acid titer profiles for 4-copy HzDST strains.
  • FIG.8 includes data characterizing SPV1994 with a fourth HzDST copy.
  • FIG. 8A shows Z11-16Acid/Z9-16Acid selectivity increases by ⁇ 1% in strain SPV1994 as compared to the control strain SPV1629.
  • FIG.8B shows C16Acids selectivity is lower in SPV1994, due to lower 16Acid titer.
  • FIG. 8C shows Z11-16Acid/products selectivity is equivalent for SPV1629 and SPV1994.
  • FIG.8D shows Z11-16Acid total selectivity is marginally higher in SPV1994.
  • FIG.8E shows the total fatty acid profiles for SPV1629 and SPV1994.
  • FIG. 9 includes fatty acid titer profiles for P. dactylifera desaturase- expressing strains.
  • FIG.9A shows C16 fatty acid titers.
  • FIG.9B shows C18 fatty acid titers.
  • FIG. 10 includes data characterizing SPV1995 expressing the P. dactylifera LPAAT.
  • FIG.10A shows Z11-16Acid/Z9-16Acid selectivity is higher in strain SPV1995 than the control strain SPV1629.
  • FIG.10B shows C16Acids selectivity is higher in SPV1995, due to lower C18 fatty acid titers.
  • FIG.10C shows Z11-16Acid/products selectivity is higher in SPV1995.
  • FIG. 10D shows Z11-16Acid total selectivity is higher in SPV1995.
  • FIG. 10E shows the total fatty acid profiles for SPV1629 and SPV1995.
  • FIG.11 includes data characterizing SPV1994 and SPV1995, relative to strain SPV1629.
  • FIG.11A shows Z11- 16Acid/Z9-16Acid selectivity is higher in strains SPV1994 and SPV1995.
  • FIG.11B shows C16Acids selectivity is higher in SPV1995, due to lower C18 fatty acid titers.
  • FIG.11C shows Z11-16Acid/products selectivity is higher in SPV1995.
  • FIG. 12 includes a representation of data showing strain improvements within the SPV458 lineage.
  • FIG. 12A shows Z11-16Acid titers for SPV458 lineage strains.
  • FIG. 12B shows the mass ratio of Z11-16Acid and Z9-18Acid titers for SPV458 lineage strains.
  • FIG.12C shows Z11-16Acid/Z9-16Acid selectivity (S Z11/Z9-16 ) for SPV458 lineage strains.
  • FIG. 13 includes data showing the Z11-16Acid and Z9-16Acid titers from strains in the SPV458 lineage.
  • FIG. 14 includes data showing the fatty acid selectivities of SPV1629 lineage strains.
  • FIG.14A shows data from a screen of 2xHzDST/1xZ9 DST strains.
  • FIG.14B shows data from a screen of 3xHzDST/1xZ9 DST strains. All strains were screened in a Biolector ® assay with an initial seeding density OD600 ⁇ 1. The black line represents selectivity of 75%.
  • FIG. 14 includes data showing the Z11-16Acid and Z9-16Acid titers from strains in the SPV458 lineage.
  • FIG. 14 includes data showing the fatty acid selectivities of SPV1629 lineage strains.
  • FIG.14A shows data from a screen of 2xHzDST/1xZ9 DST strains.
  • FIG.14B shows data from a screen of 3x
  • FIG. 15 includes data characterizing the properties of select Gen1 and Gen2 blend strains in a Biolector ® bioconversion assay.
  • FIG. 15A shows unsaturated C16 (Z11/Z9) titers and selectivity for select Gen1 and Gen2 strains in the Biolector ® bioconversion assay with low initial seeding density.
  • FIG.14B shows unsaturated C16 selectivities for select Gen1 and Gen2 strains in the same Biolector ® bioconversion assay. Small decreases in selectivity result from a 20% increase in Z9-18Acid and Z11-18Acid relative to SPV1629.
  • FIG.16 includes data showing the Z11-16Acid/Z9-16Acid selectivities of blend strains in a Biolector ® bioconversion assay.
  • FIG.16A shows the results of a repeat Biolector ® screen of select strains (initial cell density OD 600 ⁇ 0.2).
  • FIG.16B shows the results of a panel screen of select Gen1 and Gen2 blend strains in 24-well plate assay (initial cell density OD600 ⁇ 15). The black line represents selectivity of 75%.
  • FIG. 17 includes data characterizing the properties of select Gen1 and Gen2 blend strains in a 24-well plate bioconversion assay.
  • FIG.17A shows unsaturated C16 (Z11/Z9) titers and selectivity for select Gen1 and Gen2 strains in a 24-well plate assay with high initial seeding density.
  • FIG.17B shows unsaturated C16 selectivity for select Gen1 and Gen2 strains in the same 24-well plate assay.
  • FIG. 18 includes data showing the effect of culture media on the growth of particular blend strains in a Biolector ® bioconversion assay. Two media were used.
  • Solulys ® is a bioprocess medium that uses Solulys ® 95, a glycerol carbon source, and ammonium sulfate as major components.
  • FERM1 is a medium used for small-scale processes that contains yeast extract, a glucose carbon source, and ammonium sulfate as major components.
  • FIG.19 includes data showing the growth of SPV2076 in a 24-well assay with methyl oleate (Z9-18) supplementation.
  • FIG. 19A shows growth curves of SPV1629 and SPV2076 in FERM1 medium when supplemented with varying concentrations of methyl oleate.
  • FIG.19B shows fatty acid profiles of SPV2076 from the endpoint (90 h) of the same assay. Increasing methyl oleate concentrations from 0.9 g/L to 4.4 g/L did not lead to a further increase in growth rate, but did lead to increased Z9-18Acid (oleic acid) accumulation. Error bars represent the standard error for three replicates. [0047] FIG.
  • FIG. 20 includes growth curves of SPV2076 lineage strains in a Biolector ® growth assay.
  • FIG. 20A shows data for the SPV1629 control, SPV2076, and SPV2135 in Solulys ® medium.
  • FIG.20B shows data for the SPV1629 control, SPV2076, and SPV2135 in FERM1 medium.
  • FIG.20C shows data for the SPV1629 control, SPV2135, and SPV2148 in Solulys ® medium.
  • FIG.20D shows data for the SPV1629 control, SPV2135, and SPV2148 in Solulys ® medium. Seed cultures were grown in YPD and inoculated to a starting OD of ⁇ 1. Error bars represent the standard error for three replicates.
  • FIG. 21 includes data showing the fatty acid profiles for SPV2076 lineage strains in a Biolector ® growth assay.
  • FIG.21A shows data for 70 h in spent media.
  • FIG.21B shows data for 70 h in fresh YPD media. Error bars represent the standard error for three replicates.
  • FIG. 22 includes data showing the fatty acid profiles for SPV2076 lineage strains in a 24-well bioconversion assay. Fatty acid profiles show all detected fatty acids except for the saturated 16Acid substrate. This was omitted for simplicity, because excess 16Acid remains in the culture. Error bars represent the standard error for four replicates.
  • FIG. 23 shows the full profiles as titers and percentage of total fatty acid.
  • FIG.23A shows results of a Biolector ® screen of select strains (initial cell density OD 600 ⁇ 0.2).
  • FIG.23B shows results of a panel screen of select Gen1 and Gen2 blend strains in a 24-well plate assay (initial cell density OD 600 ⁇ 15). The black line represents selectivity of 75%.
  • FIG. 24 includes data showing the fatty acid selectivities of SPV2076 lineage strains in a 24-well bioconversion assay.
  • FIG.24A shows the Z11/Z9-16 selectivity.
  • FIG.24B shows the Z11-16Acid total selectivity for SPV2076 and SPV2135 is higher than that of SPV1629 in the 24-well assay.
  • FIG.24A shows results of a Biolector ® screen of select strains (initial cell density OD 600 ⁇ 0.2).
  • FIG.23B shows results of a panel screen of select Gen1 and Gen2 blend strains in a 24-well plate assay (initial cell density
  • FIG. 24C shows that the Z11-16Acid selectivity excluding 16Acid is equivalent between SPV2135 and SPV1629. Error bars represented the standard error for four replicates.
  • FIG. 25 diagrams representative strategies to produce H. zea and C. suppressalis active pheromone blend compositions, as well as pure products, by Z-selective metathesis, distillation, and/or incorporation of Z9-16.
  • FIG. 26 includes an illustration of the fatty acid profile of strain SPV2028. SPV2028 was selected as the parent of the C. suppressalis blend production strain, because it produces a Z9-16/Z11-16 ratio of 0.15, which is close to a target range of 0.091-0.12. [0053] FIG.
  • FIG. 27 illustrates the fatty acid profiles of CoA elongation expression constructs.
  • FIG. 27A includes data showing the full fatty acid profiles for the elongation overexpression library.
  • FIG. 27B includes data showing the 18CoA derived fatty acids produced by each elongation overexpression strain.
  • FIG. 27C includes the composition of target AI precursors of each CoA elongation overexpression strain.
  • FIG. 28 includes data showing titers of Z9-derived fatty acids in strains containing native and recombinant desaturases. Elongase overexpression in SPV2165 increased production of Z9-18Acid.
  • FIG. 29 includes predictions made from desaturase binding pocket models and Z9-16/18 fatty acid titers from desaturases utilized in certain embodiments herein.
  • FIG. 29A diagrams a M. sexta binding pocket model: wild-type DST2 and DST3 exert mainly Z11- 16 activity and E/Z14 activity, respectively (Middle); swapping one residue on TM4 is sufficient to switch DST2 and DST3 activity towards weak E/Z14 activity and Z11-16 activity, respectively (Bottom).
  • FIG. 29A diagrams a M. sexta binding pocket model: wild-type DST2 and DST3 exert mainly Z11- 16 activity and E/Z14 activity, respectively (Middle); swapping one residue on TM4 is sufficient to switch DST2 and DST3 activity towards weak E/Z14 activity and Z11-16 activity, respectively (Bottom).
  • FIG. 29A diagrams a M. sexta binding pocket model: wild-type DST2 and DST3 exert mainly Z11-
  • FIG. 29B diagrams a binding pocket model for wild-type DST192, DST183, and DST245.
  • DST245 has smaller side chains at three positions of TM2 and bottom positions of TM4.
  • FIG.29C shows the Z9-derived fatty acid titer for wild-type DST183, DST192, and DST245.
  • DST245 shows higher selectivity for C18.
  • FIG.30 includes data showing Z9-derived fatty acid titers in DST192 mutant strains.
  • FIG. 30A shows fatty acid titers for single point mutants of DST192 in SPV2416.
  • FIG. 30B shows fatty acid titers for DST192 double mutants in the same background.
  • FIG. 31 includes data showing the fatty acid profiles of pPXA2-driven DST192 variants in SPV2588.
  • FIG.30A shows the fatty acid titers of 2x copy DST192 variant strains and the parent SPV2588 (1x DST183, 1x DST192).
  • FIG. 30B shows the fatty acid distribution of AI precursors, Z9-18Acid, and 18Acid in the same strains.
  • FIG. 30C shows 18Acid and Z9-derived fatty acids in the same strains.
  • FIG. 32 includes growth rate curves for several C. suppressalis blend production strains in Biolector ® reactors. Overall growth rates for 2x DST192 variant strains (SPV2665, SPV2667, SPV2668, SPV2669) are reduced compared with the strain SPV2165.
  • FIG. 33 includes diagrams of pathways from 16ME to Z9-14. Referring to FIG.33A, Pathway 1 shows conversion of Z9-14Acid substrate into the CoA cognate by POX activity of the endogenous acetyl-CoA synthetase.
  • Pathway 2 acetyl-CoA synthetase converts unsaturated Z11-16Acid to the CoA analog, which can be truncated to Z9-14CoA through POX activity.
  • Pathway 3 utilizes the cheap and commercially available saturated fatty acid 16ME (methyl palmitate). 16ME is first demethylated through lipase activity, then converted to the CoA cognate by acetyl-CoA synthetase. 16CoA is converted to Z11-16CoA through the activity of HzeaDST, and Z11-16CoA is then chain-shortened to Z9- 14CoA through POX activity.
  • Z9-14 is shown as the CoA for simplicity; however, Z9-14 can be converted to the free acid, the triacyl glyceride cognate, or as another ester in the cell.
  • Z9- 14 may be converted to Z9-14ME through chemical transesterification. Referring to FIG.33B, a Z9-14-specific desaturase that redirects flux toward the desired products (dotted line) was engineered. This pathway co-opts Z11-16ME production strains and thus allows the use of 16ME, a cheap and commercially available feedstock, to access Z9-14.
  • Engineering of POX activity produces a desired ratio of Z11-16CoA:Z9-14CoA. [0060] FIG.
  • FIG. 34 includes data showing the selectivity of DST192 mutants against 14CoA, 16CoA, and 18CoA substrates by monitoring Z9-14ME, Z9-16ME, and Z9-19ME formation. Fatty acyl CoAs were converted to methyl ester (ME) form during work-up. See FIGs.38-40 for chromatograms.
  • FIG. 35 includes homology models of DST192 mutants with 14CoA (built on PDB ID: 4ymk). WT (left), G100I (middle), and G100L (right) are each predicted to accommodate 14:0 substrate without steric hindrance. The GC-MS data (FIG.34) support these models and suggest that neither mutant alters 14:0 binding compared to WT.
  • FIG.36 includes homology models of DST192 mutants with 16CoA (built on PDB ID: 4ymk). G100I (middle) and G100L (right) cannot accommodate 16:0 substrate, which abuts the tunnel of the active site. The GC-FID data (FIG.34) support these models, which suggest that these mutants occlude the active site and prevent binding of 16CoA. Notably, the substrate does not buttress the active site tunnel in the WT model.
  • FIG.37 includes homology models of DST192 mutants with 18CoA (built on PDB ID: 4ymk). G100I (middle) and G100L (right) cannot accommodate 18:0 substrate, which abuts the tunnel of the active site.
  • FIG. 34 includes GC-FID chromatograms of DST192 WT, DST192 G100I, and DST192 G100L, against a Z9-14 authentic standard (yellow box). Wild type and both mutants produce Z9-14.
  • FIG. 39 includes GC-FID chromatograms of DST192 WT, DST192 G100I, and DST192 G100L, against a Z9-16 authentic standard (yellow box).
  • FIG. 40 includes GC-FID chromatograms of DST192 WT, DST192 G100I, and DST192 G100L, against a Z9-18 authentic standard (yellow box). Wild type produces high quantities of Z9-18, whereas the mutants produce lower levels of Z9-18.
  • FIG.41 includes homology models of DST192 mutants with 14CoA (built on PDB ID: 4ymk). The guanidino group of the G100R side chain (left) occludes the active site, preventing productive binding of 14CoA and longer substrates.
  • FIG. 42 includes the Z11-16Acid titer and selectivity correlation plot for strains expressing recombinant acyl transferases when cultured with methyl palmitate supplementation.
  • the scatter plot shows Z11-16Acid titer as a function of all other fatty acid products (excluding the 16ME substrate).
  • Replicates for all screened clones are shown as points with colors corresponding to each genetic modification. Strains with improved titer lie above the data points for the SPV1629 control, and strains with improved selectivity lie above and to the left of the control points.
  • FIG. 43 includes the Z11-16Acid titer and selectivity correlation plot for strains overexpressing native lipid metabolism genes when cultured with methyl palmitate supplementation.
  • the scatter plot shows Z11-16Acid titer as a function of all other fatty acid products (excluding the 16ME substrate).
  • Replicates for all screened clones are shown as points with colors corresponding to each genetic modification. Strains with improved selectivity lie above and to the left of the control points.
  • FIG. 44 includes data showing the GC-FAME analysis of fatty acid profiles for strains expressing recombinant acyltransferases when cultured with methyl palmitate substrate.
  • FIG. 44A shows average fatty acid profiles for all clonal replicates for each acyltransferase construct. Error bars represent the standard error from 4 replicates.
  • FIG.44B includes a stacked bar graph showing the average titers of each fatty acid. The total fatty acid titer is shown in the label above the bar.
  • FIG. 45 includes the Z11-16Acid titer and selectivity correlation plot for strains expressing recombinant acyltransferases when cultured without methyl palmitate supplementation.
  • the scatter plot shows Z11-16Acid titer as a function of all other fatty acid products (excluding the 16ME substrate).
  • Replicates for all screened clones are shown as points with colors corresponding to each genetic modification. Strains with improved titer lie above the data points for the SPV1629 control, and strains with improved selectivity lie above and to the left of the control points. Lines indicating 35-60% Z11-16 total selectivity (S Z11- 16_Tot) are also shown. Higher Z11-16Acid titers were observed with expression of DGATs.
  • FIG. 46 includes data showing the GC-FAME analysis of fatty acid profiles for strains expressing recombinant acyltransferases when cultured without methyl palmitate substrate.
  • FIG. 46A shows average fatty acid profiles for all clonal replicates for each acyltransferase construct. Error bars represent the standard error from 4 replicates.
  • FIG.46B shows a stacked bar graph of the average titers of each fatty acid. The total fatty acid titer is shown in the label above the bar.
  • FIG. 47 includes the Z11-16Acid titer and selectivity correlation plot for strains overexpressing native lipid metabolism genes when cultured without methyl palmitate supplementation.
  • the scatter plot shows Z11-16Acid titer as a function of all other fatty acid products (excluding the 16ME substrate). Replicates for all screened clones are shown as points with colors corresponding to each genetic modification. Strains with improved selectivity lie above and to the left of the control points. Lines indicating 35-60% Z11-16 total selectivity (SZ11-16_Tot) are also shown. Overexpressing native lipid metabolism genes tended to increase the synthesis of fatty acids, especially C18 fatty acids. Overexpression the mutant MGA2 activator domain increased Z11-16Acid titer and significantly increased C18 fatty acid titers. [0074] FIG.
  • FIG. 48 includes data showing the GC-FAME analysis of fatty acid profiles for strains overexpressing native lipid metabolism genes when cultured without methyl palmitate substrate.
  • FIG.48A shows the average fatty acid profiles for all clonal replicates for each construct. Error bars represent the standard error from 4 replicates.
  • FIG. 48B shows a stacked bar graph of the average titers of each fatty acid. The total fatty acid titer is shown in the label above the bar.
  • FIG.49 includes data showing the fatty acid profiles of select strains in a 1 L bioconversion process at 40 hours. Fatty acid profiles displayed increased Z11-16Acid titers for SPV2473 and SPV2474, and increased Z9-18Acid content in all Gen5 strains.
  • FIG. 50 includes Z11-16Acid and lipid content time course data for select strains in a 1 L bioconversion process.
  • FIG.50A shows that increased Z11-16Acid production was most pronounced in the earlier phase of the bioconversion for SPV2474 and was sustained across the bioconversion for SPV2473.
  • Z11-16Acid titer was normalized to the initial working volume to account for differences in volume changes over the course of the bioconversions.
  • FIG. 50B shows that the lipid fraction is higher for strains expressing multiple recombinant DGATs. Lipid accumulated rapidly in SPV2474, nearly reaching peak value by 35 hours.
  • FIG. 52 includes fatty acid titer time course data for strains in a 1 L bioconversion process. All titers were normalized to the initial working volume to account for volume differences.
  • FIG. 53 includes a graph of DST192G100L strains engineered to produce Spodoptera frugiperda precursors.
  • FIG. 54 includes Pan7-F1 and F2 bioconversion profile of DO, temperature, pH, OUR and feeds. The drop in OUR profile after 38h is due to the closure of the offgas valve once the foam probe detected foaming. [0081] FIG.
  • Pan7-F1 and F2 65 L initial volume
  • FERM137-7 0.9 L initial volume
  • bioconversion profile biomass, ChiSu components Z11- 16FAME/TAG, Z9-16 FAME/TAG, Z13 FAME/TAG titers, and byproducts Z9-18 FAME/TAG, Z11-18 FAME/TAG titer.
  • SEQUENCE LISTING [0001] The nucleic acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, as defined in 37 C.F.R. ⁇ 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.
  • SEQ ID NO:1 shows an exemplary ACX1 nucleotide sequence: ATGGAGGGCATCGACCACCTGGCCGATGAGCGGAATAAGGCCGAGTTCGACGTGG AGGATATGAAGATCGTGTGGGCCGGCAGCAGACACGCCTTTGAGGTGTCCGATAG GATCGCACGCCTGGTGGCATCCGACCCCGTGTTCGAGAAGTCCAATAGGGCCCGC CTGTCTCGGAAGGAGCTGTTCAAGTCTACCCTGAGAAAGTGCCCACGCCTTTAA GAGGATCATCGAGCTGCGCCTGAACGAGGAGGAGGCAGGCCGGCTGAGACACTTC ATCGACCAGCCTGCCTACGTGGATCTGCACTGGGGCATGTTTGTGCCAGCCATCAA GGGCCAGGGCACCGAGGAGCAGCAGAAGAAGTGGCTGAGCCTGGCCAATAAGAT GCAGATCATCGGCTGTTATGCCCAGACAGAGCTGGGACACGGATCCAACGTGCAG GGACTGGAGACCACA
  • SEQ ID NO:40 shows an exemplary DST148 nucleotide sequence: ATGGAGGCCAAGCAGAACAATCTGGCACCCACCCTGGAGGAGGAGGCACAGTTCG AGAAGCTGATCGCCCCTCAGGCCTCCGATCGCAAGCACGAGATCATCTACGCCAA CCTGATCACCTTCGCCTATGGCCACATCTCTGCCCTGTACGGCCTGTATCTGTGCTT TAGCTCCGCCAAGTGGGCCACAATCATCATCATGGCCTACGTGATCCTGATCGCAGCAG AAGTGGGAGTGACCGCAGGAGCCCACAGGCTGTGGACACACCGCGCCTATAAGGC CAAGCGGCCCCTGCAGATCATCCTGATGGTCATGAACTCCTTCGCCTTTCAGAATT CTGCCATCACATGGATCAGGGACCACCGCATGCACCACAGGTACTCTGACACCGA TGCCGACCCACACAACGCCACACGCGGCTTCTTTTATAGCCACATCGGATGGCTGC TGGTGCGGAAGCACCCAGAGGTGAAGCG
  • Embodiments herein utilize at least one reaction step in an apparatus, wherein the reaction step(s) may be a biosynthetic step catalyzed by one or more enzymes of a biocatalyst (e.g., a microorganism), a chemical synthetic step, or a combination of biosynthetic step(s) and chemical synthetic step(s).
  • a biocatalyst e.g., a microorganism
  • a chemical synthetic step e.g., a microorganism
  • Particular embodiments herein include an apparatus or method wherein multiple synthetic pathways (e.g., pathways catalyzed by different biocatalysts) are utilized in tandem (e.g., simultaneously, and non-simultaneously) to produce different fatty acid products or derivatives thereof, which fatty acid products or derivatives may be blended to produce a product blend.
  • the products of the one-pot synthesis may be blended with further fatty acids or derivatives thereof, isolated (e.g., via distillation), or otherwise subjected to downstream processing to produce a biologically active synthetic insect pheromone composition.
  • TERMS [0149] Organism: As used herein with regard to naturally occurring and genetically modified organism biocatalysts, the term “organism” includes cells, where the term “cell,” unless it is clear to a person in the art from its particular context, includes in this and other contexts cells in suspension, adhered cell cultures, and other cultures and multicellular forms that make the catalytic activity available to substrate in the reaction volume.
  • One-pot refers to a chemical synthesis or reaction whereby a reactant is subjected to successive chemical reactions a single reaction volume; e.g., the working volume of a reactor.
  • a one-pot synthesis provides advantages over conventional reactions that require separation or purification of intermediates or products during the reaction process.
  • the one-pot reactions described herein include “telescoping” reactions, whereby the synthesis is sequential, and reagents or other reaction components (e.g., genetically engineered organisms) are added during the synthesis, but without isolation and purification of the product(s) of a chemical reaction.
  • Organic substrate As used herein, the term “organic substrate” specifically includes sugars, glycerol, ethanol, organic acids, alkanes. Unless it is clear from its particular context, the term also includes fatty acids. Some embodiments herein utilize an organic substrate as a carbon source for the production of a saturated or unsaturated lipid moiety. In particular embodiments, the organic substrate is glucose.
  • Exogenous The term “exogenous,” as applied to polynucleotides and polypeptides herein, refers to one or more species that are not normally present within their specific environment or context.
  • exogenous also refers to a polynucleotides or polypeptides that are identical in sequence to a species already present in a host cell, but that is located in a different cellular or genomic context than the species with the same sequence already present in the host cell.
  • a polynucleotide that is integrated in the genome of a host cell in a different location than a polynucleotide with the same sequence is normally integrated in the genome of the host cell is exogenous to the host cell.
  • a polypeptide that is present in a subcellular compartment in a host cell is exogenous to the host cell when a polypeptide with the same sequence is only normally present in a different compartment of the host cell.
  • Heterologous The term “heterologous,” as applied to nucleic acids and polypeptides herein, means of different origin.
  • a host cell is transformed with a nucleic acid molecule that does not occur in the untransformed host cell in nature, then that nucleic acid molecule and polynucleotides therein are heterologous (and exogenous) to the host cell.
  • different elements e.g., promoters, enhancers, coding sequences, and terminators
  • Apparatuses and methods of embodiments herein may therefore include biocatalysts and biosynthetic methods for producing an insect pheromone or precursor thereof.
  • Such methods may comprise, for example, culturing a biocatalyst (e.g., a microorganism) as herein described, and feeding the culture with a saturated or unsaturated substrate.
  • the method further comprises isolating an insect pheromone or precursor thereof produced from the substrate.
  • the method may comprise isolating the pheromone or precursor via distillation.
  • isolating the pheromone or precursor may comprise membrane-based separation.
  • a pheromone precursor is isolated, and is then converted into an active pheromone via chemical methods.
  • a method for producing an insect pheromone or precursor thereof does not utilize significant amounts of organic solvents, proceeds in one step, and results in high yield of a particular product isomer, providing a significant improvement upon conventional production methods.
  • microorganisms comprising at least one component of a biosynthesis pathway to produce one or more product unsaturated lipid moiety isomers.
  • a biocatalyst may be used to catalyze, for example, any combination of the following reactions, in one or more steps: synthesis of a product unsaturated lipid moiety isomer and synthesis of an intermediate selected from among a saturated lipid moiety, an unsaturated lipid moiety isomer, a fatty alcohol, a fatty aldehyde, a FFA, a FAE (e.g., a FAME), a fatty alcohol, a fatty aldehyde, or and a fatty acetate.
  • the product unsaturated lipid moiety isomer is a stereospecific and regiospecific isomer with a specific chain length.
  • an intermediate synthesized with the use of a biocatalyst herein typically is produced in a stereospecific and/or regiospecific reaction utilizing one or more enzymes of the biocatalyst.
  • the chain length of a saturated lipid moiety or stereospecific and regiospecific unsaturated lipid moiety isomer is modified by one or more enzymes of the biocatalyst.
  • the biocatalyst is a microorganism comprising at least one component of a biosynthesis pathway to produce one or more product unsaturated lipid moiety isomers.
  • the microorganism is a bacterium, yeast, or a cultured cell (e.g., a plant cell or insect cell in cell culture).
  • the microorganism is a naturally occurring organism.
  • the microorganism is a genetically engineered microorganism comprising at least one exogenous polynucleotide encoding a biosynthetic enzyme that catalyzes at least one step in the biosynthesis.
  • the biocatalyst may be a genetically engineered yeast, bacterium, insect cell, or plant cell.
  • the biocatalyst is a genetically engineered yeast selected from the group consisting of Saccharomyces, Scizosacchoromyces pombe, Pichia pastoris, Hansanula polymorpha, Yarrowia lipolytica, Candida albicans, Candida tropicalis, and Candida viswanathii.
  • the biocatalyst is a genetically engineered insect cell of the genus Amyelois (e.g., A. transitella). The biocatalyst is preferably suitable for large-scale culture in a bioreactor, for example, as shown in FIG.1.
  • a biocatalyst comprises at least one exogenous (e.g., heterologous) biosynthetic enzyme selected from the group consisting of desaturases (e.g., fatty acyl-CoA desaturases, and fatty acyl-ACP desaturases), acyl-CoA oxidases, acylglycerol lipases, flavoprotein pyridine nucleotide cytochrome reductases (e.g., cytochrome-b5 reductases, and NADPH-dependent cytochrome P450 reductases), elongases (e.g., ELO1, ELO2, and ELO3), thioesterases (e.g., acyl-ACP thioesterases, and acyl-CoA thioesterases), glycerol-3-phosphate acyltransferases (e.g., dual glycerol-3-phosphate O- acyl
  • desaturases e.
  • the biosynthetic enzyme(s) is native to an organism from a genus selected from the group consisting of Saccharomyces (e.g., S. cerevisiae), Yarrowia (e.g., Y. lipolytica), Candida (e.g., C. albicans, C. tropicalis, and C. viswanathii), Helicoverpa (e.g., H. armigera, and H. zea), Thalassiosira, (e.g., T. pseudonana), Agrotis (e.g., A. segetum), Trichoplusia (e.g., T. ni), Spodoptera (e.g., S.
  • Saccharomyces e.g., S. cerevisiae
  • Yarrowia e.g., Y. lipolytica
  • Candida e.g., C. albicans, C. tropicalis, and C. viswanathii
  • Ostrinia e.g., O. scapulalis, O. fumacalis, O. nubilalis, O. latipennis, and O. ovalipennis
  • Amyelois e.g., A. transitella
  • Lobesia e.g., L. botrana
  • Cydia e.g., C. pomonella
  • Grapholita e.g., G. molesta
  • Lampronia e.g., L. capitella
  • Sesamia e.g., S. inferens
  • Plodia e.g., P. interpunctella
  • Bombyx e.g., B.
  • a biocatalyst comprises a heterologous desaturase capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.
  • the desaturase may be capable of utilizing a C16 or C18 fatty acyl-CoA as a substrate.
  • a biocatalyst may comprise a desaturase that generates a double bond at position C5, C6, C7, C8, C9, C10, C11, C12, or C13 in the fatty acid or its derivatives, such as, for example, fatty acid CoA esters.
  • the desaturase may generate a double bond at position C9, C11, or C13.
  • the desaturase generates the Z isomer of an unsaturated fatty acid or fatty acid derivative.
  • the desaturase is at least 90% identical to SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, or SEQ ID NO:47 (e.g., SEQ ID NOs:49, 51, 53, 55, and 57).
  • the desaturase may be at least 95% or at least 98% identical to SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, or SEQ ID NO:47.
  • a biocatalyst comprises a heterologous Z11 desaturase that catalyzes the conversion of a fatty acyl-CoA into a monounsaturated or polyunsaturated product selected from Z11-13:Acyl-CoA, (Z,Z)-7,11-13:Acyl-CoA, Z11-14:Acyl-CoA, (E,Z)- 9,11-14:Acyl-CoA, (Z,Z)-9,11-14:Acyl-CoA, (E,Z)-9,11-15:Acyl-CoA, (Z,Z)-9,11-15:Acyl- CoA, Z11-16:Acyl-CoA, (E,Z)-6,11-16:Acyl-CoA, (E,Z)-7,11-16:Acyl-CoA, (E,Z)-8,11- 16:Acyl-CoA, (E,Z)-9,11-16
  • a biocatalyst comprises a heterologous Z9 desaturase that catalyzes the conversion of a fatty acyl-CoA into a monounsaturated or polyunsaturated product selected from Z9-11:Acyl-CoA, Z9-12:Acyl-CoA, (E,Z)-7,9-12:Acyl-CoA, (Z,Z)-7,9- 12:Acyl-CoA, Z9-13:Acyl-CoA, (E,Z)-5,9-13:Acyl-CoA, (Z,Z)-5,9-13:Acyl-CoA, Z9- 14:Acyl-CoA, (E,Z)-4,9-14:Acyl-CoA, (Z,E)-9,11-14:Acyl-CoA, (Z,Z)-9,11-14:Acyl-CoA, (Z,E)-9,12-14:Acyl
  • a biocatalyst comprises an exogenous acyl-CoA oxidase that catalyzes the conversion of a monounsaturated or polyunsaturated C6-C24 fatty acyl-CoA into a truncated monounsaturated or polyunsaturated fatty acyl-CoA after one or more successive cycle of acyl-CoA oxidase activity, with a given cycle producing a monounsaturated or polyunsaturated C4-C22 fatty acyl-CoA intermediate with a two carbon truncation relative to a starting monounsaturated or polyunsaturated C6-C24 fatty acyl-CoA substrate in that cycle.
  • the acyl-CoA oxidase is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.
  • the acyl-CoA oxidase may be at least 95% or at least 98% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.
  • a biocatalyst comprises at least one heterologous glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferases (LPAAT), glycerolphospholipid acyltransferase (GPLAT), or diacylglycerol acyltransferases (DGAT).
  • GPAT heterologous glycerol-3-phosphate acyltransferase
  • LPAAT lysophosphatidic acid acyltransferases
  • GPLAT glycerolphospholipid acyltransferase
  • DGAT diacylglycerol acyltransferases
  • the biocatalyst comprises a heterologous polypeptide that is at least 90% identical to a DGAT1 (e.g., SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:34) or an LPAAT (e.g., SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:32).
  • DGAT1 e.g., SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:32
  • LPAAT e.g., SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:32
  • the heterologous polypeptide may be at least 95% or at least 98% identical to SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:22, SEQ ID NO:24,
  • a biocatalyst comprises at least one exogenous enzyme that extends the length of a fatty acid or fatty acid derivative.
  • Two different types of fatty acid elongation takes place in different organisms. These elongation pathways use Coenzyme-A as acyl carrier rather than the acyl carrier protein (ACP) of fatty acid synthesis systems FASI and FASII.
  • the first type, mitochondrial fatty acid elongation is the reversal of fatty acid oxidation. This utilizes acetyl-CoA as a substrate and extends the chain length of fatty acids with two carbons. This process acts mainly on acyl-CoA shorter than C16.
  • the second process is the elongation pathway of the endoplasmic reticulum, which is present in plants, mammals, yeast, and other lower eukaryotes.
  • This is a four-step reaction, where the steps are catalyzed by individual enzymes; beta-ketoacyl-CoA synthase, beta-ketoacyl-CoA reductase, beta-hydroxyacyl-CoA dehydratase, and trans-2-enoyl-CoA reductase.
  • This pathway mainly acts with acyl-CoA of chain length C16 or larger, and it is important in the generation of very long chain fatty acids.
  • This process utilizes malonyl-CoA rather than acetyl-CoA for chain elongation.
  • the first enzyme which leads to condensation of malonyl-CoA with acyl-CoA is also called elongase.
  • elongase The first enzyme which leads to condensation of malonyl-CoA with acyl-CoA (beta-ketoacyl-CoA synthase) is also called elongase.
  • elongase Several variants of elongase exist, depending upon the host organism. In yeast, three different genes each encode an elongase (ELO1, ELO2 (FEN1), and ELO3 (FEN12)), and they have different substrate specificities.
  • ELO1 prefers shorter saturated fatty acids (C14-C16)
  • ELO2 prefers longer saturated and monounsaturated fatty acids
  • ELO3 prefers monounsaturated and polyunsaturated fatty acids.
  • the biocatalyst comprises an exogenous elongase that is at least 90% identical to an ELO1, ELO2 (e.g., SEQ ID NO:30), or ELO3.
  • the exogenous elongase may be at least 95% or at least 98% identical to SEQ ID NO:30.
  • a biocatalyst comprises at least one exogenous lipid regulator.
  • the biocatalyst comprises a lipid regulator that is at least 90% identical to a truncated transcriptional activator fragment (e.g., MGA2act (e.g., SEQ ID NO:59 and SEQ ID NO:61)), a cytochrome B5 reductase (e.g., SEQ ID NO:63), a heme-responsive zinc finger transcription factor (e.g., SEQ ID NO:65), or a diacylglycerol phosphate phosphatase (e.g., SEQ ID NO:67).
  • MGA2act e.g., SEQ ID NO:59 and SEQ ID NO:61
  • a cytochrome B5 reductase e.g., SEQ ID NO:63
  • a heme-responsive zinc finger transcription factor e.g., SEQ ID NO:65
  • a diacylglycerol phosphate phosphatase e.g., SEQ ID NO:67
  • the exogenous lipid regulator may be at least 95% or at least 98% identical to SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, or SEQ ID NO:67.
  • Fatty acid products and derivatives thereof produced by the foregoing biocatalysts and biosynthetic methods are modified by non-biological means such as chemical synthesis in the one-pot synthesis in some embodiments herein to yield final products.
  • Further details regarding microorganisms suitable for use as biocatalysts in embodiments herein, and regarding methods for utilizing such microorganisms to produce chemicals in a biosynthetic reaction, may be found in PCT International Patent Publication No.
  • Apparatuses and methods of embodiments herein may also include chemical synthetic methods.
  • Chemical synthesis is the preparation of a compound by performing various chemical reactions to change the molecular structure of a starting material by reactions with other chemicals.
  • the starting materials for a chemical synthesis may be simple compounds removed from oil and natural gas, or more complex chemicals isolated in from biological sources or biosynthetic fermentation reactions.
  • the one-pot synthesis includes the chemical synthesis of at least one free fatty acid (FFA), fatty acid alkyl ester (FAAE), fatty alcohol, or fatty aldehyde from an unsaturated lipid (e.g., monounsaturated fatty acids, polyunsaturated fatty acids, branched unsaturated fatty acids, unsaturated triacylglycerol, and unsaturated triglyceride).
  • unsaturated lipid e.g., monounsaturated fatty acids, polyunsaturated fatty acids, branched unsaturated fatty acids, unsaturated triacylglycerol, and unsaturated triglyceride.
  • unsaturated lipid e.g., monounsaturated fatty acids, polyunsaturated fatty acids, branched unsaturated fatty acids, unsaturated triacylglycerol, and unsaturated triglyceride.
  • unsaturated lipid is produced by a biocatalyst
  • an unsaturated lipid, FFA and/or FAAE is reduced to a fatty alcohol and/or a fatty aldehyde by contacting the unsaturated lipid, FFA, and/or FAAE with at least one stoichiometric reducing agent (e.g., sodium bis(2-methoxyethoxy)aluminum hydride (VitrideTM, Red-Al, SMEAH), and diisobutylaluminum hydride (DIBAL)), and/or at least one transition metal catalyst (e.g., a group VIII catalyst).
  • at least one stoichiometric reducing agent e.g., sodium bis(2-methoxyethoxy)aluminum hydride (VitrideTM, Red-Al, SMEAH), and diisobutylaluminum hydride (DIBAL)
  • at least one transition metal catalyst e.g., a group VIII catalyst
  • a bulky cyclic nitrogen Lewis base is used to modify the reducing agent to allow for selectivity of the reducing step to produce one or more fatty aldehydes.
  • a fatty alcohol is oxidized to a fatty aldehyde by an oxidation step, which oxidation step may be, for example, a partial oxidation method such as NaOCl/TEMPO, TEMPO-bleach, TEMPO-PhI(OAc) 2 , TEMPO-copper-air, copper-catalyzed aerobic oxidation, Swern oxidation, and noble metal-air oxidation.
  • a fatty alcohol may be converted into a fatty acetate using chemical methods, e.g., via chemical catalysis utilizing a chemical agent; for example, acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride, and propionic anhydride.
  • chemical methods e.g., via chemical catalysis utilizing a chemical agent; for example, acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride, and propionic anhydride.
  • Z11 desaturase from Helicoverpa zea was identified as particularly active on a 16Acid substrate from among a library of Z11 specific insect desaturases (data not shown).
  • Gen1 strain SPV458 H. zea desaturase integrated at XPR2 locus
  • FIG.2. 22 plasmid constructs were integrated into SPV739, the marker rescued descendant of SPV458 to create Gen2 strains.
  • FIG.2A Strains harboring multiple copies of the H. zea desaturase produced increased Z11-16Acid titers.
  • Gen2 strains SPV968 and SPV969, were marker rescued, and additional DNA cassettes were integrated to create a small library of Gen3 strains with 2 or 3 copies of Z11 desaturase, and deletions of key lipid metabolism genes.
  • FIG.2A Increasing desaturase copy number was found to increase both Z11-16Acid productivity and selectivity. Data from Gen1 to Gen3 strains indicated that total fatty acid content made up 50-60% of dry cell weight. Further increases in productivity may be made by expressing recombinant acyltransferases.
  • Integrating acyltransferases was found to further increase Z11-16Acid titer and selectivity.
  • Both Z9-16Ald and Z11-16Ald are recognized as active ingredients in the pheromone for Helicoverpa zea and H. armigera.
  • Z11-16Acid/Z9-16Acid synthetic pheromone for Helicoverpa it is desired to produce Z11-16Acid at ⁇ 97% of the unsaturated C16 fatty acid composition from the microbial bioprocess, for example, so that a blend of 97% Z11-16Acid, 3% Z9-16Acid can be synthesized, or a small amount of metathesis- derived Z9-16Ald can be doped into the final mixture.
  • FIG.2A the Gen4 strain expressing P. dactylifera DGAT1A (SPV1629) was selected for further engineering (96% Z11- 16Acid/Z9-16Acid selectivity at 1 L scale).
  • FIG.2A Two distinct strategies were employed to achieve this goal. First, the native Z9 desaturase encoded by the OLE1 gene was replaced with recombinant desaturases that could either reduce Z9-16 desaturase activity while maintaining Z9-18 desaturase activity or replace Z9 desaturase activity completely with Z11-18 desaturase activity (Example 1.1; FIG. 2B). Second, incremental gains in selectivity were made by increasing the H. zea desaturase copy number to four and combined expression of both P.
  • Example 1.1 Replacing OLE1 with Alternate Z9-18CoA and Z11-18CoA Desaturases [0176] The native Z9 desaturase (OLE1) was replaced with alternative Z9-18CoA and Z11-18CoA desaturases, improving Z11-16Acid/Z9-16Acid selectivity. A Y.
  • lipoltyica strain devoid of Z9 desaturase activity was engineered that can be used for a Z11-18Acid/Z13- 18Acid blend strain to produce the Z11-18Ald/Z13-18Ald pheromone blend of the rice pest Cnaphalocrocis medinalis.
  • Three lepidopteran desaturases and one fungal desaturase were integrated into the marker rescued descendant of SPV1994 (xpr2::HzDST, fat1::HzDST, fao1::HzDST, axp::DGAT1A_Pd, and pox5::HzDST) at the OLE1 locus.
  • the resulting strains had a deletion of OLE1, and the expression of the recombinant desaturase was driven by either the native OLE1 promoter or the TEF promoter.
  • Table 1. [0178] Table 1. OLE1 knockout desaturase constructs. Desaturase Source species Activity Promoter [0179] The resulting strains were screened in both a 24-well plate bioconversion assay using methyl palmitate as substrate, and a 48-well Biolector ® assay to assess growth rate. The fatty acid composition of all cultures was measured using GC-FAME analysis.
  • the lipid profiles were devoid of detectable Z9 unsaturated species, resulting in a Z11-16Acid/Z9- 16Acid selectivity of 100%.
  • Z11-16Acid titers were 1.36 ⁇ 0.12 g/L with Z11-18Acid titers of 1.62 ⁇ 0.14 g/L.
  • a C18 diene was produced at 0.30 ⁇ 0.02 g/L. Growth was significantly reduced (29% of SPV1629 growth rate in FERM1, and undetected in Solulys ® -based medium). [0182] Table 2. Performance of OLE1 knockout strains.
  • Each desaturase was subcloned from an existing expression vector into a plasmid that targets the OLE1 locus.
  • This OLE1 construct uses the native OLE1 promoter and terminator sequences as homology arms, so that upon integration the entire coding sequence of the native Z9 desaturase is removed.
  • the recombinant Z9 desaturases were cloned with two different expression approaches. In the first approach, the coding sequence of the recombinant Z9 desaturase was cloned directly downstream of the native OLE1 promoter sequence, which also served as the homology arm; the resulting constructs used the native OLE1 promoter to drive expression of the desaturase.
  • the second strategy added a TEF promoter sequence downstream of the OLE1 promoter/homology arm sequence; this second set of constructs used the TEF promoter to drive expression.
  • Positive integrants were screened in a 24-well plate assay in a semi-defined medium used previously for 24-well plate and Biolector ® assays.
  • Fatty Acid Profiling of OLE1 Replacement Strains [0187] Relative to the control, SPV2073 (pTEF-DST301, R. irregularis) produced less Z9-16Acid (60%), statistically equivalent Z11-16Acid, more 18Acid (130%), and similar unsaturated C18 fatty acid titers.
  • the Z9-16Acid titers 10 mg/L, were near the detection limit of the assay. Interestingly, 18Acid and Z11-18Acid were increased by ⁇ 5-fold and ⁇ 3.5-fold, respectively.
  • the Z11-16Acid/Z9-16Acid selectivity for SPV2074 was >99%.
  • FIG. 3A The dominant fatty acid products were Z11- 16Acid (1.2-1.6 g/L) and Z11-18Acid (1.4-1.9 g/L).
  • FIG.3B Relative titers were consistent between the two isolates; absolute titers correlated with final cell density measurements. Final cell densities were 25% (SPV2075) and 40% (SPV2076) lower than the SPV1994 control.
  • OLE1 Replacement Strains [0190] Growth rates for OLE1 replacement strains were independently measured through online cell density monitoring using a Biolector ® . Growth rates were compared to both the current production strain SPV1629 and the direct parent SPV1994 in two different media; the small-scale bioconversion medium FERM1, and a large-scale bioprocess medium using an initial batch phase with glycerol as the primary carbon source along with corn-steep liquor (Solulys ® 95) to supply both nitrogen and vitamins. Growth rates for all strains and conditions are shown in FIG. 6. In general, a greater diversity of growth rates was observed in the bioprocess medium.
  • the XPR2 construct was used as template for desaturase coding sequence subcloning into OLE1 locus integration vector pPV235. Desaturases were cloned to either remove the TEF promoter from the construct, resulting in integration directly downstream of the native OLE1 promoter, or cloned with the intact TEF promoter for integration at the same OLE1 insertion site. The constructs simultaneously remove the native OLE1 gene. The resulting constructs as listed in Table 3. [0194] Table 3. Plasmid Descriptions.
  • lipolytica strains were inoculated from YPD agar patches and grown in 1 mL YPD seed cultures for 24 hours at 28 °C and 1000 rpm (InforsTM plate incubator) to an OD600 ⁇ 15-20. Seed cultures were pelleted by centrifugation at 800 xg for 5 min. Supernatant was removed by pipetting before 1 mL bioconversion medium (FERM1) was added. Cells were resuspended before returning to the plate incubator (28 °C, 1000 rpm) for six hours. After the six-hour incubation, 25 ⁇ L warmed methyl palmitate was added to each vial. Cultures were then incubated under the same conditions for an additional 66 hours before sampling.
  • vials were transferred to 108-well aluminum racks, and placed in a convection oven for 40 minutes at 60 °C. After heating, the vials were decapped, and 29 ⁇ L 24N sulfuric acid was added. Vials were then re-sealed, arrayed in a V54 rack, and mixed again for 2 minutes at 2000 rpm using a MixMate ® plate shaker. Following this mixing, the vials were transferred to 108-well aluminum racks, and again placed in a convection oven for 40 minutes at 60 °C. After this heating, the vials were decapped, and 1 mL n-hexane was added.
  • Example 1.2 Expression of DGAT1A and LPAAT with Increased HzDST Copy Number
  • Increases in Z11-16Acid fraction with additional copies of the Z11 desaturase were observed; the addition of a second copy produced an increase in Z11/Z9-16Acid selectivity from 83% to 96%, and a third copy increased the Z11/Z9-16Acid selectivity to 97%. P.
  • the second strategy was to combine expression of the P. dactylifera DGAT1A and LPAAT. Expression of the LPAAT alone had led to a small increase in the Z11/Z9-16Acid selectivity, while also reducing the C18 acid content of the strain.
  • FIG. 10A The observed increase in Z11-16Acid titer and decrease in Z9-16Acid titer resulted in a Z11-16Acid/Z9-16Acid selectivity increase from 97.4% in SPV1629 to 98.4% in SPV1995.
  • FIG. 10A The reduction in all C18 fatty acid titers led to increases in C16 selectivity (SC16), Z11-16Acid/products selectivity (SZ11-16_prod), and Z11- 16Acid total selectivity (S Z11-16_tot ).
  • FIG. 10A-C 16Acid titers were statistically equivalent for both strains.
  • SPV1994 (4-copy HzDST) and SPV1995 (P. dactylifera LPAAT and DGAT1A) were rescreened against an SPV1629 control in an independent experiment using the same small-scale assay.
  • Selectivity metrics and Z11-16Acid titer from SPV1629 showed that SPV1629 produces a profile with S Z11/Z9-16 ⁇ 96.5-97.5%, S C16 ⁇ 80%, S Z11-16_prod ⁇ 70%, SZ11-16_tot ⁇ 50%.
  • FIG. 11A The rank order of strains determined by S Z11/Z9-16 was maintained, with SPV1994 producing the highest selectivity at 97.7%. Similar consistency was observed for C16 selectivity (SC16), Z11-16Acid/products selectivity (SZ11-16_prod), and Z11-16Acid total selectivity (SZ11-16_tot), with the highest selectivities observed for SPV1995 at 85%, 76%, and 54%, respectively.
  • FIG.11B-D [0219] Table 6. Two independent replicates showing P.
  • SPV1994 and SPV1995 Performance were constructed based on previous observations on the effect of additional H. zea Z11 desaturase copy-number and the expression of P. dactylifera acyl transferases. By examining the performance of all SPV458 lineage strains (FIG. 12B), the improved performance of SPV1994 and SPV1995 was confirmed. [0222] When a second copy of the H.
  • FIG.12B Titers of Z9-16Acid were also lower in the LPAAT-expressing strain when compared to the DGAT expressing strain (SPV1629).
  • FIG.13 The result was a higher S Z11/Z9-16 at nearly 98%.
  • FIG. 12C [0224] Based on this evidence, both addition of a fourth copy of the H. zea desaturase and combining P. dactylifera DGAT1A and LPAAT expression were predicted to result in increases in S Z11/Z9-16 .
  • FIG.12 The increase in SZ11/Z9-16 resulted from both the increase in Z11-16Acid titer and the reduction in Z9-16Acid titer relative to SPV1629.
  • FIG.13 As described above, the C18 fatty acid content was also reduced, and the resulting Z11-16Acid/Z9-18Acid ratio was between that of SPV1629 (DGAT1A only) and SPV1796 (LPAAT only).
  • E. coli EPI400 E. coli EPI400 strains were grown overnight in LB at 37 oC and 250 rpm to an OD600 ⁇ 2. Cells were back-diluted 10x into LB with copy-cutter induction solution and grown 24 hours at 37 oC and 250 rpm before miniprepping.
  • Strain Construction Strain construction, including plasmid digestion, transformation, check PCR, and colony patching was performed essentially as described in Example 1.1.
  • Biolector ® Assay Y. lipolytica strains were inoculated from YPD agar patches and grown in 2 mL YPD seed cultures for 16 hours at 28 oC and 1000 rpm (InforsTM plate incubator) to an OD600 ⁇ 15-20. Seed cultures were pelleted and concentrated to OD600 ⁇ 50 in fresh YPD. A 1.5-2.5% inoculum was used to seed 700 ⁇ L FERM1 medium in a M2Plabs ® 48-well flower plate.
  • Example 2 Z11-16 / Z9-16 Blend Production [0233] To engineer a strain that targets a Z11-16Acid, Z9-16Acid blend that matches a biologically active ratio in S.
  • incertulas (70%-75% Z11-16), expression of a variety of Z9-16 desaturases were combined with expression of the H. zea Z11 desaturase to produce strains that produce a variety of Z11/Z9 ratios.
  • the selection of Z9 desaturases was based on results of a screen of all publicly available lepidopteran desaturases (data not shown).
  • the resulting strains produced a Z9-16Acid/Z11-16Acid blend as a precursor to Scirpophaga incertulas synthetic pheromone (Z9-16Ald/Z11-16Ald) at performances suitable for commercial scale deployment.
  • Combinations of insect Z9-16Acid desaturases were integrated into the marker rescued descendant of a Z11-16Acid production strain (SPV1629: xpr2::HzDST, fat1::HzDST, fao1::HzDST, axp::DGAT1A_Pd) that was validated at 150L-scale to modify its profile of 97% Z11-16Acid, 3% Z9-16Acid.
  • Z9 desaturases were integrated at either the previously deleted POX5 locus, or replaced an existing copy of the H. zea Z11 desaturase at the XPR2 locus.
  • Nine Z9 desaturases were selected based on varying activity observed from screening 300 Lepidopteran desaturases in Y.
  • Desaturases comprising the Z9 DST motif are listed in Table 7 below. The activity level indicated is based on the increase in Z9-16Acid titer over the control strain in the desaturase screen. [0235] Table 7. Desaturases used to develop blend strains. The listed desaturases all contain the Lepidopteran Z9 DST motif (SEQ ID NO:39), a structural region within lepidopteran desaturases that correlates with enzyme activities. *Activity levels represent the g/L increase in Z9-16Acid titer over the control strain in the desaturase library screen.
  • the first set replaced one of the three copies of the H. zea Z11 desaturase (XPR2 locus) with a single Z9-16 desaturase selected from each of the three activity level groups shown in Table 7 (DST181, DST183, and DST192 (SEQ ID NO:53)).
  • the second set added a single Z9-16 desaturase at the POX5 locus, producing strains with two copies of the H. zea Z11 desaturase and one copy of a Z9-16 desaturase. All nine desaturases shown in Table 7 were transformed for this set.
  • Gen2 strains were screened in a Biolector ® assay (See Materials & Methods) with the best Gen1 candidates, SPV2044 and SPV2045 (2xHzDST, 1xDST192).
  • the Biolector ® assay was modified by reducing the initial seeding density and adding substrate later (18h) as a single bolus.
  • FIG. 15 A second 24-well plate assay was completed on a select panel of the best Gen1 and Gen2 strains to test the sensitivity of Z11-16Acid/Z9-16Acid selectivity to high seeding density, and alternative vessel format (See Materials & Methods). Under the 24-well plate assay format, the panel of strains produced Z11-16Acid/Z9-16Acid selectivities from 53%- 87%, while the control, SPV1629, had a selectivity of 96%. FIG. 17A.
  • strains displayed lower Z11-16Acid/Z9-16Acid selectivities (more Z9-16Acid) in this assay.
  • Unsaturated C16 selectivity (SC16) for SPV1629 was 14% lower than in the Biolector ® assay and was nearly identical for all strains.
  • FIG. 17B These 24-well plate results support the hypothesis that, in addition to strain modifications, changes to growth/seeding conditions can be used to adjust the selectivities of blend strains.
  • Strain Growth Rates [0246] Final cell densities in all bioconversion assays and qualitative observations of phenotype suggested that blend strains were healthy and grew as well as the parent, SPV1629.
  • FIG.18 Two different media were tested; FERM1 (small-scale assay medium that primarily uses glucose, ammonium sulfate, yeast nitrogen base, and yeast extract to supply carbon, nitrogen, and vitamins), and Solulys ® (batch version of a bioprocess medium that uses glycerol, ammonium sulfate, and Solulys ® 95 to supply carbon, nitrogen and vitamins). Growth rates for all strains in both media were within 10% of the control SPV1629.
  • Biolector ® bioconversion assays The Biolector ® bioconversion assays with Gen1 and Gen2 strains were performed essentially as described in Example 1.2. For assays including Gen1 strains, 8.4 ⁇ L liquid 37 °C methyl palmitate was added to each well after growing for 7 hours, and again after growing for 23 hours. For assays including Gen2 strains, 21 ⁇ L liquid 37 °C methyl palmitate was added to each well after growing for 18 hours. [0259] Biolector ® growth rate assay: The Biolector ® growth rate assay was performed essentially as described in Example 1.1.
  • GC processing of lyophilized samples [0261] The Biolector ® growth rate assay was performed essentially as described in Example 1.1. Samples were run on the GC in a one-pot format using the parameters (for both front and back detectors) listed in Table 4 of Example 1.1, above. The GC needle height was adjusted from 3.0 mm to 14.5 mm, as the sample is drawn from the very top of vials when run in “one-pot” format.
  • Example 3 Z9-16 / Z11-16 / Z13-18 Blend Production [0262] To engineer a one-pot synthesis for a Z9-16, Z11-16, and Z13-18 blend, such as matches a biologically active ratio in C.
  • Example 3.1 Improved Production of Z11-16
  • SPV2076 pTEF-DST148, B. mori Z11 desaturase
  • OLE1 native Z9-18/16 desaturase
  • SPV2076 is selective for Z11-16Acid and Z11-18Acid, and its growth phenotype could be improved for use as a Z11 selective production strain.
  • Two parallel approaches were used to improve the growth phenotype of SPV2076, based on the hypothesis that the absence of Z9-18CoA (oleoyl-CoA) dysregulates lipid metabolism: First, the growth of SPV2076 with methyl oleate (Z9-18ME) supplementation was improved.
  • SPV2076 was propagated in rich media (YPD) before plating the resulting population and isolating colonies with increased growth rate (colony size). SPV2076 culture was randomly mutagenized using ethyl methanesulfonate (EMS), and faster growing isolates were identified by plating. Growth rates of resulting strains were measured in two different semi-defined media with online monitoring in a Biolector ® assay.
  • YPD rich media
  • EMS ethyl methanesulfonate
  • Bioconversion phenotypes were assayed in a 24-well plate assay with methyl-palmitate (16ME) supplementation. The fatty acid composition of all cultures was measured using GC-FAME analysis.
  • Strains were evaluated, relative to SPV1629, with the following four metrics: (1) Exponential and linear phase growth rates; (2) Z11-16Acid/Z9-16Acid selectivity: (3) Z11-16Acid total selectivity: ; and (4) Z11-16Acid/products selectivity: [0267] Methyl Oleate Supplementation [0268] It was hypothesized that the absence of oleoyl-CoA (Z9-18CoA) in SPV2076 led dysregulated lipid metabolism and created a slower growth rate than parent strains still containing the Ole1p Z9 desaturase.
  • FIG.19A Supplementation of either 0.9 or 4.4 g/L methyl oleate led to an increase in growth rate and final cell density by 90 hours.
  • FIG. 19A While the exponential growth phase was slower than for SPV1629, the following linear growth phase was similar.
  • SPV2076 was treated with ethyl methanesulfonate (EMS) to increase the natural mutation rate (See Materials & Methods).
  • EMS treatment led to the isolation of five fast growing colonies, from which strain SPV2148 was selected.
  • a Biolector ® growth rate assay with two different media was used to evaluate SPV2076, SPV2135 (growth selection), SPV2148 (EMS treatment), and strain SPV1629 (See Materials & Methods).
  • the first medium, FERM1 uses glucose, glycerol, ammonium sulfate, yeast extract, and YNB as major components.
  • the second medium here referred to as Solulys ® , mimics a batch phase medium.
  • FIG. 21 Cultures were incubated for 70 hours before sampling to profile fatty acid content, and SPV2135 produced >4 g/L of Z11-16Acid without the addition of methyl palmitate. These results showed that growth rate could be improved, and that SPV2135 displayed an increased lipid capacity relative to other strains.
  • Derivative Strain Methyl Palmitate Bioconversion [0276] After identifying strains with improved growth rate, bioconversion performance was tested using a standard 24-well bioconversion assay (See Materials & Methods).
  • SPV2135 continued to display an improved lipid accumulation phenotype with >10 g/L of Z11-16Acid produced.
  • FIG. 22 This titer was 50% greater than that observed with SPV1629.
  • Cell densities measured by optical density were marginally lower for SPV2135 compared to SPV1629.
  • the SPV2076 control also produced lower fatty acid titers and lower final cell density.
  • SPV2148 produced significantly less Z11-16Acid, while producing more Z11-18Acid than SPV2148.
  • Selectivity metrics were equivalent or improved for SPV2135; Z11/Z9-16 selectivity was nearly 1.0 for SPV2135.
  • FIG.24A Total Z11-16 selectivity was marginally improved over SPV1629, and Z11-16 products selectivity was equivalent to SPV1629.
  • FIG. 24B; FIG. 24C Total and products selectivities were lower for SPV2148, due to the lower Z11-16Acid titer and higher Z11-18Acid titer.
  • the growth rates of SPV2076 lineage strains were improved relative to SPV2076; growth rates were ⁇ 80% of SPV1629 in FERM1, and ⁇ 50- 60% of SPV1629 in Solulys ® .
  • the growth rate rank order of the strains was generally consistent between both media. Table 12. Growth rates were measured with online optical density detection using the Biolector ® .
  • SPV2135 produced higher Z11-16Acid titers (2- to 3-fold) than SPV1629 in both media under growth assay conditions (no addition of methyl palmitate).
  • SPV2135 produced 50% higher Z11-16Acid titer ( ⁇ 10 g/L) than SPV1629 ( ⁇ 7 g/L), while only producing saturated acids (16Acid, 18Acid) and Z11 acids (Z11-16Acid, Z11-18Acid, Z13-18Acid, and Z11Z14- 18Acid).
  • the measured cell density for SPV2135 was lower than that observed for SPV1629.
  • This Example shows that adaptive evolution and chemical mutagenesis of SPV2076 produced strains with an improved growth rate phenotype (SPV2135 and SPV2148), and additionally an increased lipid accumulation phenotype and high Z11-16Acid selectivity (SPV2135).
  • FAD2 ⁇ 12
  • SPV2135 lipid accumulation phenotype and high Z11-16Acid selectivity
  • the 1 mL culture was transferred to a sterile microcentrifuge tube and pelleted before washing twice with 100 mM phosphate buffer (pH 7). After the second wash, the cell pellet was resuspended in 1.5 mL 100 mM phosphate buffer (pH 7), and 700 ⁇ L was transferred to a 14 mL round-bottom culture tube. 25 ⁇ L ethyl methanesulfonate was added to the 700 ⁇ L resuspended culture, and the tube was returned to the incubator for 35 minutes at 28 oC.
  • GC sample processing of lyophilized samples was performed essentially as described in Example 1.1. Samples were run on the GC in a one-pot format, using the parameters (for both front and back detectors) listed in Table 4 of Example 1.1. The GC needle height was adjusted from 3.0 mm to 14.5 mm.
  • Example 3.2 Production of Z13-18 in a Z11-16 / Z9-16 Background [0290] To develop strains that produce blends of unsaturated fatty acids that can be processed to a synthetic Chilo suppressalis pheromone from our Z11-16 platform, the Gen1 blend strain expressing the recombinant Z9 desaturase DST183 from Helicoverpa assulta (SPV2028) was selected for further development. A target composition was selected for an active C. suppressalis pheromone a blend containing 81% Z11-16Ald : 9% Z9-16Ald : 10% Z13-18Ald after distillation.
  • the native Y. lipolytica fatty acid synthase predominantly produces 18CoA (stearoyl-Coenzyme A), and the native Z9 desaturase (OLE1) has a selective preference for 18CoA, converting it to the unsaturated product Z9-18CoA.
  • Z9-18Acid (oleic acid), one of our byproducts, is required for rapid cell growth, because it is a key regulator of native lipid metabolism and is a major cellular membrane component.
  • a strain devoid of desaturase activity that can act on 18CoA leads to a build-up of saturated 18Acid, which reduces membrane fluidity and renders the strain inviable.
  • OLE1 was replaced with a Lepidopteran Z11-18 desaturase (DST148) from Bombyx mori in our Z11-16Acid producing strain, SPV1994.
  • DST148 Lepidopteran Z11-18 desaturase
  • Z11- 18Acid can serve as a substitute for Z9-18Acid and provide some degree of membrane fluidity, but ultimately lacks recognition in the regulatory pathway, which hampers the efficient regulation of cell growth.
  • the native Z12 desaturase FAD2 was deleted to eliminate Z9Z12-18Acid synthesis.
  • the native elongase, ELO2 was overexpressed in SPV2028 to test for increased Z13-18Acid production from Z11-16Acid (SPV2366).
  • the resulting marker rescued strain (SPV2416) served as a clean background for screening Z9 desaturase selectivity.
  • SPV2028 generated in the Scirpophaga incertulas blend strain library, was selected as a parent for the C. suppressalis blend production strain, because it produced a Z9- 16/Z11-16 ratio of 0.15, close to our target range of 0.091-0.12. FIG.26.
  • a 14-strain combinatorial library of CoA elongation overexpression constructs in the SPV2028 background was tested.
  • the lepidopteran Z9 desaturase DST192 SEQ ID NO:47 was engineered for improved selectivity to enable independent control of Z9-16 desaturase and Z9-18 desaturase activities.
  • DST192 variants were incorporated into the lead strain to reduce the Z9-18Acid byproduct titer.
  • All strains were screened in a 1 mL 24-well plate assay format, and all growth rates were measured through a Biolector ® assay. Fatty acid composition was measured using GC- FAME analysis.
  • Elongase Overexpression in a FAD2 Deletion Background [0297] To increase the Z13-18Acid content, a library of DNA constructs overexpressing key genes of the native CoA elongation pathway (ELO2, IFA38, and TSC13) were integrated into the SPV2028 background.
  • TSC13 overexpression can be observed when comparing performance to SPV2322, SPV2166, and SPV2324.
  • a second copy of TSC13 in SPV2327 also reduced the Z13-18Acid titer relative to SPV2325.
  • FIG.27C [0298]
  • the overexpression of ELO2 in the SPV2028 background increased elongation of Z11-16Acid to Z13-18Acid, and 16Acid substrate to 18Acid, which was subsequently converted to Z9-18Acid.
  • SPV2165 was selected as Gen1 C. suppressalis blend production strain.
  • OLE1 was therefore deleted in the SPV2165 background to reduce the Z9-18Acid content.
  • the resulting strain utilizes DST183 as the sole producer of Z9-18Acid.
  • DST183 still produced excess Z9-18Acid, suggesting strong activity on 18CoA.
  • FIG.28 To further reduce Z9-18Acid production, DST183 was replaced with a more selective Z9 desaturase. [0301] In a DST library screen, a range of Z9-16Acid/Z9-18Acid ratios were observed for Lepidopteran desaturases.
  • DST192 produced a similar Z9-18Acid titer to DST183, while producing higher Z9-16Acid titers, which suggests a higher selectivity for the 16CoA substrate.
  • FIG.29C It is also possible that the higher activity of DST192 draws more flux of 16CoA away from elongation by producing Z9-16CoA, which lowers the local concentration of 18CoA. We hypothesized that its high wild type activity and potential higher selectivity made DST192 the best choice for protein engineering.
  • DST192 Engineering [0303] A simplified but accurate binding pocket model of DST192 was created using structural information from mammalian Z9-18 desaturases, and sequence-function data for Lepidopteran desaturases. FIG.29(A-B).
  • the ⁇ -tail of the fatty acyl-CoA substrates sits in a pocket between two transmembrane helices of the desaturase that were labeled as Transmembrane Helix 2 (TM2) and Transmembrane Helix 4 (TM4), based on the order they appear in the primary sequence of the enzyme.
  • TM2 Transmembrane Helix 2
  • TM4 Transmembrane Helix 4
  • a correlation was identified between predictions from the binding pocket model and our data observed in the DST screen; Z9 desaturases producing higher relative titers of Z9-18Acid compared to Z9-16Acid had smaller amino acids at positions toward the bottom of the binding pocket (FIG.29(B-C)).
  • the smaller side chains likely reduce the binding constant K m for the longer substrate 18CoA.
  • a library of point mutants at 8 positions split across both TM2 and TM4 was created to improve substrate selectivity for 16CoA.
  • Three positions (F244, I245, and Y96) at the bottom of the binding pocket were mutated to bulkier amino acids to create steric hindrance against the binding of an 18CoA substrate.
  • DBK degenerative codon libraries were created for six-positions (Y96, V97, A98, S99, G100, and I101) along the center of the binding pocket to test if bulkier amino acids at positions in the center of the pocket would also increase selectivity for 16CoA (See Materials & Methods for constructs).
  • FIG. 30A Data from the DBK libraries identified the G100 position of TM2 as a key determinant of substrate selectivity. Mutations at G100 produced a trend in substrate selectivity that correlated well with amino acid size, indicating that bulkier substitutions hinder longer chain length substrates from entering and accessing the binding pocket (FIG.30A; FIG. 30C).
  • a G100V mutation produced a highly C16-selective desaturase (SEQ ID NO:55), whereas smaller amino acids like alanine (A) and serine (S) were less selective for 16CoA. Larger side chains like phenylalanine (F) and arginine (R) eliminated Z9 activity on both 16CoA and 18CoA (FIG.30A; FIG.30C). [0306] From the DST192 mutant data, a double mutant library was generated, combining the I245F mutation with four different G100 mutations. DST192 double mutants were also screened in SPV2416.
  • the results support a model where the G100 position is a structural determinant of substrate specificity, whereby bulky residues limit the binding of 18CoA without drastically impacting 16CoA binding.
  • a residue that is too large at position 100 on TM2 will sterically hinder even a 16CoA substrate (FIG.30C).
  • Assaying these mutants with a 14Acid substrate would elucidate whether mutations of G100 to amino acids bulkier than valine retain activity on shorter chain length substrates.
  • the DST192_I245F point mutant improved the 16/18 selectivity to 1:1 in vivo.
  • the DST192_G100V point mutant resulted in a highly C16 selective Z9 desaturase (>16:1 in vivo).
  • DST192 and DST192_I245F were cloned behind pPXA1 and pAMO2 at the POX5 locus, and resulting plasmids were transformed into the SPV2588 background (1xELO2, 1xIFA38, ⁇ ole1, 1xDST192-I245F), replacing the previously integrated DST183.
  • Resulting strains expressing two copies of DST192 variants reduced Z9-18 titers by 52% (SPV2665) and 70% (SPV2667).
  • FIG. 31C While these strains were successful in restricting Z9-18Acid titer, the overall fatty acid productivity and growth rate were reduced.
  • Example 4 Selective Biocatalysis of Z9-14 from Substrate [0319] To demonstrate how the Lepidopteran desaturase active site structural determinants identified in Example 3 that confer substrate and regioselectivity can be used to rationally engineer desaturases at will, a Z9-14 specific desaturase was engineered as a tool for increasing the Z9-14 fraction in production strains. Z9-14 is an active ingredient or valuable intermediate in insect pheromones; for example, Spodoptera frugiperda (83% Z9-14Ac and 13% Z11-16Ac). However, a Z9-14-specific desaturase has not been previously reported.
  • FIG.33 POX catalyzing conversion of 16CoA to 14CoA.
  • a desaturase operating on 14CoA was believed to drive flux toward Z9-14CoA in a pathway circuit even when 16ME (the substrate of choice for many biocatalysts and blend strains) is fed.
  • FIG. 33 A Z9-16 specific mutant desaturase (DST192 G100V) was engineered by modeling the active site of Z9-16/Z9-18 desaturase DST192. See Example 3.2.
  • a DST192 G100(DBK) library (SEQ ID NO:49) was used to engineer a Z9- 16-specific desaturase, wherein the degenerate DBK codon encodes the following set of canonical amino acids: alanine (A), arginine (R), cysteine (C), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), serine (S), threonine (T), tryptophan (W), and valine (V).
  • FIG. 34 ore mutants were not screened, as two variants (DST192 G100I and DST192 G100L) with the desired Z9-14 selectivity were discovered.
  • DST192 G100I and DST192 G100L were found to not impede the binding of 14CoA (FIG. 35).
  • FIG.36 and FIG.37 show that the G100I and G100L occlude the active site, and prevent the binding of 16CoA and 18CoA, respectively.
  • Models of the bulkier mutants DST192 G100F and DST192 G100R show at least two reasons why these enzymes completely lose activity against 14CoA, 16CoA, and 18CoA.
  • FIG. 41 shows that the phenol abuts the CoA substrate in at least one hypothetical rotamer of the G100F mutant.
  • Such strains are preferable for production of a synthetic Helicoverpa sp. pheromone, where ⁇ 97% of the unsaturated C16 fatty acid composition is Z11-16 (Both Z9-16Ald ( ⁇ 3%) and Z11-16Ald ( ⁇ 97%) are recognized as active ingredients in the pheromone for H. zea and H. armigera).
  • Z11-16Ald Bottom Z9-16Ald ( ⁇ 3%) and Z11-16Ald ( ⁇ 97%) are recognized as active ingredients in the pheromone for H. zea and H. armigera.
  • dactylifera DGAT1A Macadamia tetraphylla DGAT1 (SEQ ID NO:18); Thalassiosira pseudonana DGAT1 (SEQ ID NO:20); Vernicia fordii DGAT1 (SEQ ID NO:26); V. fordii DGAT2 (SEQ ID NO:28); T. pseudonana LPAAT1 (SEQ ID NO:22); and T. pseudonana LPAAT2 (SEQ ID NO:24). Overexpressing native Y.
  • lipolytica lipid regulators MGA2act (SEQ ID NO:59) (a truncated transcriptional activator fragment), MGA2act G643R (SEQ ID NO:61) (enhanced activity mutant), HAP1 (SEQ ID NO:65) (Heme-responsive zinc finger transcription factor), the diacylglycerol phosphate (PA) phosphatase PAH1 (SEQ ID NO:67), and the cytochrome B5 reductase CBR1 (SEQ ID NO:63) were also screened in SPV1629.
  • MGA2act SEQ ID NO:59
  • MGA2act G643R SEQ ID NO:61
  • HAP1 SEQ ID NO:65
  • PA diacylglycerol phosphate
  • PAH1 SEQ ID NO:67
  • cytochrome B5 reductase CBR1 SEQ ID NO:63
  • PAH1 phosphatidate phosphatase catalyzes the first committed step in TAG synthesis by removing the sn-3 phosphate, exposing the final hydroxyl group which forms an ester with a third acyl group in the final TAG.
  • Post-translational phosphorylation of PAH1 in S. cerevisiae has been implicated in down-regulation of PAH1 activity. Since many phosphorylation sites are not conserved in the Y. lipolytica variant, we set out to test whether simple overexpression would be sufficient. However, overexpressing Y. lipolytica PAH1 was not expected to to enhance activity. See Choi et al. (2011) J. Biol. Chem.
  • CBR1 microsomal cytochrome B5 reductase
  • the fatty acid composition of all cultures was measured using GC-FAME analysis. Strains were evaluated, relative to SPV1629, with the following metrics: (1) Exponential and linear phase growth rates; (2) Z11-16Acid titer and productivity; (3) Z11-16Acid/Z9-16Acid selectivity: (4) Z11-16Acid total selectivity: ; and (5) Z11-16Acid/products selectivity: . [0332] Growth rates of promising strains based on lipid profile in the bioconversion assay were measured in two different semi-defined media with online monitoring in a Biolector ® assay. Select promising strains were screened at 1 L in fermentors.
  • Z11- 16Acid titers for SPV1629 ranged from 5.4 to 10.7 g/L. FIG.42; FIG.54. The average value was 9.6 g/L with 16 of 24 replicates producing titers from 9.2 - 10.1 g/L. Five samples were low outliers, which were verified to be the result of variations in substrate loading (data not shown). [0336] Surprisingly, different lipid phenotypes were observed for strains expressing a second recombinant acyl transferase. Clones expressing the two T.
  • pseudonana LPAAT variants produced a wider range of Z11-16Acid titer that spanned the range of the non-outlier SPV1629 titers.
  • FIG.42 Both DGAT variants from V. fordi produced a split distribution of Z11-16Acid titer with two higher and two lower replicates. The higher values exceeded the control distribution, with the lower values at the upper end of the control distribution. The titers of byproduct fatty acids relative to the Z11-16Acid titer were marginally lower than those observed for SPV1629, indicating a potential increase in selectivity.
  • FIG. 42 The clearest phenotypes were observed for M. tetraphylla and T. pseudonana DGAT1 overexpression.
  • DGAT1_Tp byproducts were 64% higher with Z9-18Acid and 18Acid titers, accounting for 73% and 13% of the increase, respectively.
  • Strains overexpressing native lipid metabolism regulators and enzymes also generated distinct phenotypes, although none of the strains produced higher Z11-16Acid titers than the SPV1629 control. Overexpression of the full-length MGA2 gene led to a reduction in all fatty acid titers, including Z11-16Acid.
  • FIG. 43. The Z11-16 product selectivity was comparable to the SPV1629 control.
  • FIG.46. [0342] A distinct phenotype was observed for all four native lipid metabolism overexpression constructs.
  • FIG.47 CBR1 overexpression reduced the Z11- 16Acid titer, so that the total fatty acid titer was equivalent to that of SPV1629.
  • FIG.48 The activator domains MGA2act and MGA2act G643R produced higher total fatty acid titers, with MGA2act G643R also producing higher Z11-16Acid titers.
  • Table 17. Increase in fatty acid titers with overexpression of select native lipid metabolism genes. Percent increases are relative to the SPV1629 control. 1 L data taken from ⁇ 40-hour timepoint to best differentiate between productivity of strains.
  • SPV2473 (DGAT1_Mt) has an increased lipid accumulation phenotype, while maintaining similar selectivity to SPV1629.
  • SPV2473 has 13% higher peak Z11-16Acid productivity, equivalent S Z11/Z9-16 , and marginally lower S Z11-16_Prod , due to an increase in 18Acid and Z9-18Acid titer.
  • SPV2474 (DGAT1_Tp) has the highest lipid accumulation phenotype, while sacrificing selectivity for Z11-16Acid.
  • SPV2474 has a 30% higher peak Z11-16Acid productivity, ⁇ 5% lower SZ11/Z9-16, and 20-25% lower SZ11-16_Prod, due to increases in C18 fatty acid titers, especially Z9-18Acid.
  • Table 15; Table 18; FIG.49. SPV2477 (DGAT1_Vf) produced a lipid profile nearly identical to that of SPV1629, with marginally higher titers early in the fermentation.
  • FIG. 49; FIG. 50. did maintain a linear increase in lipid content through the later stages of the bioconversion, leading to a final lipid fraction equivalent to SPV2473. Table 18; FIG.50.
  • the SPV2477 data suggests either lower expression or slower kinetics of DGAT1_Vf in the Y. lipolytica background, relative to DGAT1_Mt and DGAT1_Tp.
  • SPV2479 CBR1
  • a higher Z9-18Acid fraction was observed at 1 L confirming the phenotype observed at 1 mL scale.
  • FIG.49 [0349] Table 18. Performance of select strains in 1 L bioconversion process at 70 hours.
  • Tuning the methyl palmitate feeding schedule for SPV2473 or SPV2474 resulted in increases in titer.
  • the feeding schedule of the bioconversion process was tuned for the lipid accumulation rate of SPV1629, and both SPV2473 and SPV2474 display an increased rate of both total lipid and product lipid (all fatty acids excluding 16Acid substrate) accumulation during peak productivity between 20 and 40 hours (FIG. 51; FIG. 52).
  • the product lipid accumulation rate does not exceed substrate loading for SPV2473, but the rate is higher than SPV1629.
  • the product lipid accumulation rate is higher than substrate loading for SPV2474.
  • SPV2473 produces a 96:4 ratio of Z11-16Acid:Z9-16Acid, which is equivalent to SPV1629 selectivity. Increased lipid productivity and lipid content was achieved through increased expression of DGAT activity in these two strains. Identifying DGATs or other acyl transferases that are selective for Z11-16Acid and Z9-16Acid further improves the performance of Helicoverpa sp. blend producing strains. [0352] In summary, growth rates for the best performing strains were at least 95% of the SPV1629 growth rate in both media. Table 19. All strains exhibited two growth phases in FERM1 base medium with a true exponential growth period followed by a linear growth phase. In Solulys ® base medium, all strains exhibited exponential growth. [0353] Table 19. Growth rates of strains expressing multiple acyltransferases or overexpressing native lipid metabolism genes.
  • Table 17 Three strains overexpressing native lipid metabolism genes produced higher titers of at least one fatty acid, altering the observed fatty acid profile. Table 17. At 1 mL scale, increased titers were generally more pronounced for native C18 fatty acids. SPV2479 was selected for 1 L scale fermentation. Lower biomass was observed compared to acyl transferase and SPV1629, and relatively higher titers of C18 fatty acids were observed. [0355] Table 20 and Table 21 show complete titer and selectivity summaries for all strains (uncertainty in the selectivity measurements less than 0.01).
  • a FAD2 targeting expression vector was created by cloning homology arms from existing vector pPV1132 into expression vector pPV0601. Y. lipolytica genes were then cloned into the resulting vector, pPV1295, using AvrII and NotI cloning sites. DNA constructs were transformed into E. coli EPI400 to propagate the plasmid. E. coli EPI400 strains were grown overnight in LB at 37 °C and 250 rpm to an OD600 ⁇ 2. Cells were back diluted 10x into LB with copy-cutter induction solution and grown 24 hours at 37 °C and 250 rpm before miniprepping. [0362] Table 22. Construct summary.
  • Strain construction Strain construction, including plasmid digestion, transformation, check PCR, and colony patching was performed essentially as described in Example 1.1.
  • 24-well bioconversion assay The 24-well bioconversion assay was performed essentially as described in Example 1.1.
  • Biolector ® growth rate assay The Biolector ® growth rate assay was performed essentially as described in Example 1.1.
  • GC processing of lyophilized samples One-pot format GC assays and lyophilized sample processing was performed essentially as described in Example 2.1. Example 6.
  • the substrate plates were pre-loaded by adding a methyl palmitate solution in ethanol and letting the ethanol evaporate overnight.
  • the co-substrate plates were kept in liquid form by incubating at 37 ⁇ C until feeding. Cultures were sampled after 48 hours of bioconversion. OD600 measurements taken.
  • Example 7. Developing a Spodoptera frugiperda blend [0372] We sought to build a biocatalyst that makes Spodoptera frugiperda blend precursors Z9-14 and Z11-16. We overexpressed DST192G100L, a Z9-14-specific desaturase, in Yarrowia lipolytica background SPV298.
  • the one-copy variant (SPV3212) produced greater than 3 g/L Z9-14 and approximately 0.4 g/L of the elongation product Z11-16.
  • the two-copy variant (SPV3217) produced almost 5 g/L Z9-14 and approximately 0.8 g/L Z11-16.
  • this biocatalyst produces these precursors in a ratio close to the optimal blend of 87:13 (Z9- 14: Z11-16) ( Figure 53 and Table 24).
  • Table 24 Table of DST192G100L-dependent analytes. Values are shown in grams per liter.
  • Strain construction Strain construction, including plasmid digestion, transformation, check PCR, and colony patching was performed essentially as described in Example 1.1.
  • Example 8 Large-scale Production of Biologically Active Pheromone Blends
  • Engineered Y. lipolytica strain SPV2777 were produced as an example of a biocatalyst for the bioconversion of methyl palmitate (MP) to mixtures of Z11-16FAME/TAG (Z11-16:1), Z9-16 FAME/TAG (Z9-16:1) and Z13-18 FAME/TAG (Z13-18:1) that serve as precursors for ingredients in a biologically active Chilo suppressalis pheromone blend. This bioconversion process was successfully used at increased scale ( ⁇ 150 L).
  • DSP Downstream processing
  • TM centrifugation
  • GEA NiroTM FSD 4.0 spray drying
  • SPV2777 Biodesaturation of methyl palmitate with SPV2777 for blend production was performed in two 150 L Frings ® reactors. The culture broth was processed to dry cell pellet, and OD 600 cell-dry-weight and broth metabolites were quantified via HPLC. The lipid profile was quantified via GC-FID.
  • FIG.54 shows the major fermentation parameters, DO, temperature, pH, oxygen utilization rate (OUR), feed addition and fermentor weight of these two reactions.
  • Table 25 Methyl palmitate and AF204 additions during bioconversion.
  • DO dissolved oxygen
  • OUR oxygen utilization rate
  • FIG.55 shows the bioconversion profile (biomass, Z11-16:1, Z9-16:1, Z13-18:1 titers, and Z9-18:1, Z11-18:1 titers) of Reaction 1, Reaction 2 (65 L initial volume), and Reaction 3 (0.9 L initial volume).
  • the profiles of these three reactions are very similar, with the exception of Z9-16:1 and Z9-18:1.
  • the biomass accumulation of these three reactions shows the same profile reaching final biomass values of 70, 71, and 77 for Reaction 1, Reaction 2, and Reaction 3, respectively.
  • the difference in final biomass between the lab scale reaction to the 65 L reactions is mostly due to a difference in volume increase of 23% in the lab scale reaction compared to 41% in the pilot reactions. Correcting for the volume increase, the normalized biomass (on the initial volume basis) would be 98, 99, and 95 for Reaction 1, Reaction 2, and Reaction 3, respectively. [0385] The accumulation of Z11-16:1, Z9-16:1, and Z13-18:1 across these three reactions exhibited only a difference with Z9-16:1. The identical accumulation of Z11-16 / Z9- 16 / Z13-18 between Reaction 1 and Reaction 2, which differed in MP loading (45 g/L vs.48 g/L) indicates that the additional MP was not utilized for product formation.
  • the broths were combined and stirred in a jacketed (40 oC) holding tank as feed for the centrifuge.
  • the combined feed had a dry matter (DM) content of 12.6 wt%, a total lipid content of 3.1 wt%, and Z11-16 / Z9-16 / Z13-18 content of 1.8 wt%.
  • An Alfa Laval Clara 20TM centrifuge was used for biomass recovery at a feed rate of 200-220 L/h and 2 min discharge cycle.
  • the loss of biomass as indicated by the pack cell volume (PCV) of the supernatant stream at the start and end of the discharge cycle was constant at a value of ⁇ 5 % (2 mL pellet from a 40 mL sample).
  • the air at the outlet was set to a target value of 80 oC.
  • the feed rate of the sludge was under feedback control to maintain the outlet temperature setpoint.
  • An average feed rate of 21.7 kg/h was maintained over the 1.7 hours of spray drying operation.
  • a total of 10.7 kg spray dried powder (98.6 wt% DM, 37.2 wt% total lipid, 22.5 wt% Z11-16 / Z9-16 / Z13-18) was produced with an average production rate of 6.3 kg/h.
  • Based on the sludge feed rate an average evaporation rate of 15 kg/h was maintained during spray drying operation.
  • Table 26 shows the DM and lipid composition of the major DPS streams, inactivated broth, sludge, and dried biomass.
  • the 10.7 kg final dried product with 22.5 wt% Z11- 16 / Z9-16 / Z13-18 contain 2.4 kg crude Z11-16 / Z9-16 / Z13-18. This represent 70.5% recovery of the 3.4 kg crude Z11-16 / Z9-16 / Z13-18 present in the 189 kg of inactivated broth. Based on the Z11-16 / Z9-16 / Z13-18 content of the supernatant stream, only 11% of the product loss could be attributed to centrifugation. The remaining 18.5% of product loss during DSP is likely the result of handling at pilot scale. For example, only 37 kg of the 40 kg sludge produced from centrifugation was processed through spray drying. This loss of 3 kg during handling represents an 8% product loss.
  • Table 27 Composition of DSP materials from Reaction 1 and Reaction 2 DSP.
  • Table 28 Summary of overall performance of bioconversion reactions carried out in 150 L reactors. *MP loading was varied between Reaction 1 (45 g/L) and Reaction 2 (48 g/L)
  • Reactions performed in 150 L reactors produced 18.0 g/L and 18.3 g/L Z11- 16 / Z9-16 / Z13-18, with Z11-16:Z9-16:Z13-18 ratios of 82:8:10 and 81:9:10.

Abstract

This disclosure concerns methods and apparatuses for the synthesis of insect pheromones and precursors thereof in a scalable and eco-friendly fermentation reaction; for example, by converting saturated or unsaturated substrate feedstocks utilizing exogenous and endogenous metabolic machinery.

Description

SYNTHESIS OF COMPLEX INSECT PHEROMONE BLENDS AND REACTION APPARATUSES FOR THE SAME CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63/043,113 filed June 23, 2021, the disclosure of which is hereby incorporated by this reference in its entirety. FIELD OF THE DISCLOSURE [0002] The present disclosure relates to the production of complex blend compositions containing one or more fatty acids and/or derivatives thereof (e.g., fatty alcohols, fatty acetates, and fatty aldehydes) in a single reaction volume. Many aspects of the disclosure relate to the production of complex blends of regiospecific and stereospecific unsaturated fatty aldehydes that are biologically active as insect pheromone compositions, and/or complex blends of precursors thereof. More specifically, the disclosure relates to the coordination of multiple catalytic activities in a single reaction volume to produce a plurality of particular fatty acids and/or derivatives thereof (e.g., fatty alcohols, fatty acetates, and fatty aldehydes) in a ratio that elicits a behavior response an insect; for example, to disrupt mating and confer crop protection in a host plant of the insect, or to produce a plurality of precursors of the active forms in relative amounts corresponding to the amounts of the active forms in the biologically active blend compositions. BACKGROUND [0003] Producing more food to meet a growing demand requires effective pest control. Conventional insecticides are among the most popular chemical insect pest control agents because they are readily available, fast-acting, and highly reliable. However, the overuse, misuse, and abuse of these chemicals leads to resistant pests, alters natural ecology, and damages the environment. [0004] Pheromones are central to mate-finding behavior in insects, and they show promise for the eco-friendly protection of a wide range of crops. These molecules have proven effective in reducing insect populations through a variety of methods including mass trappings, attract and kill, and mating disruption. The latter method,in particular, represents a non-toxic means of pest control and utilizes synthetic pheromones to mask naturally occurring pheromones, thereby confusing insects and disrupting their mating. Furthermore, pheromones are biodegradable and are non-toxic to humans. These properties make pheromones ideal candidates for modern eco-friendly crop protection. [0005] In many insect species of interest to agriculture, females emit an airborne trail of a specific chemical blend constituting that species’ sex pheromone. This aerial trail is referred to as a pheromone plume. Males of that species use the information contained in the pheromone plume to locate the emitting female. Mating disruption exploits the male insects’ natural response to follow the plume by introducing a synthetic pheromone blend into the insects’ habitat. The blend is designed to mimic the sex pheromone produced by the female insect. [0006] Insect sex pheromones contain a diverse group of chemical compounds including regiospecific and stereospecific unsaturated fatty aldehyde isomers. The sex pheromones of different insect species are specific blends of the constituent compounds in characteristic ratios, which in the current state of the art requires for effective synthetic pheromone blends to be assembled from isolated and purified compounds according to the requirements of a particular target insect species. The constituent compounds of biologically active synthetic insect pheromones are regiospecific and stereospecific unsaturated fatty aldehyde isomers comprising lipid moieties with specific chain lengths, so the independent synthesis and work-up of the constituent compounds typically requires the expenditure of considerable resources. Therefore, although pheromones have significant potential in agricultural insect control, the cost of synthesizing pheromones using currently available techniques is very high, and the widespread use of this effective, eco-friendly, and sustainable technology in other than high-value crops is effectively prohibited. BRIEF SUMMARY OF THE DISCLOSURE [0007] Described herein are apparatuses and methods for producing complex compositions in a single reaction volume that comprise particular amounts of specific fatty acids, fatty alcohols, fatty aldehydes, and/or fatty acetates. Such complex compositions may be biologically active in an insect pest (for example, by mimicking the natural pheromone of the insect), or may contain corresponding amounts of chemical precursors of the biologically active composition compounds. Alternatively, the fatty acids and/or derivatives thereof (e.g., fatty alcohols, fatty acetates, and fatty aldehydes) produced according to methods and compositions described herein may be fragrances, flavoring agents, and/or polymer intermediates. [0008] In embodiments herein, a complex blend comprising particular amounts of regiospecific and stereospecific product isomers (i.e., fatty acids and/or derivatives thereof comprising lipid moieties with specific chain lengths) is produced from organic substrate(s) (e.g., molecules comprising saturated or unsaturated lipid moieties) by combining activities representing steps in the synthesis of each of the products. In some embodiments, the activities are comprised in a biocatalyst (a naturally occurring cell or organism, or genetically modified cell or organism), with or without conversion by non-biological means of reaction intermediates (e.g., regiospecific and/or stereospecific isomers comprising saturated or unsaturated lipid moieties) as one or more of the successive reaction steps. The substrate(s) are subjected to successive chemical reactions in a single reaction volume, for example, without separation or purification of intermediates or products during the reaction process. In some embodiments, reagents or other reaction components (e.g., catalysts, biocatalysts, and substrates) may be added during a sequential reaction process, without isolation and purification of the product(s) of the chemical reaction process. [0009] In some embodiments herein, an apparatus for producing a complex composition in a single reaction volume is assembled for the one-pot synthesis of a complex blend comprising particular amounts of regiospecific and stereospecific product isomers comprising lipid moieties with specific chain lengths. In such a one-pot synthesis, one or more unsaturated lipid moieties utilized as a reactant in one or more step(s) of the synthesis may be introduced into the reaction volume, or produced therein by a biocatalyst from a saturated lipid moiety or organic substrate (e.g., glucose). In these and further one-pot synthesis according to embodiments herein, such unsaturated lipid moiety-containing reactants may be reduced in the reaction volume directly to produce one or more fatty alcohols, one or more fatty aldehydes, and/or one or more fatty acetates. Alternatively, such unsaturated lipid moieties may be chemically converted in the reaction volume to one or more free fatty acids (FFAs), fatty acid alkyl esters (FAAEs) (e.g., fatty acid methyl esters (FAMEs)), fatty alcohols, fatty acetates, and/or fatty aldehydes. For example, one or more FFAs may be esterified to produce one or more corresponding fatty acid alkyl esters (FAAEs), one or more FFAs and/or FAAEs may be reduced to produce one or more corresponding fatty alcohols and/or fatty aldehydes, and/or one or more fatty alcohols may be acetylated to produce one or more corresponding fatty acetates. [0010] In particular embodiments herein, an apparatus for producing a complex composition in a single reaction volume comprises the reaction volume within a single compartment in the apparatus. In alternative embodiments, an apparatus for producing a complex composition in a single reaction volume may comprise more than one compartment in the apparatus, disposed therein such that they are integrally connected to comprise the reaction volume. In certain examples, one or more compartment of the apparatus may comprise one or more points of access for the addition and/or removal of fluids, gases, chemicals, reactants, catalysts, biocatalysts, etc. In certain examples, one or more compartment of the apparatus may comprise a working station, for example, adapted to facilitate observation or monitoring of the reaction process. [0011] According to the foregoing, a biocatalyst may be used in embodiments herein to catalyze, for example, any combination of the following reactions, in one or more steps: synthesis of a saturated lipid moiety or unsaturated lipid moiety isomer (e.g., from an organic substrate); production of an unsaturated lipid moiety isomer (e.g., from a saturated lipid moiety); production of a fatty alcohol (e.g., from an unsaturated lipid moiety isomer); production of a fatty aldehyde (e.g., from an unsaturated lipid moiety isomer); production of a FFA (e.g., from an unsaturated lipid moiety isomer); production of a FAE (e.g., from an unsaturated lipid moiety isomer, and from a FFA); production of a FAME (e.g., from an unsaturated lipid moiety isomer, and from a FFA); production of a fatty alcohol (e.g., from a FFA, from a FAAE, and from a FAME); production of a fatty aldehyde (e.g., from a FFA, from a FAAE, and from a FAME); and production of a fatty acetate (e.g., from a FFA, from a FAAE, from a FAME, from a fatty alcohol, and from a fatty aldehyde). [0012] A biocatalyst according to embodiments herein may be an organism (e.g., a genetically modified organism). In some embodiments, the organism is a fungus or bacteria. In particular embodiments, the organism is a yeast; for example, an oleaginous yeast. For example, the organism may an oleaginous yeast from the genus Yarrowia or Candida; e.g., Yarrowia lipolytica, Candida viswanathii, and Candida tropicalis. In such embodiments, the biocatalyst may be a genetically modified strain of Y. lipolytica. In other particular embodiments, the organism is a bacteria, for example, a gram-negative bacteria or cyanobacteria. For example, the organism may be a gram-negative bacteria from the genus Myxococcus, e.g., M. xanthus. In another example, the organism is a cyanobacteria species from the genus Synechococcus, e.g., S. elongatus. In alternative embodiments, the organism is an insect, such as Amyelois transitella or Pyralis farinalis in one specific example. In other alternative embodiments, the organism is a plant, or algae, or bacteria. In specific examples, the organism is a plant from the family Proteaceae, or the family Rhamnaceae. In certain examples, the organism is a plant species from the genus Gevuina, Calendula, Limnanthes, Lunaria, Carum, Daucus, Conandrum, Macadamia, Myristica, Licania, Aleurites, Ricinus, Corylus, Kermadecia, Asclepias, Cardwellia, Grevillea, Orites, Ziziphus, Hicksbeachia, Hippophae, Ephedra, Placospermum, Xylomelum, or Simmondsia. For example, the organism may be a plant species selected from the group consisting of Gevuina avellana, Corylus avellana, Kermadecia sinuata, Asclepias syriaca, Cardwellia sublimis, Grevillea exul var. rubiginosa, Orites diversifolius, Orites revoluta, Ziziphus jujube, Hicksbeachia pinnatifolia, and Grevillea decora. In some examples, the organism is an algae, for example, a green algae. For example, the organism may be a green algae species from the genus Pediastrum, e.g., Pediastrum simplex. [0013] The apparatuses and methods herein may specifically be utilized to produce complex compositions of a synthetic pheromone of the insect, In embodiments herein, a complex composition is created as a synthetic pheromone in an insect selected from the group consisting of the Asiatic rice borer or striped rice stemborer (i.e, Chilo suppressalis), corn earworm (i.e, Helicoverpa zea), cotton bollworm (i.e, Helicoverpa armigera), rice leafroller (i.e, Cnaphalocrocis medinalis), yellow stem borer or rice yellow stem borer (i.e, Scirpophaga incertulas, fall armyworm (i.e, Spodoptera frugiperda), soybean looper moth (i.e, Pseudoplusia includens), and jasmine bud borer (i.e, Trichophysetis cretacea). Apparatuses and methods according to the embodiments herein provide significant advantages over the prior art by enabling the one-pot synthesis of complex compositions comprising the constituent compounds in relative amounts that are within a biologically relevant range, or compositions comprising precursors of the constituent compounds in relative amounts that are immediately able to be purified and processed without separation into a biologically active complex composition comprising the constituent compounds in relative amounts within the biologically active range. [0014] In some embodiments, a complex composition is produced as a synthetic pheromone, wherein the composition comprises a combination of constituent compounds selected from the group consisting of Z9-14Acid, Z9-14OH, Z9-14Ald, Z9-14Ac, Z9-16Acid, Z9-16OH, Z9-16Ald, Z9-16Ac, Z11-16Acid, Z11-16OH, Z11-16Ald, Z11-16Ac, Z9-18Acid, Z9-18OH, Z9-18Ald, Z9-18Ac, Z11-18Acid, Z11-18OH, Z11-18Ald, Z11-18Ac, Z13-18Acid, Z13- 18OH, Z13-18Ald, and Z13-18Ac. [0015] In particular embodiments, a complex composition is produced as a synthetic pheromone, wherein the composition comprises Z9-16Ald. In some examples, such a complex composition is biologically active as a synthetic pheromone in Chilo suppressalis, Scirpophaga incertulas, Helicoverpa zea, or Helicoverpa armigera, wherein the composition comprises 0.5- 40% (e.g., 0.5-10%, 5-40%, 3-15%, 1-20%, 1-4%, 5-12%, 5-15%, 15-25%, 15-33%, about 3%, about 7%, about 9%, about 19%, and about 25%) Z9-16Ald. For example, a complex composition that is biologically active as a synthetic pheromone in C. suppressalis, may comprise 7-9% (e.g, 7%, 8%, and 9%) Z9-16Ald. [0016] In particular embodiments, a complex composition is produced that is biologically active as a synthetic pheromone, wherein the composition comprises Z11-16Ald. In some examples, such a complex composition is biologically active as a synthetic pheromone in Chilo suppressalis, Scirpophaga incertulas, Trichophysetis cretacea, Helicoverpa zea, or Helicoverpa armigera, wherein the composition comprises at least 40% Z11-16Ald. For example, a complex composition that is biologically active as a synthetic pheromone in Chilo suppressalis, Scirpophaga incertulas, Trichophysetis cretacea, Helicoverpa zea, or H. armigera may comprise 40-99.5% (e.g., 40-90%, 40-97%, 60-80%, 60-90%, 66-85%, 90-99.5%, 47-97%, 70-80%, 70-94%, 75-90%, 96-99%, about 74%, about 75%, about 81%, and about 97%) Z11- 16Ald. In particular examples, the complex composition is biologically active as a synthetic pheromone in T. cretacea, wherein the composition comprises 46%-49% Z11-16Ald. In particular examples, the complex composition is biologically active as a synthetic pheromone in C. suppressalis, wherein the composition comprises 74-85% (e.g., 74-81%, about 74%, about 75%, and about 81%) Z11-16Ald. In particular examples, the complex composition is biologically active as a synthetic pheromone in H. zea, or H. armigera, wherein the composition comprises about 97% Z11-16Ald. In particular examples, the complex composition is biologically active as a synthetic pheromone in S. incertulas, wherein the composition comprises about 75% Z11-16Ald. [0017] In particular embodiments, a complex composition is produced that is biologically active as a synthetic pheromone, wherein the composition comprises Z9-16Ald and at least 40% Z11-16Ald. In some examples, such a complex composition is biologically active as a synthetic pheromone in Chilo suppressalis, Scirpophaga incertulas, Helicoverpa zea, or Helicoverpa armigera, wherein the composition comprises 3%-25% Z9-16Ald and 40%-97% Z11-16Ald. In particular examples, the complex composition is biologically active as a synthetic pheromone in C. suppressalis, wherein the composition comprises 7-9% (e.g, 7%, 8%, and 9%) Z9-16Ald and 74-85% (e.g., 74-81%, about 74%, about 75%, and about 81%) Z11-16Ald. In particular examples, the complex composition is biologically active as a synthetic pheromone in H. zea, or H. armigera, wherein the composition comprises about 3% Z9-16Ald and about 97% Z11-16Ald. In particular examples, the complex composition is biologically active as a synthetic pheromone in S. incertulas, wherein the composition comprises about 25% Z9-16Ald and about 75% Z11-16Ald. [0018] In particular embodiments, a complex composition is produced that is biologically active as a synthetic pheromone, wherein the composition comprises Z13-18Ald. In some examples, such a complex composition is biologically active as a synthetic pheromone in Cnaphalocrocis medinalis or Chilo suppressalis, wherein the composition comprises 8%-90% Z13-18Ald. In particular examples, the complex composition is a synthetic pheromone in C. medinalis, wherein the composition comprises about 90% Z13-18Ald. In particular examples, the complex composition is a synthetic pheromone in C. suppressalis, wherein the composition comprises 8-11% (e.g., 8.5%, 9.5%, and 10%) Z13-18Ald. [0019] In the foregoing and further embodiments, a complex composition may be produced that comprises at least one additional constituent compound selected from the group consisting of Z9-14Acid, Z9-14OH, Z9-14Ald, Z9-14Ac, Z9-16Acid, Z9-16OH, Z9-16Ald, Z9- 16Ac, Z11-16Acid, Z11-16OH, Z11-16Ald, Z11-16Ac, Z9-18Acid, Z9-18OH, Z9-18Ald, Z9- 18Ac, Z11-18Acid, Z11-18OH, Z11-18Ald, Z11-18Ac, Z13-18Acid, Z13-18OH, Z13-18Ald, and Z13-18Ac. For example, the complex composition may be biologically active as a synthetic pheromone in Chilo suppressalis, wherein the composition comprises 7-9% (e.g, 7%, 8%, and 9%) Z9-16Ald, 74-85% (e.g., 74-81%, about 74%, about 75%, and about 81%) Z11-16Ald, and 8-11% (e.g., 8.5%, 9.5%, and 10%) Z13-18Ald. For example, such a composition may comprise about 81% Z11-16Ald, about 9% Z9-16Ald, and about 10% Z13-18Ald. In another example, the complex composition may be biologically active as a synthetic pheromone in Cnaphalocrocis medinalis, wherein the composition comprises 90% Z13-18Ald and 10% Z11-18Ald. In another example, the complex composition may be biologically active as a synthetic pheromone in Trichophysetis cretacea, wherein the composition comprises 47% Z11-16Ald, 47% Z11-16Ac, and 5% Z11-16OH. [0020] In these and further embodiments, a complex composition may be biologically active as a synthetic pheromone in an insect, wherein the composition comprises compounds that are not active ingredients in the pheromone of that insect. For example, a complex composition may be biologically active as a synthetic pheromone in Chilo suppressalis, wherein the composition comprises the active pheromone ingredients Z11-16Ald, Z9-16Ald, and Z13- 18Ald, and further comprises Z9-18Ald. In examples of such a composition, the composition comprises 7-9% (e.g, 7%, 8%, and 9%) Z9-16Ald, 74-85% (e.g., 74-81%, about 74%, about 75%, and about 81%) Z11-16Ald, 8-11% (e.g., 8.5%, 9.5%, and 10%) Z13-18Ald, and 7-11% (e.g., about 7.5% and about 10.5%) Z9-18Ald. By way of additional example, a complex composition may be biologically active as a synthetic pheromone in C. suppressalis, wherein the composition comprises the active pheromone ingredients Z11-16Ald, Z9-16Ald, and Z13-18Ald, and further comprises Z9-18Ald and Z11-18Ald. Particular embodiments include a complex composition that is biologically active as a synthetic pheromone in a plurality of insects (e.g., 2, at least 2, and 3), wherein constituent compounds of the composition are active ingredients in the pheromones of the plurality of insects, but at least one of the constituent compounds is not an active ingredient in the pheromone of at least one of the plurality of insects. For example, a composition comprising Z11-16Ald, Z9-16Ald, Z13-18Ald, Z9-18Ald, and Z11-18Ald may be biologically active as a synthetic pheromone in C. suppressalis (Z11-16Ald, Z9-16Ald, Z13-Ald) and Cnaphalocrocis medinalis (Z13-18Ald, Z11-18Ald). [0021] In still further embodiments, a complex composition may be produced that comprises a combination of constituent compounds selected from the group consisting of Z9- 14Acid, Z9-14OH, Z9-14Ald, Z9-14Ac, Z9-16Acid, Z9-16OH, Z9-16Ac, Z11-16Acid, Z11- 16OH, Z11-16Ac, Z9-18Acid, Z9-18OH, Z9-18Ald, Z9-18Ac, Z11-18Acid, Z11-18OH, Z11- 18Ac, Z13-18Acid, Z13-18OH, and Z13-18Ac, wherein the composition does not comprise Z9-16Ald, Z11-16Ald, Z11-18Ald, or Z13-18Ald. For example, the complex composition may be biologically active as a synthetic pheromone in Spodoptera frugiperda, wherein the composition comprises 82.8% Z9-14Ac and 12.9% Z11-16Ac. [0022] In the foregoing and further embodiments, a complex composition may be produced that comprises at least one additional constituent compound that is saturated lipid moiety, unsaturated lipid moiety isomer, fatty alcohol, fatty aldehyde, FFA, FAE, or fatty aldehyde. For example, the complex composition may be biologically active as a synthetic pheromone in Spodoptera frugiperda, wherein the composition comprises 82% Z9-14Ac, 12% Z11-16Ac, and 1% 14Ac. [0023] The apparatuses and methods herein may also and alternatively specifically be utilized to produce complex compositions consisting essentially of chemical precursors of the biologically active synthetic pheromone constituent compounds in corresponding amounts to that of the biologically active synthetic pheromone constituent compounds. It is immediately understood by those in the art that a complex composition according to embodiments herein may comprise precursors of the constituent compounds in corresponding amounts to that of the synthetic pheromone constituent compounds that are nonetheless not precisely those of the synthetic pheromone, for example, because a process of purifying the biologically active complex composition from the reaction volume may be expected to result in a change in the relative amounts of the constituent compounds. By way of representative example, a complex composition of synthetic pheromone in C. suppressalis may comprise 81% Z11-16Ald, 9% Z9- 16Ald, and 10% Z13-18Ald, but a complex composition comprising precursors of these compounds in corresponding amounts may comprise Z11-16Acid, Z9-16Acid, and Z13-18Acid in the relative amounts of 1 : 0.09 : 0.14-0.2 (Z11-16Acid : Z9-16Acid : Z13-18Acid), because a relative loss of Z13-18Acid would be anticipated and expected during a distillation of these compounds. [0024] The foregoing and other features will become more apparent from the following Detailed Description, which proceeds with reference to the accompanying Figures, and according to the following Definitions and Rules, which are provided to clearly convey the meaning of the disclosure: Definitions and Rules [0025] Unless specifically indicated or implied, the terms “a,” “an,” and “the” signify “at least one,” as used herein. For example, a composition comprising “an” element may of course contain two, three, or more such elements, and a composition comprising “the” element may of course contain two, three, or more copies or occasions of the reference element. [0026] Unless otherwise noted, absolute or relative numerical values set forth herein in the description and claims follow the normal and common usage of significant figures, by which numbers are rounded to avoid conveying insignificant figures; numerical values include those that would be rounded to the number set forth, with its particular significant figures. For example, the absolute or relative number of 2 includes those values between 1.5 and 2.4, while the absolute or relative number of 2.0 includes those values between 1.95 and 2.04. Specifically with regard to absolute or relative numerical values set forth herein in Examples, numbers may be reported including insignificant figures; for example, as a mathematical operation on data from an instrument. All percentages are by weight, all solvent mixture proportions are by volume, and all temperatures are in degrees Celsius, unless otherwise noted. [0027] Unless otherwise specifically explained, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology can be found in, for example, Lewin’s Genes X, Jones & Bartlett Publishers, 2009 (ISBN 100763766321); Krebs et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Meyers R.A. (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). BRIEF DESCRIPTION OF THE FIGURES [0028] FIGs. 1A-1C include diagrams of exemplary apparatuses of particular embodiments. [0029] FIG. 2 includes the lineage of genetically engineered strains of the production platform. FIG.2A shows the abridged SPV1994 and SPV1995 strain lineage. OLE1 knockout strains are descendants of SPV1994, which is the 4-copy descendant of SPV1629. FIG.2B shows the abridged SPV2072, SPV2073, SPV2074, SPV2075, and SPV2076 strain lineage. FIG.2C shows the lineage of strains that are descendants of strain SPV1629 through the 4-copy strain SPV1994 and OLE replacement strain SPV2076. FIG.2D shows the lineage of strains that are descendants of strain SPV1629. FIG. 2E shows the lineage of strains that are descendants of strain SPV2028 (See FIG. 2C). FIG. 2F shows the lineage of strains that are descendants of strain SPV1629. [0030] FIG. 3 includes a representation of Z9, Z9Z12, and Z13 fatty acid profiles of OLE1 replacement strains. FIG. 3A shows Z9 and Z9 derived fatty acid titers for each OLE1 replacement strain. FIG. 3B shows Z11 and Z11 derived fatty acids produced by each OLE1 replacement strain. Z13-18Acid is derived from elongation of Z11-16Acid. SPV1994 is the parental control. All fatty acids are labeled with “ME,” because each were quantified as methyl ester derivatives after esterification of cellular triacylglycerides. [0031] FIG.4 includes the full fatty acid profiles for OLE1 replacement strains. [0032] FIG.5 includes a representation of the final cell densities obtained in a 24-well plate bioconversion assay. [0033] FIG.6 includes growth rates of OLE1 replacement strains. Strains were grown at 32 °C and 1500 rpm in a 48-well flower plate. Growth rates are shown in both Solulys® and FERM1 media. [0034] FIG.7 includes fatty acid titer profiles for 4-copy HzDST strains. All detected fatty acid species titers are shown for the control strain, SPV1629, and two 4-copy isolates, SPV1993 and SPV1994. [0035] FIG.8 includes data characterizing SPV1994 with a fourth HzDST copy. FIG. 8A shows Z11-16Acid/Z9-16Acid selectivity increases by ~1% in strain SPV1994 as compared to the control strain SPV1629. FIG.8B shows C16Acids selectivity is lower in SPV1994, due to lower 16Acid titer. FIG. 8C shows Z11-16Acid/products selectivity is equivalent for SPV1629 and SPV1994. FIG.8D shows Z11-16Acid total selectivity is marginally higher in SPV1994. FIG.8E shows the total fatty acid profiles for SPV1629 and SPV1994. [0036] FIG. 9 includes fatty acid titer profiles for P. dactylifera desaturase- expressing strains. FIG.9A shows C16 fatty acid titers. FIG.9B shows C18 fatty acid titers. [0037] FIG. 10 includes data characterizing SPV1995 expressing the P. dactylifera LPAAT. FIG.10A shows Z11-16Acid/Z9-16Acid selectivity is higher in strain SPV1995 than the control strain SPV1629. FIG.10B shows C16Acids selectivity is higher in SPV1995, due to lower C18 fatty acid titers. FIG.10C shows Z11-16Acid/products selectivity is higher in SPV1995. FIG. 10D shows Z11-16Acid total selectivity is higher in SPV1995. FIG. 10E shows the total fatty acid profiles for SPV1629 and SPV1995. [0038] FIG.11 includes data characterizing SPV1994 and SPV1995, relative to strain SPV1629. FIG.11A shows Z11- 16Acid/Z9-16Acid selectivity is higher in strains SPV1994 and SPV1995. FIG.11B shows C16Acids selectivity is higher in SPV1995, due to lower C18 fatty acid titers. FIG.11C shows Z11-16Acid/products selectivity is higher in SPV1995. FIG. 11D shows Z11-16Acid total selectivity is higher in SPV1995. FIG.11E shows the total fatty acid profiles for SPV1629, SPV1994, and SPV1995. [0039] FIG.12 includes a representation of data showing strain improvements within the SPV458 lineage. FIG. 12A shows Z11-16Acid titers for SPV458 lineage strains. FIG. 12B shows the mass ratio of Z11-16Acid and Z9-18Acid titers for SPV458 lineage strains. FIG.12C shows Z11-16Acid/Z9-16Acid selectivity (SZ11/Z9-16) for SPV458 lineage strains. All data are averages across 6 experiments, run using the Biolector® assay protocol as described in Materials & Methods. [0040] FIG. 13 includes data showing the Z11-16Acid and Z9-16Acid titers from strains in the SPV458 lineage. [0041] FIG. 14 includes data showing the fatty acid selectivities of SPV1629 lineage strains. FIG.14A shows data from a screen of 2xHzDST/1xZ9 DST strains. FIG.14B shows data from a screen of 3xHzDST/1xZ9 DST strains. All strains were screened in a Biolector® assay with an initial seeding density OD600 ≈ 1. The black line represents selectivity of 75%. [0042] FIG. 15 includes data characterizing the properties of select Gen1 and Gen2 blend strains in a Biolector® bioconversion assay. FIG. 15A shows unsaturated C16 (Z11/Z9) titers and selectivity for select Gen1 and Gen2 strains in the Biolector® bioconversion assay with low initial seeding density. FIG.14B shows unsaturated C16 selectivities for select Gen1 and Gen2 strains in the same Biolector® bioconversion assay. Small decreases in selectivity result from a 20% increase in Z9-18Acid and Z11-18Acid relative to SPV1629. [0043] FIG.16 includes data showing the Z11-16Acid/Z9-16Acid selectivities of blend strains in a Biolector® bioconversion assay. FIG.16A shows the results of a repeat Biolector® screen of select strains (initial cell density OD600 ≈ 0.2). FIG.16B shows the results of a panel screen of select Gen1 and Gen2 blend strains in 24-well plate assay (initial cell density OD600 ≈ 15). The black line represents selectivity of 75%. [0044] FIG. 17 includes data characterizing the properties of select Gen1 and Gen2 blend strains in a 24-well plate bioconversion assay. FIG.17A shows unsaturated C16 (Z11/Z9) titers and selectivity for select Gen1 and Gen2 strains in a 24-well plate assay with high initial seeding density. FIG.17B shows unsaturated C16 selectivity for select Gen1 and Gen2 strains in the same 24-well plate assay. [0045] FIG. 18 includes data showing the effect of culture media on the growth of particular blend strains in a Biolector® bioconversion assay. Two media were used. Solulys® is a bioprocess medium that uses Solulys® 95, a glycerol carbon source, and ammonium sulfate as major components. FERM1 is a medium used for small-scale processes that contains yeast extract, a glucose carbon source, and ammonium sulfate as major components. [0046] FIG.19 includes data showing the growth of SPV2076 in a 24-well assay with methyl oleate (Z9-18) supplementation. FIG. 19A shows growth curves of SPV1629 and SPV2076 in FERM1 medium when supplemented with varying concentrations of methyl oleate. FIG.19B shows fatty acid profiles of SPV2076 from the endpoint (90 h) of the same assay. Increasing methyl oleate concentrations from 0.9 g/L to 4.4 g/L did not lead to a further increase in growth rate, but did lead to increased Z9-18Acid (oleic acid) accumulation. Error bars represent the standard error for three replicates. [0047] FIG. 20 includes growth curves of SPV2076 lineage strains in a Biolector® growth assay. FIG. 20A shows data for the SPV1629 control, SPV2076, and SPV2135 in Solulys® medium. FIG.20B shows data for the SPV1629 control, SPV2076, and SPV2135 in FERM1 medium. FIG.20C shows data for the SPV1629 control, SPV2135, and SPV2148 in Solulys® medium. FIG.20D shows data for the SPV1629 control, SPV2135, and SPV2148 in Solulys® medium. Seed cultures were grown in YPD and inoculated to a starting OD of ~1. Error bars represent the standard error for three replicates. [0048] FIG. 21 includes data showing the fatty acid profiles for SPV2076 lineage strains in a Biolector® growth assay. FIG.21A shows data for 70 h in spent media. FIG.21B shows data for 70 h in fresh YPD media. Error bars represent the standard error for three replicates. [0049] FIG. 22 includes data showing the fatty acid profiles for SPV2076 lineage strains in a 24-well bioconversion assay. Fatty acid profiles show all detected fatty acids except for the saturated 16Acid substrate. This was omitted for simplicity, because excess 16Acid remains in the culture. Error bars represent the standard error for four replicates. FIG. 23 shows the full profiles as titers and percentage of total fatty acid. FIG.23A shows results of a Biolector® screen of select strains (initial cell density OD600 ≈ 0.2). FIG.23B shows results of a panel screen of select Gen1 and Gen2 blend strains in a 24-well plate assay (initial cell density OD600 ≈ 15). The black line represents selectivity of 75%. [0050] FIG. 24 includes data showing the fatty acid selectivities of SPV2076 lineage strains in a 24-well bioconversion assay. FIG.24A shows the Z11/Z9-16 selectivity. FIG.24B shows the Z11-16Acid total selectivity for SPV2076 and SPV2135 is higher than that of SPV1629 in the 24-well assay. FIG. 24C shows that the Z11-16Acid selectivity excluding 16Acid is equivalent between SPV2135 and SPV1629. Error bars represented the standard error for four replicates. [0051] FIG. 25 diagrams representative strategies to produce H. zea and C. suppressalis active pheromone blend compositions, as well as pure products, by Z-selective metathesis, distillation, and/or incorporation of Z9-16. [0052] FIG. 26 includes an illustration of the fatty acid profile of strain SPV2028. SPV2028 was selected as the parent of the C. suppressalis blend production strain, because it produces a Z9-16/Z11-16 ratio of 0.15, which is close to a target range of 0.091-0.12. [0053] FIG. 27 illustrates the fatty acid profiles of CoA elongation expression constructs. FIG. 27A includes data showing the full fatty acid profiles for the elongation overexpression library. FIG. 27B includes data showing the 18CoA derived fatty acids produced by each elongation overexpression strain. FIG. 27C includes the composition of target AI precursors of each CoA elongation overexpression strain. [0054] FIG. 28 includes data showing titers of Z9-derived fatty acids in strains containing native and recombinant desaturases. Elongase overexpression in SPV2165 increased production of Z9-18Acid. Deletion of native OLE1 in SPV2480 shows that DST183 as the sole Z9-18Acid producer still produces Z9-18Acid at titers higher than a target of 2 g/L. [0055] FIG. 29 includes predictions made from desaturase binding pocket models and Z9-16/18 fatty acid titers from desaturases utilized in certain embodiments herein. FIG. 29A diagrams a M. sexta binding pocket model: wild-type DST2 and DST3 exert mainly Z11- 16 activity and E/Z14 activity, respectively (Middle); swapping one residue on TM4 is sufficient to switch DST2 and DST3 activity towards weak E/Z14 activity and Z11-16 activity, respectively (Bottom). FIG. 29B diagrams a binding pocket model for wild-type DST192, DST183, and DST245. Compared to DST192 and DST183, DST245 has smaller side chains at three positions of TM2 and bottom positions of TM4. FIG.29C shows the Z9-derived fatty acid titer for wild-type DST183, DST192, and DST245. DST245 shows higher selectivity for C18. [0056] FIG.30 includes data showing Z9-derived fatty acid titers in DST192 mutant strains. FIG. 30A shows fatty acid titers for single point mutants of DST192 in SPV2416. FIG. 30B shows fatty acid titers for DST192 double mutants in the same background. FIG. 30C shows includes a DST192 binding pocket model: G100 mutations produce a trend in selectivity based on amino acid size (Left); bulkier side chains restrict the length of substrate able to fit in the binding pocket (Right). [0057] FIG. 31 includes data showing the fatty acid profiles of pPXA2-driven DST192 variants in SPV2588. FIG.30A shows the fatty acid titers of 2x copy DST192 variant strains and the parent SPV2588 (1x DST183, 1x DST192). FIG. 30B shows the fatty acid distribution of AI precursors, Z9-18Acid, and 18Acid in the same strains. FIG. 30C shows 18Acid and Z9-derived fatty acids in the same strains. Replacing DST183 with DST192 and DST192_I245F reduced Z9-18Acid titers by 52% and 70%, respectively. [0058] FIG. 32 includes growth rate curves for several C. suppressalis blend production strains in Biolector® reactors. Overall growth rates for 2x DST192 variant strains (SPV2665, SPV2667, SPV2668, SPV2669) are reduced compared with the strain SPV2165. [0059] FIG. 33 includes diagrams of pathways from 16ME to Z9-14. Referring to FIG.33A, Pathway 1 shows conversion of Z9-14Acid substrate into the CoA cognate by POX activity of the endogenous acetyl-CoA synthetase. In Pathway 2, acetyl-CoA synthetase converts unsaturated Z11-16Acid to the CoA analog, which can be truncated to Z9-14CoA through POX activity. Finally, Pathway 3 utilizes the cheap and commercially available saturated fatty acid 16ME (methyl palmitate). 16ME is first demethylated through lipase activity, then converted to the CoA cognate by acetyl-CoA synthetase. 16CoA is converted to Z11-16CoA through the activity of HzeaDST, and Z11-16CoA is then chain-shortened to Z9- 14CoA through POX activity. Z9-14 is shown as the CoA for simplicity; however, Z9-14 can be converted to the free acid, the triacyl glyceride cognate, or as another ester in the cell. Z9- 14 may be converted to Z9-14ME through chemical transesterification. Referring to FIG.33B, a Z9-14-specific desaturase that redirects flux toward the desired products (dotted line) was engineered. This pathway co-opts Z11-16ME production strains and thus allows the use of 16ME, a cheap and commercially available feedstock, to access Z9-14. Engineering of POX activity produces a desired ratio of Z11-16CoA:Z9-14CoA. [0060] FIG. 34 includes data showing the selectivity of DST192 mutants against 14CoA, 16CoA, and 18CoA substrates by monitoring Z9-14ME, Z9-16ME, and Z9-19ME formation. Fatty acyl CoAs were converted to methyl ester (ME) form during work-up. See FIGs.38-40 for chromatograms. [0061] FIG. 35 includes homology models of DST192 mutants with 14CoA (built on PDB ID: 4ymk). WT (left), G100I (middle), and G100L (right) are each predicted to accommodate 14:0 substrate without steric hindrance. The GC-MS data (FIG.34) support these models and suggest that neither mutant alters 14:0 binding compared to WT. [0062] FIG.36 includes homology models of DST192 mutants with 16CoA (built on PDB ID: 4ymk). G100I (middle) and G100L (right) cannot accommodate 16:0 substrate, which abuts the tunnel of the active site. The GC-FID data (FIG.34) support these models, which suggest that these mutants occlude the active site and prevent binding of 16CoA. Notably, the substrate does not buttress the active site tunnel in the WT model. [0063] FIG.37 includes homology models of DST192 mutants with 18CoA (built on PDB ID: 4ymk). G100I (middle) and G100L (right) cannot accommodate 18:0 substrate, which abuts the tunnel of the active site. The GC-MS data (FIG. 34) support these models, which suggest that these mutants occlude the active site and prevent binding of 18CoA. Notably, the substrate does not buttress the active site tunnel in the WT model. [0064] FIG. 38 includes GC-FID chromatograms of DST192 WT, DST192 G100I, and DST192 G100L, against a Z9-14 authentic standard (yellow box). Wild type and both mutants produce Z9-14. [0065] FIG. 39 includes GC-FID chromatograms of DST192 WT, DST192 G100I, and DST192 G100L, against a Z9-16 authentic standard (yellow box). Wild type produces high quantities of Z9-16, whereas the mutants produce lower levels of Z9-16. [0066] FIG. 40 includes GC-FID chromatograms of DST192 WT, DST192 G100I, and DST192 G100L, against a Z9-18 authentic standard (yellow box). Wild type produces high quantities of Z9-18, whereas the mutants produce lower levels of Z9-18. [0067] FIG.41 includes homology models of DST192 mutants with 14CoA (built on PDB ID: 4ymk). The guanidino group of the G100R side chain (left) occludes the active site, preventing productive binding of 14CoA and longer substrates. The phenyl group of the G100F side chain (right) buttresses the opposing helix of the substrate tunnel. [0068] FIG. 42 includes the Z11-16Acid titer and selectivity correlation plot for strains expressing recombinant acyl transferases when cultured with methyl palmitate supplementation. The scatter plot shows Z11-16Acid titer as a function of all other fatty acid products (excluding the 16ME substrate). Replicates for all screened clones are shown as points with colors corresponding to each genetic modification. Strains with improved titer lie above the data points for the SPV1629 control, and strains with improved selectivity lie above and to the left of the control points. Lines indicating 50%, 60%, and 70% Z11-16 products selectivity (SZ11-16_Prod) are also shown. Higher Z11-16Acid titers were observed with expression of DGATs. [0069] FIG. 43 includes the Z11-16Acid titer and selectivity correlation plot for strains overexpressing native lipid metabolism genes when cultured with methyl palmitate supplementation. The scatter plot shows Z11-16Acid titer as a function of all other fatty acid products (excluding the 16ME substrate). Replicates for all screened clones are shown as points with colors corresponding to each genetic modification. Strains with improved selectivity lie above and to the left of the control points. Lines indicating 50%, 60%, and 70% Z11-16 products selectivity (SZ11-16_Prod) are also shown. Overexpressing native lipid metabolism genes tended to increase synthesis of fatty acids, especially C18 fatty acids. [0070] FIG. 44 includes data showing the GC-FAME analysis of fatty acid profiles for strains expressing recombinant acyltransferases when cultured with methyl palmitate substrate. FIG. 44A shows average fatty acid profiles for all clonal replicates for each acyltransferase construct. Error bars represent the standard error from 4 replicates. FIG.44B includes a stacked bar graph showing the average titers of each fatty acid. The total fatty acid titer is shown in the label above the bar. [0071] FIG. 45 includes the Z11-16Acid titer and selectivity correlation plot for strains expressing recombinant acyltransferases when cultured without methyl palmitate supplementation. The scatter plot shows Z11-16Acid titer as a function of all other fatty acid products (excluding the 16ME substrate). Replicates for all screened clones are shown as points with colors corresponding to each genetic modification. Strains with improved titer lie above the data points for the SPV1629 control, and strains with improved selectivity lie above and to the left of the control points. Lines indicating 35-60% Z11-16 total selectivity (SZ11- 16_Tot) are also shown. Higher Z11-16Acid titers were observed with expression of DGATs. [0072] FIG. 46 includes data showing the GC-FAME analysis of fatty acid profiles for strains expressing recombinant acyltransferases when cultured without methyl palmitate substrate. FIG. 46A shows average fatty acid profiles for all clonal replicates for each acyltransferase construct. Error bars represent the standard error from 4 replicates. FIG.46B shows a stacked bar graph of the average titers of each fatty acid. The total fatty acid titer is shown in the label above the bar. [0073] FIG. 47 includes the Z11-16Acid titer and selectivity correlation plot for strains overexpressing native lipid metabolism genes when cultured without methyl palmitate supplementation. The scatter plot shows Z11-16Acid titer as a function of all other fatty acid products (excluding the 16ME substrate). Replicates for all screened clones are shown as points with colors corresponding to each genetic modification. Strains with improved selectivity lie above and to the left of the control points. Lines indicating 35-60% Z11-16 total selectivity (SZ11-16_Tot) are also shown. Overexpressing native lipid metabolism genes tended to increase the synthesis of fatty acids, especially C18 fatty acids. Overexpression the mutant MGA2 activator domain increased Z11-16Acid titer and significantly increased C18 fatty acid titers. [0074] FIG. 48 includes data showing the GC-FAME analysis of fatty acid profiles for strains overexpressing native lipid metabolism genes when cultured without methyl palmitate substrate. FIG.48A shows the average fatty acid profiles for all clonal replicates for each construct. Error bars represent the standard error from 4 replicates. FIG. 48B shows a stacked bar graph of the average titers of each fatty acid. The total fatty acid titer is shown in the label above the bar. [0075] FIG.49 includes data showing the fatty acid profiles of select strains in a 1 L bioconversion process at 40 hours. Fatty acid profiles displayed increased Z11-16Acid titers for SPV2473 and SPV2474, and increased Z9-18Acid content in all Gen5 strains. The most significant Z9-18Acid increase is observed for SPV2474 (12.7 g/L). [0076] FIG. 50 includes Z11-16Acid and lipid content time course data for select strains in a 1 L bioconversion process. FIG.50A shows that increased Z11-16Acid production was most pronounced in the earlier phase of the bioconversion for SPV2474 and was sustained across the bioconversion for SPV2473. Z11-16Acid titer was normalized to the initial working volume to account for differences in volume changes over the course of the bioconversions. FIG. 50B shows that the lipid fraction is higher for strains expressing multiple recombinant DGATs. Lipid accumulated rapidly in SPV2474, nearly reaching peak value by 35 hours. Lipid content was calculated as the total mass of product fatty acids (all fatty acids except 16Acid) per gram dry cell weight. [0077] FIG. 51 includes data showing substrate loading and lipid accumulation for strains in a 1 L bioconversion process. SPV2473 and SPV2474 accumulated more lipid and product TAG over the fermentation. Total lipid accumulation and product lipid accumulation (Total TAG) approaches or exceeds the substrate loading rate for these strains, and additional improvements in titer are achieved with an alternative feeding schedule. Titers were normalized to the initial working volume to adjust for volume differences. Total lipids = all fatty acids. Total TAG = all fatty acids except 16Acid substrate. [0078] FIG. 52 includes fatty acid titer time course data for strains in a 1 L bioconversion process. All titers were normalized to the initial working volume to account for volume differences. [0079] FIG. 53 includes a graph of DST192G100L strains engineered to produce Spodoptera frugiperda precursors. [0080] FIG. 54 includes Pan7-F1 and F2 bioconversion profile of DO, temperature, pH, OUR and feeds. The drop in OUR profile after 38h is due to the closure of the offgas valve once the foam probe detected foaming. [0081] FIG. 55 includes a comparison of Pan7-F1 and F2 (65 L initial volume) and FERM137-7 (0.9 L initial volume) bioconversion profile: biomass, ChiSu components Z11- 16FAME/TAG, Z9-16 FAME/TAG, Z13 FAME/TAG titers, and byproducts Z9-18 FAME/TAG, Z11-18 FAME/TAG titer. SEQUENCE LISTING [0001] The nucleic acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, as defined in 37 C.F.R. § 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. In the accompanying sequence listing: [0002] SEQ ID NO:1 shows an exemplary ACX1 nucleotide sequence: ATGGAGGGCATCGACCACCTGGCCGATGAGCGGAATAAGGCCGAGTTCGACGTGG AGGATATGAAGATCGTGTGGGCCGGCAGCAGACACGCCTTTGAGGTGTCCGATAG GATCGCACGCCTGGTGGCATCCGACCCCGTGTTCGAGAAGTCCAATAGGGCCCGC CTGTCTCGGAAGGAGCTGTTCAAGTCTACCCTGAGAAAGTGCGCCCACGCCTTTAA GAGGATCATCGAGCTGCGCCTGAACGAGGAGGAGGCAGGCCGGCTGAGACACTTC ATCGACCAGCCTGCCTACGTGGATCTGCACTGGGGCATGTTTGTGCCAGCCATCAA GGGCCAGGGCACCGAGGAGCAGCAGAAGAAGTGGCTGAGCCTGGCCAATAAGAT GCAGATCATCGGCTGTTATGCCCAGACAGAGCTGGGACACGGATCCAACGTGCAG GGACTGGAGACCACAGCCACCTTCGACCCAAAGACAGATGAGTTTGTGATCCACA CACCAACCCAGACAGCATCCAAGTGGTGGCCAGGAGGCCTGGGCAAGGTGTCTAC CCACGCCGTGGTGTACGCCAGGCTGATCACAAATGGCAAGGATTATGGCATCCAC GGCTTCATCGTGCAGCTGCGCTCTCTGGAGGATCACAGCCCCCTGCCTAACATCAC CGTGGGCGACATCGGCACAAAGATGGGCAACGGCGCCTACAATAGCATGGACAAC GGCTTCCTGATGTTTGATCACGTGCGGATCCCCAGAGACCAGATGCTGATGCGGCT GAGCAAGGTGACCAGAGAGGGCGAGTATGTGCCATCCGATGTGCCCAAGCAGCTG GTGTACGGCACCATGGTGTATGTGCGGCAGACAATCGTGGCAGACGCAAGCAACG CACTGTCCAGGGCCGTGTGCATCGCAACACGCTACTCTGCCGTGCGGAGACAGTTT GGCGCCCACAATGGCGGCATCGAGACCCAGGTCATCGACTATAAGACACAGCAGA ACAGGCTGTTCCCACTGCTGGCATCCGCCTACGCCTTCCGCTTTGTGGGCGAGTGG CTGAAGTGGCTGTATACCGATGTGACAGAGAGGCTGGCCGCCTCTGACTTTGCCAC CCTGCCTGAGGCACACGCATGTACAGCAGGCCTGAAGAGCCTGACCACAACCGCA ACCGCAGATGGAATCGAGGAGTGCAGGAAGCTGTGCGGAGGACACGGATACCTGT GGTGCAGCGGACTGCCCGAGCTGTTTGCCGTGTACGTGCCTGCCTGTACCTATGAG GGCGACAATGTGGTGCTGCAGCTGCAGGTGGCCAGATTCCTGATGAAGACAGTGG CCCAGCTGGGCTCTGGCAAGGTGCCTGTGGGAACAACCGCATACATGGGAAGGGC AGCACACCTGCTGCAGTGCCGCAGCGGAGTGCAGAAGGCCGAGGACTGGCTGAAC CCAGATGTGGTGCTGGAGGCCTTCGAGGCAAGGGCCCTGCGCATGGCCGTGACCT GCGCCAAGAATCTGAGCAAGTTCGAGAACCAGGAGCAGGGCTTTCAGGAGCTGCT GGCAGATCTGGTGGAGGCAGCAATCGCCCACTGTCAGCTGATCGTGGTGTCCAAG TTTATCGCCAAGCTGGAGCAGGACATCGGCGGCAAGGGCGTGAAGAAGCAGCTGA ACAATCTGTGCTACATCTATGCCCTGTACCTGCTGCACAAGCACCTGGGCGATTTC CTG TCCACCAATTGTATCACACCCAAGCAGGCTAGCCTGGCCAACGACCAGCTGCGGA GCCTGTATACCCAGGTGAGACCTAACGCCGTGGCCCTGGTGGACGCCTTTAATTAC ACAGATCACTATCTGAACTCCGTGCTGGGCCGGTACGATGGCAACGTGTACCCAA AGCTGTTCGAGGAGGCCCTGAAGGACCCTCTGAACGATTCTGTGGTGCCAGACGG CTACCAGGAGTATCTGAGACCCGTGCTGCAGCAGCAGCTGAGGACCGCAAGACTG TGA [0003] SEQ ID NO:2 shows the ACX1 amino acid sequence encoded by SEQ ID NO:1: MEGIDHLADERNKAEFDVEDMKIVWAGSRHAFEVSDRIARLVASDPVFEKSNRARL SRKELFKSTLRKCAHAFKRIIELRLNEEEAGRLRHFIDQPAYVDLHWGMFVPAIKGQ GTEEQQKKWLSLANKMQIIGCYAQTELGHGSNVQGLETTATFDPKTDEFVIHTPTQT ASKWWPGGLGKVSTHAVVYARLITNGKDYGIHGFIVQLRSLEDHSPLPNITVGDIGT KMGNGAYNSMDNGFLMFDHVRIPRDQMLMRLSKVTREGEYVPSDVPKQLVYGTM VYVRQTIVADASNALSRAVCIATRYSAVRRQFGAHNGGIETQVIDYKTQQNRLFPLL ASAYAFRFVGEWLKWLYTDVTERLAASDFATLPEAHACTAGLKSLTTTATADGIEE CRKLCGGHGYLWCSGLPELFAVYVPACTYEGDNVVLQLQVARFLMKTVAQLGSGK VPVGTTAYMGRAAHLLQCRSGVQKAEDWLNPDVVLEAFEARALRMAVTCAKNLS KFENQEQGFQELLADLVEAAIAHCQLIVVSKFIAKLEQDIGGKGVKKQLNNLCYIYA LYLLHKHLGDFLSTNCITPKQASLANDQLRSLYTQVRPNAVALVDAFNYTDHYLNS VLGRYDGNVYPKLFEEALKDPLNDSVVPDGYQEYLRPVLQQQLRTARL [0004] SEQ ID NO:3 shows an exemplary PxACX1 nucleotide sequence: ATGTCCGCCAAGGTGAACCCTGATCTGCAGAGGGAGCGCGACAAGTGTACCTTCA ACGTGACCGAGCTGACAAATCTGATCGATGGCGGCGTGCAGAATACAGAGGAGCG GAAGAGACAGGAGGACCTGCTGCTGAAGGAGGGCATCCACATCGAGGAGGTGCC ATCCGAGTACCTGTCTCACAAGGAGAAGTATGAGCTGGCCGTGAAGAAGGCCTGC CTGCTGTTCAAAGTGATCCGGAGAATGCAGGAGGAGGAGAACACCGGCATGGAGA ATTACCGGTCCGTGCTGGGCGGAAACCTGGGCTCCGCCATCCTGGCAGATGGATCT CCTCTGACCCTGCACTATGTGATGTTCATCCCAACCATCCTGGGCCAGGCAACAGT GGAGCAGCAGGCATACTGGATCGGAAGGGCCTTTAATCTGGATATCATCGGCACC TATGCCCAGACAGAGCTGGGCCACGGCACCTTCATCAGAGGCCTGGAGACCACAG CCACCTACGACCCAAGCACAAAGGAGTTTGTGCTGCACTCCCCCACCCTGACCAGC TACAAGTGGTGGCCAGGAGGACTGGCCCACACCGCCAACTACTGTATCGTGATGG CCCAGCTGTATACAAAGGGCCAGTGCCACGGCCTGCACCCCTTCATCGTGCAGCTG CGGGACGAGGAGACCCACATGCCTCTGAAGGGCATCAAGATCGGCGAGATCGGCG TGAAGCTGGGCATGAATGGCACAAACAATGGCTTCCTGGGCTTTGAGCACGTGAG GATCCCCAGAGAGAACATGCTGATGAAGAATTCCAAGGTGCTGGAGGATGGCACC TACGTGCACTCTCTGAGCTCCAAGCTGACCTATGGCACAATGATGTTCGTGAGGGT GGTGCTGGTGACAGATATGTGCAACTACATGGCCAAGGCCGTGACCATCGCCACA CGCTATAGCGCCGTGAGGCGCCAGTCCCAGCCAAAGCCCGATGAGCCTGAGCCAC AGATTCTGGACTACGTGACCCAGCAGCACAAGCTGATGATCGGCATCAGCACAGT GCACGCCTTTAGGCTGTCCGCCAATTGGCTGTGGCATATGTACAACAATGTGATCG CAGAGCTGGACACCGGCGATCTGGAGCGCCTGCCAGAGCTGCACGCACTGTCTTG CTGTCTGAAGGCAGTGACCACAGCCGATGCCGCCGAGTGCGTGGAGAGGTGTCGC CTGAGCTGCGGAGGACACGGATACATGCTGTCTAGCTCCCTGCCAACCACATACG GCCTGGTGACCGCAGCCTGTACATATGAGGGCGAGAACACCGTGCTGCTGCTGCA GACAGCCAGGTACCTGGTGAAGGCATGGCAGCAGGCAATGGGAGGAAATGCACT GACCCCTACAGTGTCCTATATCTCTGAGGTGAGCACCGGCCGGAGATCTCCCCCTT GGGATAACAGCGTGCAGGGCGTGATCCACGGATTCCACAGGGTGGCAGCAGGCAA GATCGGAATGTGCGTGGCCAACATCGAGAAGAGACAGAAGAGCGGCCTGTGCTAC GAGGACGCCTGGAATATGACCTCCGTGCAGCTGGCATCTGCCAGCGAGTCCCACT GCAGGGCAATCCTGCTGAGCACCTACTATTCCGAGACAGAGAAGCTGGCCAGCAA GGTGACCCCCGCCCTGAAGACAGTGCTGCTGCAGCTGGTGGATCTGTACGTGGTGT ATTGGGCACTGCAGAGGGTGGGCGACCTGCTGCGCTTTACCTCTATCAGCGAGCGG GATATCGAGCAGCTGCAGAACTGGTACGAGGACCTGCTGACACGGCTGAGACCTA ATGCCGTGGGCCTGGTGGACGCCTTCGACTTCAGAGACGAGATCCTGCACTCTGCC CTGGGCAGCTACGATGGCCGGGTGTATGAGAGACTGATGGAGGAGGCCCTGAAGT CTCCCCTGAACGCCCAGCCTGTGAATGACAGCTTCCACAAGTATCTGAAGCCCTTT ATGCAGGGCAAGCTGTGA [0005] SEQ ID NO:4 shows the PxACX1 amino acid sequence encoded by SEQ ID NO:3: MSAKVNPDLQRERDKCTFNVTELTNLIDGGVQNTEERKRQEDLLLKEGIHIEEVPSEYL SHKEKYELAVKKACLLFKVIRRMQEEENTGMENYRSVLGGNLGSAILADGSPLTLHY VMFIPTILGQATVEQQAYWIGRAFNLDIIGTYAQTELGHGTFIRGLETTATYDPSTKEFV LHSPTLTSYKWWPGGLAHTANYCIVMAQLYTKGQCHGLHPFIVQLRDEETHMPLKGI KIGEIGVKLGMNGTNNGFLGFEHVRIPRENMLMKNSKVLEDGTYVHSLSSKLTYGTM MFVRVVLVTDMCNYMAKAVTIATRYSAVRRQSQPKPDEPEPQILDYVTQQHKLMIGI STVHAFRLSANWLWHMYNNVIAELDTGDLERLPELHALSCCLKAVTTADAAECVERC RLSCGGHGYMLSSSLPTTYGLVTAACTYEGENTVLLLQTARYLVKAWQQAMGGNAL TPTVSYISEVSTGRRSPPWDNSVQGVIHGFHRVAAGKIGMCVANIEKRQKSGLCYEDA WNMTSVQLASASESHCRAILLSTYYSETEKLASKVTPALKTVLLQLVDLYVVYWALQ RVGDLLRFTSISERDIEQLQNWYEDLLTRLRPNAVGLVDAFDFRDEILHSALGSYDGRV YERLMEEALKSPLNAQPVNDSFHKYLKPFMQGKL [0006] SEQ ID NO:5 shows an exemplary BaACX3 nucleotide sequence: ATGGAGGACCTGTCTTTTGTGCCCGAGCTGCCTAGCGGACCACTGGACGTGTACCG GAACAATGCCTCCTTCAACTGGAAGAGACTGAAGCTGGCCCTGGAGGGCGACTTT GATCTGCTGAAGCTGAAGTACAAGATCTGGAGGACCCTGGAGAAGGACCCCCTGT TCGCCCACAGCCCCATCACCCTGCCTGTGGAGGAGCAGAAGCGCATCACACAGCT GCAGCTGAAGAAGATCAACGAGTACAAGTTCATCCCCAAGGAGATGTTTAATGCC TCCTATTCTAAGCGGACCAGAACAATCATGACCATCAACGAGGCCGTGCAGTCCCT GAATCCTAGCGTGTCCGTGAAGATGGCCATCGGCATCTACCTGTTTTCCAATGCCC TGCTGTCTCTGGGCACCGAGAGGCACCTGAAGTTCTATGAGGCCACAATCAAGCA CCGCGAGATCCTGGCCTCTTTTAGCCTGACCGAGATCGCACACGGAAGCGACGCA AGGCTGATGAGAACCACAGCCACCTATGATCCCTCCACACGGGAGTTCGTGGTGC ACACACCTGATTTTGAGGCAGCAAAGTGCTGGGTGGGAAACCTGGGCAGAACCTG TACACACACCCTGCTGTTCGCCCAGCTGATCACACCAGACGGCACCAATCACGGCC TGCACGGCTTTGTGGTGCCAATCAGGAAGCCCGATACACTGGAGACCTACCCCGG CCTGATCGTGGGCGACATGGGAGAGAAGATCGGCGTGAACGGCATCGATAATGGC TTCATCATGTTTAACCAGTATAGGATCCCTCGCGAGAACCTGCTGAATCGCACAGC CGACGTGACCGAGGATGGCACATACGAGAGCTCCTTCTCCGAGCCATCTAGGATC CTGGGCGCCGCCCTGGAGAATCTGAGCGCCGGCCGCATCGGCATCATGCAGGAGA GCTGCCACTCCCTGTCTAGCGCCGTGACCATCGCCGTGAGGTACGCAGCAACAAG GACCCAGTTCGGAGAGAGGGACAGGGAGACACCCCTGATCGAGTACGAGCTGCAC CAGTGGCGGCTGTTTGGCTACGTGAGCGCCGCCATCGTGTTCAGACTGTACATCGA CCTGTTTGCCCACACCTATCTGAACATCGTGGAGAAGAGCAATGCCGGCCACCGG GTGGATAACCTGTCTGAGGCCGTGAGCGAGATCCACGCCATGGTGTCCTCTAGCAA GCCACTGCTGACCTGGACCACACTGAGGGCAGCACAGCAGTGCAGAGAGGCATGT GGAGGACACGGATACCTGAAGTGTGCCAACCTGGGCGACATCCGGTCCAATCACG AGCCCACAGTGACCTATGAGGGCGATAACAATGTGCTGTCTCAGCAGGCCGGCAA TTGGCTGCTGAGACAGTACGAGGCAGCAATCGCAGGCAAGCCTGTGGACTCCCCA CTGGGCACCGTGGCCTTCCTGAAGGACCTGCGGACAATCAAGGATCGGACCTTCA CCTGCAAGACCACAAAGGAGCTGAAGGATCCTCAGTTCATCATCTCTACCTACAAG TGGCTGCTGTGCTGGATGCTGAAGTATACACACGAGAAGAACGAGGCCAATCTGT CCCAGGGCCTGACCAAGGTGCAGGCCAGGACAAAGTCTCAGGTGTATCGCTGGAA GACACTGACCAAGGTGTACGCCGAGTATATCACCCTGGTGTTCTGCCTGGACAACA TCGATAAGAAGGAGAGAGAGCTGCAGCCTATGCTGCTGAAGCTGTTTTGTCTGTAC GGCCTGTGGAGCCTGGACGAGAATCTGGTGGAGCTGTATCAGGGAGGATTCGCAA AGGGAGAGGAGTGCGCACGGCTGGTGAGAGATTCTGTGCTGGAGCTGTGCGGCGA GGTGAAGAACGAGATCGTGAGCGTGGCAGACGCACTGGCACCAACCGATTTTGTG CTGAACAGCGTGCTGGCCAAGTCCGACGGCAAGCTGTACCAGAATCTGCAGAAGG CCTTCTTTAGCCACCCCGGCGTGTTCGAGAGGACACCTTGGTGGCGCGATGTGGTG CCAGCCTCCAAGCTGTGA [0007] SEQ ID NO:6 shows the BaACX3 amino acid sequence encoded by SEQ ID NO:5: MEDLSFVPELPSGPLDVYRNNASFNWKRLKLALEGDFDLLKLKYKIWRTLEKDPLFA HSPITLPVEEQKRITQLQLKKINEYKFIPKEMFNASYSKRTRTIMTINEAVQSLNPSVSVK MAIGIYLFSNALLSLGTERHLKFYEATIKHREILASFSLTEIAHGSDARLMRTTATYDPS TREFVVHTPDFEAAKCWVGNLGRTCTHTLLFAQLITPDGTNHGLHGFVVPIRKPDTLE TYPGLIVGDMGEKIGVNGIDNGFIMFNQYRIPRENLLNRTADVTEDGTYESSFSEPSRIL GAALENLSAGRIGIMQESCHSLSSAVTIAVRYAATRTQFGERDRETPLIEYELHQWRLF GYVSAAIVFRLYIDLFAHTYLNIVEKSNAGHRVDNLSEAVSEIHAMVSSSKPLLTWTTL RAAQQCREACGGHGYLKCANLGDIRSNHEPTVTYEGDNNVLSQQAGNWLLRQYEAA IAGKPVDSPLGTVAFLKDLRTIKDRTFTCKTTKELKDPQFIISTYKWLLCWMLKYTHEK NEANLSQGLTKVQARTKSQVYRWKTLTKVYAEYITLVFCLDNIDKKERELQPMLLKL FCLYGLWSLDENLVELYQGGFAKGEECARLVRDSVLELCGEVKNEIVSVADALAPTD FVLNSVLAKSDGKLYQNLQKAFFSHPGVFERTPWWRDVVPASKL [0008] SEQ ID NO:7 shows an exemplary PxACX3 nucleotide sequence: ATGGCCGACCTGTCTTTCATCCCAGATCTGCCCAGCGGCCCTCTGGACTGCTATAG GTCCACAGCCTCTTTTGATTGGAAGCAGCTGAAGCTGTCCCTGGAGGGCGACCTGG ATCTGCTGAAGCTGAAGTACAAGATCTGGAAGACCCTGGAGAAGGACCTGCTGTT CGCCCCCCCTTCTGTGACACCTAGCGTGGAGGAGCAGAAGCGCATCACCCAGCTG CAGCTGAAGAAGATCAACAATTACAAGTTCATGACCTATGAGATGTTTAAGAGCT CCTACTCTAAGAGGACACGCGCCATCATGACCATCAACGAGGCCGTGCAGAGCGT GAATCCAAGCGTGTCCGTGAAGATGGCCATCGGCATCTACCTGTTCAGCAACGCCC TGCTGTCCCTGGGCACAGAGAGGCACTATAAGTTCTTTGAGGCCACCATCTGGAAT CGCGAGATCCTGGCCTCCTTTGCCCTGACAGAGGTGGCACACGGATCTGACGCAA GGCTGATGTGCACCACAGCCTCTTATGATCCTAAGAGCAGAGAGTTCGTGGTGCAC ACCCCAGACTTCAAGGCAGCAAAGTGCTGGGTGGGAAACCTGGGCAGGACCTGTA CACACACCCTGCTGTTCGCCCAGCTGATCACACCTGACGGCACCAATCACGGCCTG CACGGCTTTGTGGTGCCAGTGAGAGATCCCGCCACACTGGAGACCTACCCAGGCCT GGTGGTGGGCGACATGGGAGAGAAGATCGGCGTGAACGGCATCGATAATGGCTTC ATCATGTTTAACCAGTATCGGATCCCCAGAGAGAACCTGCTGAATCGGACAGCCG ACGTGACCGAGGATGGCGTGTACGAGTCTAGCTTCAGCGAGCCTTCCAAGATGCT GGGAGCCGCCCTGGAGAATCTGAGCGCCGGCAGGATCGGCATCATGCAGGAGTCC TGCCACACAATCTCCTCTGCCGTGGCAATCGCAATCAGATACGCAGCAACCCGGA GACAGTTCGGCGCCAAGGACGATGAGATCCCCCTGCTGGAGTACGAGCTGCACCA GTGGAGACTGTTTGGATACGTGAGCGCCGCAGTGGTGTTCCGGGTGTACATCGAG AAGTTTGCCAGAATCTATCTGAACATCGTGGAGAAGAGCAATGCCGGCCTGAAGG TGGACAACCTGTCCGCCGCCGTGTCTGAGATCCACGCCATGGTGTCTTGTAGCAAG CCTCTGCTGACCTGGACCACACTGGATGCAGTGCAGCAGTGCAGGGAGGCATGTG GAGGACACGGCTACCTGAAGTGCGCCAACCTGGGCGACCTGAGATGTAATCACGA GCCAACAGTGACCTATGAGGGCGATAACAATGTGCTGAGCCAGCAGGCCGGCAAT TGGCTGCTGAGGCAGTACGAGCAGGCAGCAGGAGGAGTGGACTCCCCCCTGGGCA CAGTGACCTTCCTGACAAGGTGCCAGGACATTCTGGAGGATAAGTTTTCCTGTGAG CGCACCGACCAGCTGAAGGATGTGAAGTTCATCACATCTACCTACCAGTGGCTGCT GTGCTGGCTGCTGAAGGAGACCCACCAGCAGTATGAGTCTGAGGTGAGCAAGGGC CAGAGCAAGTTCGAGGCCAAGTCCAAGTGCCAGGTGTATAGGTGGAAGACACTGT CCCGCGTGTACGCCGAGTATCTGGCCATCATCTTTTGTTCCGAGTCTGTGAGCCAG GAGGAGACCGGACTGAGACCCGTGCTGAACAAGCTGCTGGTCATGTACGGCCTGT GGAGCCTGGACCAGCACCTGGTGGAGCTGTATCAGGGCGGCTTTGCCTCCGGCGA TTCTCTGGCAAGGCTGCTGAGGGCAGCAATTCTGGAGATGTGCGCACAGCTGAAG CCAGAGGTGGTGTCCGTGGTGGACGCACTGGCACCTACCGATTTCGTGCTGAACTC CGTGCTGGGCAAGTCTGACGGCAACCTGTACCAGAATCTGCAGAAGGCCCTGTTTG GAGGCCCAGGCGCAATGGAGAGGGCAAGCTGGTGGAGAGAGGTGGTGAATAGCG CCCCCTCCAAGCTGTGA [0009] SEQ ID NO:8 shows the PxACX3 amino acid sequence encoded by SEQ ID NO:7: MADLSFIPDLPSGPLDCYRSTASFDWKQLKLSLEGDLDLLKLKYKIWKTLEKDLLFAPP SVTPSVEEQKRITQLQLKKINNYKFMTYEMFKSSYSKRTRAIMTINEAVQSVNPSVSVK MAIGIYLFSNALLSLGTERHYKFFEATIWNREILASFALTEVAHGSDARLMCTTASYDP KSREFVVHTPDFKAAKCWVGNLGRTCTHTLLFAQLITPDGTNHGLHGFVVPVRDPAT LETYPGLVVGDMGEKIGVNGIDNGFIMFNQYRIPRENLLNRTADVTEDGVYESSFSEPS KMLGAALENLSAGRIGIMQESCHTISSAVAIAIRYAATRRQFGAKDDEIPLLEYELHQW RLFGYVSAAVVFRVYIEKFARIYLNIVEKSNAGLKVDNLSAAVSEIHAMVSCSKPLLT WTTLDAVQQCREACGGHGYLKCANLGDLRCNHEPTVTYEGDNNVLSQQAGNWLLR QYEQAAGGVDSPLGTVTFLTRCQDILEDKFSCERTDQLKDVKFITSTYQWLLCWLLKE THQQYESEVSKGQSKFEAKSKCQVYRWKTLSRVYAEYLAIIFCSESVSQEETGLRPVL NKLLVMYGLWSLDQHLVELYQGGFASGDSLARLLRAAILEMCAQLKPEVVSVVDAL APTDFVLNSVLGKSDGNLYQNLQKALFGGPGAMERASWWREVVNSAPSKL [0010] SEQ ID NO:9 shows an exemplary Y. lipolytica POX2 nucleotide sequence: ATGAATCCTAACAATACCGGCACAATCGAGATCAACGGCAAGGAGTATAATACCT TCACAGAGCCACCTGTGGCAATGGCACAGGAGAGGGCCAAGACAAGCTTCCCTGT GCGCGAGATGACCTACTTTCTGGATGGCGGCGAGAAGAACACACTGAAGAATGAG CAGATCATGGAGGAGATCGAGCGGGACCCACTGTTTAACAACGATAACTACTACG ACCTGAATAAGGAGCAGATCAGGGAGCTGACCATGGAGCGCGTGGCCAAGCTGAG CCTGTTCGTGCGGGATCAGCCAGAGGACGATATCAAGAAGAGATTTGCCCTGATC GGCATCGCCGACATGGGCACCTACACAAGGCTGGGCGTGCACTATGGACTGTTCTT TGGAGCCGTGCGCGGAACCGGAACAGCAGAGCAGTTCGGACACTGGATCTCCAAG GGAGCAGGCGATCTGAGAAAGTTCTATGGCTGCTTTTCCATGACAGAGCTGGGCC ACGGCTCTAATCTGGCCGGCCTGGAGACCACAGCCATCTACGATGAGGAGACCGA CGAGTTTATCATCAACACACCACACATCGCCGCCACCAAGTGGTGGATCGGAGGA GCCGCCCACACCGCCACACACACCGTGGTGTTCGCAAGGCTGATCGTGAAGGGCA AGGACTATGGCGTGAAGACCTTTGTGGTGCAGCTGAGAAACATCAATGATCACAG CCTGAAGGTCGGCATCTCCATCGGCGACATCGGCAAGAAGATGGGCAGGGATGGC ATCGACAACGGCTGGATCCAGTTCACCAATGTGAGGATCCCCAGACAGAACCTGC TGATGAAGTATACAAAGGTGGATAGGGAGGGAAACGTGACCCAGCCACCACTGGC ACAGCTGACATACGGCTCTCTGATCACCGGCAGAGTGAGCATGGCAAGCGACTCC CACCAAGTGGGCAAGCGGTTCATCACAATCGCCCTGAGATACGCCTGCATCCGGA GACAGTTTTCCACCACACCCGGCCAGCCTGAGACCAAGATCATCGATTACCCTTAT CACCAGAGGCGCCTGCTGCCACTGCTGGCATACGTGTATGCCCTGAAGATGACAG CCGACGAAGTGGGCGCCCTGTTCTCTAGGACCATGCTGAAGATGGACGATCTGAA GCCCGACGATAAGGCCGGCCTGAATGAGGTGGTGTCTGACGTGAAGGAGCTGTTC TCTGTGAGCGCCGGCCTGAAGGCCTTTAGCACATGGGCCTGCGCCGATGTGATCGA CAAGACCAGGCAGGCATGTGGAGGACACGGCTACTCCGGCTATAACGGCTTTGGC CAGGCCTATGCCGATTGGGTGGTGCAGTGTACATGGGAGGGCGACAACAATATCC TGACCCTGAGCGCCGGCAGGGCCCTGATCCAGTCCGCCGTGGCCCTGAGGAAGGG AGAGCCAGTGGGCAATGCCGTGTCCTACCTGAAGCGGTATAAGGATCTGGCCAAC GCCAAGCTGAATGGCAGATCTCTGACAGACCCTAAGGTGCTGGTGGAGGCATGGG AGGTGGCAGCAGGCAACATCATCAATCGGGCCACCGATCAGTACGAGAAGCTGAT CGGAGAGGGCCTGAACGCAGACCAGGCCTTCGAGGTGCTGAGCCAGCAGAGGTTT CAGGCCGCCAAGGTGCACACACGGAGACACCTGATCGCCGCCTTCTTTTCCCGCAT CGATACCGAGGCCGGCGAAGCCATCAAGCAGCCACTGCTGAATCTGGCCCTGCTG TTCGCCCTGTGGTCCATCGAGGAGGACTCTGGCCTGTTCCTGAGGGAGGGCTTTCT GGAGCCCAAGGATATCGACACAGTGACCGAGCTGGTGAACAAGTACTGCACCACA GTGCGCGAGGAAGTGATCGGCTACACCGATGCCTTCAATCTGTCTGACTACTTCAT CAACGCCCCCATCGGCTGTTACGATGGCGACGCCTACCGGCACTATTTCCAGAAGG TGAACGAGCAGAATCCAGCCCGGGATCCCAGACCTCCATACTATGCCAGCACCCT GAAGCCTTTCCTGTTTAGAGAGGAGGAGGACGATGACATCTGTGAGCTGGACGAG GAGTGA [0011] SEQ ID NO:10 shows the POX2 amino acid sequence encoded by SEQ ID NO:9: MNPNNTGTIEINGKEYNTFTEPPVAMAQERAKTSFPVREMTYFLDGGEKNTLKNEQIM EEIERDPLFNNDNYYDLNKEQIRELTMERVAKLSLFVRDQPEDDIKKRFALIGIADMGT YTRLGVHYGLFFGAVRGTGTAEQFGHWISKGAGDLRKFYGCFSMTELGHGSNLAGLE TTAIYDEETDEFIINTPHIAATKWWIGGAAHTATHTVVFARLIVKGKDYGVKTFVVQL RNINDHSLKVGISIGDIGKKMGRDGIDNGWIQFTNVRIPRQNLLMKYTKVDREGNVTQ PPLAQLTYGSLITGRVSMASDSHQVGKRFITIALRYACIRRQFSTTPGQPETKIIDYPYHQ RRLLPLLAYVYALKMTADEVGALFSRTMLKMDDLKPDDKAGLNEVVSDVKELFSVS AGLKAFSTWACADVIDKTRQACGGHGYSGYNGFGQAYADWVVQCTWEGDNNILTL SAGRALIQSAVALRKGEPVGNAVSYLKRYKDLANAKLNGRSLTDPKVLVEAWEVAA GNIINRATDQYEKLIGEGLNADQAFEVLSQQRFQAAKVHTRRHLIAAFFSRIDTEAGEA IKQPLLNLALLFALWSIEEDSGLFLREGFLEPKDIDTVTELVNKYCTTVREEVIGYTDAF NLSDYFINAPIGCYDGDAYRHYFQKVNEQNPARDPRPPYYASTLKPFLFREEEDDDICE LDEE [0012] SEQ ID NO:11 shows an exemplary AtACX2 nucleotide sequence: ATGGAATCAAGACGCGAGAAGAACCCTATGACCGAGGAGGAATCTGACGGACTG ATCGCCGCACGCCGCATCCAGCGCCTGAGCCTGCACCTGTCTCCCAGCCTGACACC ATCCCCCTCTCTGCCTCTGGTGCAGACCGAGACATGCAGCGCCCGGTCCAAGAAGC TGGACGTGAACGGCGAGGCCCTGAGCCTGTACATGAGAGGCAAGCACATCGACAT TCAGGAGAAGATCTTCGATTTCTTTAACTCCAGGCCAGACCTGCAGACCCCCATCG AGATCTCTAAGGACGATCACCGGGAGCTGTGCATGAATCAGCTGATCGGCCTGGT GAGAGAGGCAGGCGTGCGGCCTTTCAGATACGTGGCCGACGATCCAGAGAAGTAT TTTGCCATCATGGAGGCCGTGGGCTCTGTGGATATGAGCCTGGGCATCAAGATGGG CGTGCAGTACTCTCTGTGGGGCGGCAGCGTGATCAACCTGGGCACCAAGAAGCAC CGCGACAAGTACTTCGACGGCATCGATAATCTGGACTATACAGGCTGCTTTGCCAT GACCGAGCTGCACCACGGCTCCAACGTGCAGGGCCTGCAGACCACAGCCACATTC GATCCACTGAAGGACGAGTTTGTGATCGATACCCCCAACGACGGCGCCATCAAGT GGTGGATCGGCAATGCCGCCGTGCACGGCAAGTTCGCCACAGTGTTTGCAAGGCT GATCCTGCCAACCCACGACTCCAAGGGCGTGTCTGATATGGGCGTGCACGCCTTCA TCGTGCCTATCAGAGATATGAAGACACACCAGACCCTGCCAGGCGTGGAGATTCA GGACTGTGGCCACAAAGTGGGCCTGAACGGCGTGGACAATGGCGCCCTGCGGTTC AGATCTGTGAGGATCCCACGCGACAACCTGCTGAATCGGTTTGGCGATGTGAGCA GAGACGGCACCTACACAAGCTCCCTGCCCACAATCAATAAGAGGTTTGGAGCCAC CCTGGGAGAGCTGGTGGGCGGCAGAGTGGGCCTGGCATACGCATCTGTGGGCGTG CTGAAGATCAGCGCCACCATCGCCATCCGGTATAGCCTGCTGAGACAGCAGTTCG GCCCCCCTAAGCAGCCCGAGGTGAGCATTCTGGACTATCAGTCCCAGCAGCACAA GCTGATGCCTATGCTGGCCTCCACATACGCCTATCACTTTGCCACCGTGTACCTGGT GGAGAAGTATTCTGAGATGAAGAAGACCCACGACGAGCAGCTGGTGGCAGATGTG CACGCACTGAGCGCCGGCCTGAAGAGCTACGTGACATCCTATACCGCAAAGGCCC TGTCCGTGTGCAGGGAGGCATGTGGAGGACACGGCTACGCAGCAGTGAACAGGTT CGGCTCCCTGCGCAATGACCACGATATCTTCCAGACATTTGAGGGCGATAACACCG TGCTGCTGCAGCAGGTGGCAGCAGACCTGCTGAAGAGGTATAAGGAGAAGTTTCA GGGCGGCACCCTGACAGTGACCTGGTCCTACCTGCGCGAGTCTATGAACACATATC TGTCTCAGCCTAATCCAGTGACCGCAAGATGGGAGGGAGAGGATCACCTGCGCGA CCCTAAGTTCCAGCTGGACGCCTTTAGGTACCGCACAAGCAGGCTGCTGCAGAAC GTGGCAGCAAGACTGCAGAAGCACTCCAAGACCCTGGGAGGATTCGGAGCATGGA ACCGGTGCCTGAATCACCTGCTGACACTGGCCGAGTCTCACATCGAGACAGTGATC CTGGCCAAGTTTATCGAGGCCGTGAAGAATTGCCCAGATCCTAGCGCCAAGGCCG CCCTGAAGCTGGCATGTGATCTGTACGCCCTGGACAGGATCTGGAAGGATATCGG CACATACCGCAACGTGGACTATGTGGCCCCTAATAAGGCCAAGGCCATCCACAAG CTGACCGAGTATCTGTCCTTCCAGGTGAGAAATGTGGCCAAGGAGCTGGTGGACG CCTTTGAGCTGCCAGATCACGTGACCAGAGCCCCTATTGCTATGCAGTCTGATGCT TATTCACAGTACACCCAGGTCGTCGGATTCTGA [0013] SEQ ID NO:12 shows the AtACX2 amino acid sequence encoded by SEQ ID NO:11: MESRREKNPMTEEESDGLIAARRIQRLSLHLSPSLTPSPSLPLVQTETCSARSKKLDVNG EALSLYMRGKHIDIQEKIFDFFNSRPDLQTPIEISKDDHRELCMNQLIGLVREAGVRPFR YVADDPEKYFAIMEAVGSVDMSLGIKMGVQYSLWGGSVINLGTKKHRDKYFDGIDN LDYTGCFAMTELHHGSNVQGLQTTATFDPLKDEFVIDTPNDGAIKWWIGNAAVHGKF ATVFARLILPTHDSKGVSDMGVHAFIVPIRDMKTHQTLPGVEIQDCGHKVGLNGVDN GALRFRSVRIPRDNLLNRFGDVSRDGTYTSSLPTINKRFGATLGELVGGRVGLAYASV GVLKISATIAIRYSLLRQQFGPPKQPEVSILDYQSQQHKLMPMLASTYAYHFATVYLVE KYSEMKKTHDEQLVADVHALSAGLKSYVTSYTAKALSVCREACGGHGYAAVNRFGS LRNDHDIFQTFEGDNTVLLQQVAADLLKRYKEKFQGGTLTVTWSYLRESMNTYLSQP NPVTARWEGEDHLRDPKFQLDAFRYRTSRLLQNVAARLQKHSKTLGGFGAWNRCLN HLLTLAESHIETVILAKFIEAVKNCPDPSAKAALKLACDLYALDRIWKDIGTYRNVDYV APNKAKAIHKLTEYLSFQVRNVAKELVDAFELPDHVTRAPIAMQSDAYSQYTQVVGF [0014] SEQ ID NO:13 shows an exemplary RnACOX1-1 (also referred to herein as “ACOI-I”) nucleotide sequence: ATGAATCCAGATCTGAGGAAGGAGAGGGCAAGCGCCACATTCAACCCAGAGCTGA TCACCCACATTCTGGACGGCTCCCCCGAGAATACCCGGAGAAGGCGCGAGATCGA GAACCTGATCCTGAATGACCCTGATTTCCAGCACGAGGATTACAACTTTCTGACAC GGTCTCAGAGATATGAGGTGGCCGTGAAGAAGAGCGCCACCATGGTGAAGAAGAT GAGGGAGTACGGCATCTCCGACCCAGAGGAGATCATGTGGTTCAAGAAGCTGTAC CTGGCCAATTTTGTGGAGCCCGTGGGCCTGAACTATTCTATGTTCATCCCTACCCTG CTGAACCAGGGCACCACAGCCCAGCAGGAGAAGTGGATGCGGCCCAGCCAGGAG CTGCAGATCATCGGCACCTACGCCCAGACAGAGATGGGACACGGAACCCACCTGA GAGGACTGGAGACCACAGCCACCTATGATCCTAAGACACAGGAGTTTATCCTGAA TAGCCCTACCGTGACATCCATCAAGTGGTGGCCAGGCGGCCTGGGCAAGACAAGC AACCACGCCATCGTGCTGGCCCAGCTGATCACCCAGGGAGAGTGCTACGGACTGC ACGCCTTCGTGGTGCCCATCAGAGAGATCGGCACACACAAGCCCCTGCCTGGCATC ACCGTGGGCGATATCGGCCCTAAGTTTGGCTATGAGGAGATGGATAACGGCTACC TGAAGATGGACAATTATAGGATCCCACGCGAGAACATGCTGATGAAGTACGCCCA GGTGAAGCCCGACGGCACATATGTGAAGCCTCTGTCCAATAAGCTGACCTACGGC ACAATGGTGTTCGTGAGGTCTTTTCTGGTGGGCAACGCCGCCCAGTCTCTGAGCAA GGCCTGTACCATCGCCATCCGCTATTCTGCCGTGCGGAGACAGTCTGAGATCAAGC AGAGCGAGCCAGAGCCCCAGATTCTGGACTTCCAGACACAGCAGTACAAGCTGTT TCCACTGCTGGCCACCGCCTATGCCTTCCACTTTGTGGGCAGGTACATGAAGGAGA CCTATCTGCGCATCAATGAGAGCATCGGACAGGGCGACCTGTCCGAGCTGCCCGA GCTGCACGCACTGACAGCAGGCCTGAAGGCCTTCACCACATGGACCGCAAACGCA GGAATCGAGGAGTGCAGGATGGCATGTGGAGGACACGGATACAGCCACAGCTCCG GCATCCCAAATATCTATGTGACCTTCACACCCGCCTGCACCTTTGAGGGCGAGAAC ACAGTGATGATGCTGCAGACCGCCAGGTTTCTGATGAAGATCTACGATCAGGTGC GCTCCGGCAAGCTGGTGGGAGGAATGGTGTCTTATCTGAATGACCTGCCTAGCCAG AGAATCCAGCCACAGCAGGTGGCCGTGTGGCCAACAATGGTGGATATCAACTCTC TGGAGGGACTGACCGAGGCCTACAAGCTGAGGGCAGCAAGACTGGTGGAGATCGC AGCCAAGAATCTGCAGACACACGTGTCCCACCGGAAGTCTAAGGAGGTGGCCTGG AACCTGACCTCCGTGGACCTGGTGAGAGCCTCTGAGGCCCACTGCCACTATGTGGT GGTGAAGGTGTTCAGCGATAAGCTGCCCAAGATTCAGGACAAAGCCGTGCAGGCC GTGCTGAGGAACCTGTGCCTGCTGTACTCCCTGTATGGCATCTCTCAGAAGGGCGG CGATTTTCTGGAGGGCAGCATCATCACAGGAGCACAGCTGTCCCAAGTGAATGCA AGGATTCTGGAGCTGCTGACCCTGATCAGACCTAACGCCGTGGCCCTGGTGGACGC CTTCGACTTCAAGGACATGACACTGGGCTCCGTGCTGGGCCGCTACGACGGAAAC GTGTATGAGAATCTGTTCGAGTGGGCCAAGAAGTCCCCTCTGAACAAGACCGAGG TGCACGAGTCTTACCACAAGCACCTGAAGCCACTGCAGAGCAAGCTGTGA [0015] SEQ ID NO:14 shows the RnACOX1-1 amino acid sequence encoded by SEQ ID NO:13: MNPDLRKERASATFNPELITHILDGSPENTRRRREIENLILNDPDFQHEDYNFLTRSQRY EVAVKKSATMVKKMREYGISDPEEIMWFKKLYLANFVEPVGLNYSMFIPTLLNQGTT AQQEKWMRPSQELQIIGTYAQTEMGHGTHLRGLETTATYDPKTQEFILNSPTVTSIKW WPGGLGKTSNHAIVLAQLITQGECYGLHAFVVPIREIGTHKPLPGITVGDIGPKFGYEE MDNGYLKMDNYRIPRENMLMKYAQVKPDGTYVKPLSNKLTYGTMVFVRSFLVGNA AQSLSKACTIAIRYSAVRRQSEIKQSEPEPQILDFQTQQYKLFPLLATAYAFHFVGRYM KETYLRINESIGQGDLSELPELHALTAGLKAFTTWTANAGIEECRMACGGHGYSHSSGI PNIYVTFTPACTFEGENTVMMLQTARFLMKIYDQVRSGKLVGGMVSYLNDLPSQRIQP QQVAVWPTMVDINSLEGLTEAYKLRAARLVEIAAKNLQTHVSHRKSKEVAWNLTSV DLVRASEAHCHYVVVKVFSDKLPKIQDKAVQAVLRNLCLLYSLYGISQKGGDFLEGSI ITGAQLSQVNARILELLTLIRPNAVALVDAFDFKDMTLGSVLGRYDGNVYENLFEWAK KSPLNKTEVHESYHKHLKPLQSKL [0016] SEQ ID NO:15 shows an exemplary RnACOX1-2 (also referred to herein as “ACOI-II”) nucleotide sequence: ATGAATCCAGATCTGAGGAAGGAGCGCGCCTCTGCCACATTCAACCCAGAGCTGA TCACCCACATTCTGGACGGCAGCCCCGAGAATACCCGGAGAAGGCGCGAGATCGA GAACCTGATCCTGAATGACCCTGATTTCCAGCACGAGGATTACAACTTTCTGACAC GGTCTCAGAGATATGAGGTGGCCGTGAAGAAGAGCGCCACCATGGTGAAGAAGAT GAGGGAGTACGGCATCTCCGATCCAGAGGAGATCATGTGGTTCAAGAATTCTGTG CACAGGGGACACCCAGAGCCTCTGGACCTGCACCTGGGCATGTTTCTGCCCACCCT GCTGCACCAGGCAACAGCAGAGCAGCAGGAGAGGTTCTTTATGCCTGCCTGGAAC CTGGAGATCACCGGCACATACGCCCAGACCGAGATGGGACACGGAACACACCTGA GGGGACTGGAGACCACAGCAACCTATGACCCCAAGACACAGGAGTTTATCCTGAA TAGCCCCACCGTGACATCCATCAAGTGGTGGCCTGGCGGCCTGGGCAAGACAAGC AACCACGCCATCGTGCTGGCCCAGCTGATCACCCAGGGAGAGTGCTACGGACTGC ACGCCTTCGTGGTGCCAATCAGGGAGATCGGCACACACAAGCCACTGCCCGGCAT CACCGTGGGCGATATCGGACCCAAGTTTGGCTATGAGGAGATGGATAACGGCTAC CTGAAGATGGACAATTATAGGATCCCTCGCGAGAACATGCTGATGAAGTACGCCC AGGTGAAGCCAGACGGCACCTATGTGAAGCCCCTGAGCAATAAGCTGACCTACGG CACAATGGTGTTCGTGCGGTCCTTTCTGGTGGGCAACGCCGCCCAGTCTCTGAGCA AGGCCTGTACCATCGCCATCAGATATTCCGCCGTGCGGAGACAGTCTGAGATCAA GCAGAGCGAGCCTGAGCCACAGATTCTGGACTTCCAGACACAGCAGTACAAGCTG TTTCCACTGCTGGCCACCGCCTATGCCTTCCACTTTGTGGGCAGGTACATGAAGGA GACCTATCTGCGCATCAATGAGAGCATCGGACAGGGCGACCTGTCCGAGCTGCCC GAGCTGCACGCACTGACAGCAGGCCTGAAGGCCTTCACCACATGGACCGCCAACG CCGGCATCGAGGAGTGCAGGATGGCATGTGGAGGACACGGATACAGCCACAGCTC CGGCATCCCTAATATCTATGTGACCTTCACACCAGCCTGCACCTTTGAGGGCGAGA ACACAGTGATGATGCTGCAGACCGCCAGGTTTCTGATGAAGATCTACGATCAGGT GCGCTCCGGCAAGCTGGTGGGAGGAATGGTGTCTTATCTGAATGACCTGCCCAGCC AGAGGATCCAGCCTCAGCAGGTGGCCGTGTGGCCTACAATGGTGGATATCAACTC TCTGGAGGGACTGACCGAGGCCTACAAGCTGAGGGCAGCAAGACTGGTGGAGATC GCAGCCAAGAATCTGCAGACACACGTGTCCCACCGGAAGTCTAAGGAGGTGGCCT GGAACCTGACCTCCGTGGACCTGGTGAGAGCCTCTGAGGCCCACTGCCACTATGTG GTGGTGAAGGTGTTCTCCGATAAGCTGCCCAAGATTCAGGACAAAGCCGTGCAGG CCGTGCTGAGGAACCTGTGCCTGCTGTACTCCCTGTATGGCATCTCTCAGAAGGGC GGCGATTTTCTGGAGGGCAGCATCATCACAGGAGCACAGCTGTCCCAAGTGAATG CAAGGATTCTGGAGCTGCTGACCCTGATCAGACCTAACGCCGTGGCCCTGGTGGAC GCCTTCGACTTCAAGGACATGACACTGGGCAGCGTGCTGGGCAGATACGACGGCA ACGTGTATGAGAATCTGTTCGAGTGGGCCAAGAAGTCCCCTCTGAACAAGACCGA GGTGCACGAGTCTTACCACAAGCACCTGAAGCCACTGCAGAGCAAGCTGTGA [0017] SEQ ID NO:16 shows the RnACOX1-2 amino acid sequence encoded by SEQ ID NO:15: MNPDLRKERASATFNPELITHILDGSPENTRRRREIENLILNDPDFQHEDYNFLTRSQRY EVAVKKSATMVKKMREYGISDPEEIMWFKNSVHRGHPEPLDLHLGMFLPTLLHQATA EQQERFFMPAWNLEITGTYAQTEMGHGTHLRGLETTATYDPKTQEFILNSPTVTSIKW WPGGLGKTSNHAIVLAQLITQGECYGLHAFVVPIREIGTHKPLPGITVGDIGPKFGYEE MDNGYLKMDNYRIPRENMLMKYAQVKPDGTYVKPLSNKLTYGTMVFVRSFLVGNA AQSLSKACTIAIRYSAVRRQSEIKQSEPEPQILDFQTQQYKLFPLLATAYAFHFVGRYM KETYLRINESIGQGDLSELPELHALTAGLKAFTTWTANAGIEECRMACGGHGYSHSSGI PNIYVTFTPACTFEGENTVMMLQTARFLMKIYDQVRSGKLVGGMVSYLNDLPSQRIQP QQVAVWPTMVDINSLEGLTEAYKLRAARLVEIAAKNLQTHVSHRKSKEVAWNLTSV DLVRASEAHCHYVVVKVFSDKLPKIQDKAVQAVLRNLCLLYSLYGISQKGGDFLEGSI ITGAQLSQVNARILELLTLIRPNAVALVDAFDFKDMTLGSVLGRYDGNVYENLFEWAK KSPLNKTEVHESYHKHLKPLQSKL [0018] SEQ ID NO:17 shows an exemplary DGAT1_Mt nucleotide sequence: ATGAGCATCTCCAACTCTCCCGAGAATCTGGACCAGAAGCCTAACATCCGCAATG AGAGCGAGGATCGGAGCTTTTCCGTGCGGAGAAGGACCAACTCCAATGTGGTGAA CGCCGTGCCTGACTCTTGCAGCAAAGTGGGCACAATCGAGACCGTGACAAAGTTC AGCTCCGTGGACTCCGCCGCCGAGATGTCTAGCGGCGAGGATAGAAACAGGATCG GCAATGGCGACGATAGAAAGGAGGAGGCCAACTCCCTGTCTAATGGCCAGGTGTA CAACGAGAGAGTGGGAATCGGAGAGGGCAGGAGCCCTGAGGCCGAGGTGCCATT TCGCTTCTCTTATAGGGCAAGCGCCCCAGCACACAGGAGGGTGAAGGAGTCTCCTC TGTCCTCTGACGCCATCTTTCGCCAGAGCCACGCCGGCCTGTTCAACCTGTGCATC CTGGTGCTGGTGGCAGTGAACGGCCGGCTGATCATCGAGAATCTGATGAAGTACG GCCTGCTGATCCGCGCCGGCTTTTGGTTCAGCTCCAAGTCCCTGCGGGATTGGCCC CTGCTGATGTGCTGTCTGTCTCTGCCAATCTTTCCCCTGGGCGCCTTCACCGTGGAG AAGTCCGCCAAGCACAAGTTCATCCCCGAGCCTGTGGTCATGAGCCTGCACGTGCT GATCTGCACAGCCTCCCTGCTGTACCCTATCTTTGTGATCCTGAGATGTGGAAGCG CCGTGCTGTTCGGAATCGCCCTGATGCTGCTGGCCTCTATCGTGTGGCTGAAGCTG GTGAGCTATGCCCACACCAATTGCGACATGCGGGCCCTGGCCAAGCTGACAGATA AGGTGGCCTCCAAGTCTAGCGACATGGATTACTCTTATGACGTGAGCTTTGAGTCC CTGGCCTACTTCATGGTGGCCCCAACCCTGTGCTACCAGGCCTCCTATCCCCGCTCT ACCTGCATCCGGAAGGGCTGGGTCATCAGACAGCTGGTGAAGCTGGTCATCTTCAC CGGCGTGATGGGCTTCATCGTGGAGCAGTACATCAACCCTATCGTGCAGCACTCTC AGCACCCACTGAAGGGCAATCTGCTGTATGCCATCGAGAGGGTGCTGAAGCTGAG CGTGCCAACACTGTACGTGTGGCTGTGCATGTTCTATTGTTTCTTTCACCTGTGGCT GAACATCCTGGCCGAGCTGCTGAGATTTGGCGACAGGGAGTTCTACAAGGATTGG TGGAACGCCAAGACCGTGGAGGAGTATTGGAGAATGTGGAATATGCCCGTGCACA AGTGGATGGTGAGGCACATCTACTTTCCATGTCTGAGAAATGGCCTGCCCAGGGA GGTGACCCTGGTCATCGTGTTCTTCCTGTCCGCCATCTTCCACGAGCTGTGCATCGG CGTGCCTTGTCACATCTTTAAGTTCTGGGCCTTTATCGGCATCATGTTCCAGGTGCC ACTGGTGCTGATCACCAACTATCTGCAGAATAAGTTTAGATCCTCTATGGTGGGCA ACATGGTGTTCTGGTTCTTTTTCAGCATCCTGGGCCAGCCCATGTCCGTGCTGCTGT ACTATCACGATGTGATGAACAGGAAGGAGAATGGCGTGAGCAAGTGA [0019] SEQ ID NO:18 shows the DGAT1_Mt amino acid sequence encoded by SEQ ID NO:17: MSISNSPENLDQKPNIRNESEDRSFSVRRRTNSNVVNAVPDSCSKVGTIETVTKFSSVDS AAEMSSGEDRNRIGNGDDRKEEANSLSNGQVYNERVGIGEGRSPEAEVPFRFSYRASA PAHRRVKESPLSSDAIFRQSHAGLFNLCILVLVAVNGRLIIENLMKYGLLIRAGFWFSSK SLRDWPLLMCCLSLPIFPLGAFTVEKSAKHKFIPEPVVMSLHVLICTASLLYPIFVILRCG SAVLFGIALMLLASIVWLKLVSYAHTNCDMRALAKLTDKVASKSSDMDYSYDVSFES LAYFMVAPTLCYQASYPRSTCIRKGWVIRQLVKLVIFTGVMGFIVEQYINPIVQHSQHP LKGNLLYAIERVLKLSVPTLYVWLCMFYCFFHLWLNILAELLRFGDREFYKDWWNAK TVEEYWRMWNMPVHKWMVRHIYFPCLRNGLPREVTLVIVFFLSAIFHELCIGVPCHIF KFWAFIGIMFQVPLVLITNYLQNKFRSSMVGNMVFWFFFSILGQPMSVLLYYHDVMN RKENGVSK [0020] SEQ ID NO:19 shows an exemplary DGAT1_Tp nucleotide sequence: ATGGACTCCACACCCTCTGAGACCGAGGACGATCTGCGGAGAGAGATCCAGCGGC TGAGACAGCAGCTGCACGAGGCCAGCCGCGCCTCCGGCAACTCTGACGCCGCCCT GCCTACCCTGGGACAGACCGATAGCACAATCTCCACCGCACCACCTGAGAAGAGC GGATACCTGTTCAAGTGGCAGGACAGGACCATCGGATGGGGAGGAACAAAGTGG GCACTGAGATATGTGCGCCTGAACCACGGCCAGCTGAGCTACTATAAGTCCCACG AGGAGCGGAGCCCAAGATACATCATGACACTGAAGAATTGCGCCGTGAGAGATGA GGGCTCCAAGGTGAACAAGAGGCACGGCAGCGCCAAGAATGGAGAGGACTCCGA TCACCACGCAGTGGGATCCCGGTTCTACGTGTTCAGCGTGTACAGGCGCGTGAAGA ACTGGGGCGACTCTGATGCCCAGTATGATAATGAGGACGATATCATCCCCCTGCTG AGGTTTTCTACCCAGAGCCTGGCCGAGAAGATCCTGTGGGTGGACCTGATCTCTGA GAGCTGCGCCTACTGTGATTCCGAGGAGTTCGCCCTGTATCAGCAGCAGCAGCAG GAGATCCAGCAGAAGCAGCAGGAGCAGCAGATCAGAACAGAGAAGGGCACCCTG CCAGCCCTGGTGTTTGAGGCACCAAGGCTGACACACAAGCGGCTGCCTAGCGGCC ACAAGCTGAACGAGATGGGCAAGTCTTTCCGCAAGAAGAGCGTGGACAAGGATGC CGCCCGGTCTAACAAGATCAGCTACCCACCCTCCAAGCCTATGCACAGGCAGTCCA ATCCATCTTACCTGAGCGACGGCTCCCACGTGCAGAACTATAGAGGCCTGTTTAAT CTGCTGCTGCTGATCCTGGTGCTGTCCAATTTCAGGCTGCTGCTGGACACCGTGGC ACAGCACGGCTTCATTCTGGACAAGCTGGCCACACTGCAGGGCTTCTCTCAGGCCC CCCTGGATTTTCCTTTCGTGAGCGGCCTGCTGATCGTGCAGGCCTTTGTGGTGGGC GCCTACGCCATCGAGAAGATGCTGTCTGTGGGCCTGATCGGCAACCAGTTCGGCAT GCTGCTGCACGTGATCAACAGCAATGCCACCCTGGGAGTGGTGGTGGCAATCGTG TGGTACCTGATCGACCAGCCTTTTGTGGGAGCAGGACTGATCATGCAGGCAACCAT CACATGGCTGAAGCTGATCTCCTATGCCCACGCCAATTACGACTATAGAACATCTC CAGATACCCAGAAGGTGACAGTGGCCCTGGTGAAGGACCTGGACGATGGCCAGAA CGTGTCCTACCCTCAGAATGTGACCCTGAAGGATATCTACTATTTCTGGCTGGCCC CAACCCTGACATATCAGATCGCCTTTCCTAGATCTCCATTCATCAGGTGGCCCAAG GTGTTTTCTCTGACCCTGCAGCTGTTCATCAGCGTGACACTGGCCGTGTTTCTGTGC GCCCAGGTGGTGGCCCCTAACCTGGACAGCCTGGTGAAGAACCTGGAGGCCAATA AGGGCGAGGTGCGCACCCAGCAGATCTTCGATTACCTGCTGAAGCTGTCTATCACC AGCACATACATCTGGCTGCTGGGCTTTTATGGCTTCTTTCACTGTTTCATGAATCTG GCAGCAGAGCTGCTGAGGTTTGGCGACCGCGTGTTCTACCGGGATTGGTGGAACG CCTCCGAGGTGTCTGCCTATTGGCGGCTGTGGAATATGCCCGTGCACTACTGGCTG GTGCGCCACGTGTATTTTCCTTGCATCCGGGTGGGCATGAGCAAGAAGGGCGCCAC CTTCGTGGTGTTCTTTTTCAGCGCCGTGCTGCACGAGGTGCTGATCTCCGTGCCCTG TCACATGATCCGCGCCTGGTCCTTTCTGGCCATGATGGGCCAGATCCCACTGATCA TCCTGACCAAGATCATCGACAAGAGGGTGCCCGGCAGCTCCATCGGCAACATCAT CTTTTGGATCAGCTTCTGTCTGGTGGGCCAGCCAATGGCCATGCTGCTGTACACAA TCGACTATTGGGAGGTGCACTTCAATGCCGCCATCACAGAGTCCACCATCGAGGTG CCCAGAAAGTCTTTTAGGTTCGATAAGATCGGCAGGTTTTTCGGCGCCCACTCCGA GCTGTGA [0021] SEQ ID NO:20 shows the DGAT1_Tp amino acid sequence encoded by SEQ ID NO:19: MDSTPSETEDDLRREIQRLRQQLHEASRASGNSDAALPTLGQTDSTISTAPPEKSGYLF KWQDRTIGWGGTKWALRYVRLNHGQLSYYKSHEERSPRYIMTLKNCAVRDEGSKVN KRHGSAKNGEDSDHHAVGSRFYVFSVYRRVKNWGDSDAQYDNEDDIIPLLRFSTQSL AEKILWVDLISESCAYCDSEEFALYQQQQQEIQQKQQEQQIRTEKGTLPALVFEAPRLT HKRLPSGHKLNEMGKSFRKKSVDKDAARSNKISYPPSKPMHRQSNPSYLSDGSHVQN YRGLFNLLLLILVLSNFRLLLDTVAQHGFILDKLATLQGFSQAPLDFPFVSGLLIVQAFV VGAYAIEKMLSVGLIGNQFGMLLHVINSNATLGVVVAIVWYLIDQPFVGAGLIMQATI TWLKLISYAHANYDYRTSPDTQKVTVALVKDLDDGQNVSYPQNVTLKDIYYFWLAPT LTYQIAFPRSPFIRWPKVFSLTLQLFISVTLAVFLCAQVVAPNLDSLVKNLEANKGEVRT QQIFDYLLKLSITSTYIWLLGFYGFFHCFMNLAAELLRFGDRVFYRDWWNASEVSAY WRLWNMPVHYWLVRHVYFPCIRVGMSKKGATFVVFFFSAVLHEVLISVPCHMIRAW SFLAMMGQIPLIILTKIIDKRVPGSSIGNIIFWISFCLVGQPMAMLLYTIDYWEVHFNAAI TESTIEVPRKSFRFDKIGRFFGAHSEL [0022] SEQ ID NO:21 shows an exemplary LPAAT1_Tp nucleotide sequence: ATGGTGAAGCAGAACCCTAGCTCCACCACAGCCAGCGCCTTCGTGGCTCTGCTGGC CTTCTTCAGCGTGGTGTCTGGCCTGGTGCCATCTAGCACCCTGCCCAGGCCTAACA ATCTGAGCGCCTGCCAGCAGTGGAGGACCACAAGCGCCACAATGGCCAGCCGGAA CAATATCCTGCTGCGCAAGAATACCCAGCTGATGTCCTCTGCCACAGCCGCCCTGA GCTCCTCTCTGGAGCTGCCCAGAGTGGAGTCTCAGAGAACCCTGAGCAAGCTGCA GAGCTCCATCGTGAGGACACTGATGATCGCCTACATCCTGTCCATGTGCATCGCCC TGCCTGTGACCATGCTGCCAGTGTACATCCTGTATAAGCTGCGGCTGATCAACAGA GTGCAGAAGGAGAAGATGTCTCTGCAGGTGGGCCAGTTCTGCAGCAGGTGGCTGA TGCGCCTGTTCCCTTTTGCCAGGAAGCGCGTGATCGTGGACACAGACGATGAGAAC TATAAGAATCCACAGCCCTCCATCTGGGTGTGCAATCACATCTCTCTGCTGGACCT GTTCTTTGTGCTGGCCCTGGATAAGCAGATGCGGGGCGTGAACCGGAGACCCATC AAGATCCTGTACTGGAAGGGCCTGGAGAGCAATCCTGTGACCAGACTGCTGTGCA AGATGTGCGGCTTTATCCCAGTGGACATGGCCGATAACGGCAATGGCAACGCCAA TCAGTATGATCCCAAGAGCTTCAAGCAGATGCTGAAGTCCACAAAGGCCGCCATC GACGAGGGCTTTGATATCGGACTGCTGCCCGAGGGACAGCCTAACCCAACCCCAC ACCTGGGCCTGCAGCCTATCTTCAGCGGCGCCTTTACACTGGCCAAGATGTCCAGG CGCCCAATCCAGATGATCGGGCTGTACGGCCTGCACAATATGTGGCACCCCGACG AGGATGTGGGCATGGAGTGCGCCGCCAATGACATGGCCGTGCGGGTGTACACCGG AGCAAGAGTGTATAAGGAGGCCGATGAGTTCGCCGCCACATTTGAGGCAGTGGTG GGACACTTTGGAGCACACGGCAAGGACATGCAGGAGGAGGAGCTGCAGATGTGG CTGGATGGCACCATGTGGGAGACAGAGCTGAGCCGGCGGGCAGCAAATAGGATG GAGGTGGAGGACGTGAAGGAGACCATCGCCGAGTCTAAGAGCGACGCCTCCATCG AGGAGGATAAGTCTATCCTGTGA [0100] SEQ ID NO:22 shows the LPAAT1_Tp amino acid sequence encoded by SEQ ID NO:21: MVKQNPSSTTASAFVALLAFFSVVSGLVPSSTLPRPNNLSACQQWRTTSATMASRNNI LLRKNTQLMSSATAALSSSLELPRVESQRTLSKLQSSIVRTLMIAYILSMCIALPVTMLP VYILYKLRLINRVQKEKMSLQVGQFCSRWLMRLFPFARKRVIVDTDDENYKNPQPSIW VCNHISLLDLFFVLALDKQMRGVNRRPIKILYWKGLESNPVTRLLCKMCGFIPVDMAD NGNGNANQYDPKSFKQMLKSTKAAIDEGFDIGLLPEGQPNPTPHLGLQPIFSGAFTLAK MSRRPIQMIGLYGLHNMWHPDEDVGMECAANDMAVRVYTGARVYKEADEFAATFE AVVGHFGAHGKDMQEEELQMWLDGTMWETELSRRAANRMEVEDVKETIAESKSDA SIEEDKSIL [0101] SEQ ID NO:23 shows an exemplary LPAAT2_Tp nucleotide sequence: ATGGCACCCGTGCTGCTGTTCCTGGCCCTGCTGGTGCACATCAGCATGCTGAGCTC CACCGCCACAGCCTTCCAGCTGCCTTCTAGCTCCTCTCGCTTTACCGCCAACCGGC AGATCTGCAATTCTGGCCACCGCCACAAGCACCACCAGCACTGTAGGACCAGCCC TCGCCTGGAGCCACTGTTCGCAAGCGTGGAGCGGACATCCGAGCCTATGTCCTTTT CTCTGCAGGAGGAGGACGATAGCTCCCAGCAGCTGCACGTGAGCGCCTCCACCTTT GACACAGATGAGAAGTCTCACCACGGCAACGTGAGCCTGATCAACACAAATGAGA TCGAGGTGTCTCTGAGCCAGCAGCCCCCTACCACAAGCAAGACCACATCTAGCATC CAGAACCAGCCCTCCCACAAGCTGACCATCCCTGAGATGGGCGGCTTCCAGTTTAC AAAGAATGATAAGCCTAAGGTGCTGAACCTGTACGGCCTGTATAATCTGTTCACCA TCGCCGTGACAATGCCATTTTGGCTGGCCGCCATGGAGATCCTGCAGTGGCTGGGC GATAACATCGAGGGCTTCGACAAGGATAGAGCCATGTTTGACTACAGCGGCAAGG TCTGGTGCAGGGTGTACCTGACCCTGGTGGACTCCTATCCCGAGATCGCCGGCGAT GTGGAGAGGCTGAAGAATAAGAAGAGCCTGCTGGGCGATGGAGGAGGAGAGAAC CAGGCCTGTATGTATGTGGCCAATCACGCCAGCTTTCTGGACATCGCCGTGCTGTG CTGCGTGCTGGACCCAGTGTTCAAGTTTATCGCCAAGGACAGCCTGAAGAAGTTTC CCGGCGTGGGCAAGCAGCTGTGCGGAGGAGAGCACGTGCTGATCGACCGGTCCAA CAAGAGATCTCAGCTGAGGACCTTCAAGCAGGCCATCACATACCTGCAGAATGGC GTGTCCGTGATGGCCTTTCCAGAGGGAGCAAGATCTCCAGACGGCAGGCTGATGG ATTTCAAGCCAGGCCTGTTTTCCATGGCCACCAAAGCCAACGTGCCAATCGTGCCC CTGTCTATCGCCAATACCCACGCCGTGATGCCTACAGTGGGCTTCCTGCCAGTGCA GAGGGGCAAGGGCAAGCTGCGGGTGTACGTGCACGAGCCCATCGAGGTGGAGGG CAAGTCTGAGGAGGCCATCGCCCGGGAGGTGAGAGAGGTGCTGCTGTCCGAGCTG CCACTGGACCAGCACCCTCTGGAGTATGGCGAGGAGGAGATCTGA [0102] SEQ ID NO:24 shows the LPAAT2_Tp amino acid sequence encoded by SEQ ID NO:23: MAPVLLFLALLVHISMLSSTATAFQLPSSSSRFTANRQICNSGHRHKHHQHCRTSPRLE PLFASVERTSEPMSFSLQEEDDSSQQLHVSASTFDTDEKSHHGNVSLINTNEIEVSLSQQ PPTTSKTTSSIQNQPSHKLTIPEMGGFQFTKNDKPKVLNLYGLYNLFTIAVTMPFWLAA MEILQWLGDNIEGFDKDRAMFDYSGKVWCRVYLTLVDSYPEIAGDVERLKNKKSLLG DGGGENQACMYVANHASFLDIAVLCCVLDPVFKFIAKDSLKKFPGVGKQLCGGEHVL IDRSNKRSQLRTFKQAITYLQNGVSVMAFPEGARSPDGRLMDFKPGLFSMATKANVPI VPLSIANTHAVMPTVGFLPVQRGKGKLRVYVHEPIEVEGKSEEAIAREVREVLLSELPL DQHPLEYGEEEI [0103] SEQ ID NO:25 shows an exemplary DGAT1_Vf nucleotide sequence: ATGACCATCCCCGAGACACCTGACAACTCTACCGATGCAACCACAAGCGGAGGAG CAGAGAGCTCCTCTGACCTGAACCTGTCCCTGCGGAGAAGGCGCACAGCCAGCAA TTCCGATGGAGCAGTGGCTGAGCTGGCCAGCAAGATCGACGAGCTGGAGTCCGAT GCAGGAGGAGGACAGGTCATCAAGGACCCCGGCGCAGAGATGGATAGCGGCACC CTGAAGTCCAATGGCAAGGACTGCGGCACAGTGAAGGATAGGATCGAGAACCGG GAGAATAGAGGCGGCTCCGACGTGAAGTTTACCTACAGGCCATCTGTGCCCGCCC ACCGCGCCCTGAAGGAGAGCCCTCTGAGCTCCGATAACATCTTTAAGCAGTCCCAC GCCGGCCTGTTCAACCTGTGCATCGTGGTGCTGGTGGCCGTGAACTCTAGGCTGAT CATCGAGAATATCATGAAGTATGGCTGGCTGATCAAGACCGGCTTCTGGTTTTCTA GCAGGAGCCTGCGCGACTGGCCTCTGCTGATGTGCTGTCTGACACTGCCAATCTTC TCCCTGGCCGCCTACCTGGTGGAGAAGCTGGCCTGCCGCAAGTATATCTCTGCCCC CACCGTGGTGTTCCTGCACATCCTGTTTTCCTCTACAGCCGTGCTGTACCCCGTGAG CGTGATCCTGTCTTGCGAGAGCGCCGTGCTGAGCGGAGTGGCCCTGATGCTGTTTG CCTGTATCGTGTGGCTGAAGCTGGTGAGCTATGCCCACACCAACTTCGACATGCGG GCCATCGCCAATTCTGTGGACAAGGGCGATGCACTGAGCAACGCAAGCTCCGCCG AGTCTAGCCACGACGTGAGCTTCAAGTCTCTGGTGTACTTCATGGTGGCCCCCACC CTGTGCTACCAGCCTTCCTATCCAAGGACAGCCTCTATCCGCAAGGGCTGGGTGGT GCGGCAGTTTGTGAAGCTGATCATCTTTACAGGCTTCATGGGCTTTATCATCGAGC AGTACATCAACCCAATCGTGCAGAATAGCCAGCACCCCCTGAAGGGCGACCTGCT GTATGCCATCGAGAGAGTGCTGAAGCTGTCCGTGCCTAACCTGTACGTGTGGCTGT GCATGTTCTATTGTTTCTTTCACCTGTGGCTGAATATCCTGGCAGAGCTGCTGAGGT TCGGCGACAGAGAGTTTTACAAGGATTGGTGGAACGCCCGGACCGTGGAGGAGTA TTGGAGAATGTGGAATATGCCAGTGCACAAGTGGATGGTGCGGCACATCTACTTTC CCTGCCTGCGGCACAAGATCCCTAGAGGCGTGGCCCTGCTGATCACCTTCTTCGTG AGCGCCGTGTTCCACGAGCTGTGCATCGCCGTGCCATGTCACATCTTCAAGCTGTG GGCCTTTATCGGCATCATGTTCCAGATCCCCCTGGTGGGCATCACAAACTATCTGC AGAATAAGTTCAGATCCTCTATGGTGGGCAACATGATCTTCTGGTTTATCTTCTGC ATCCTGGGCCAGCCTATGTGCCTGCTGCTGTACTATCACGATCTGATGAATAGGAA GGGCACCACAGAGAGCCGCTGA [0104] SEQ ID NO:26 shows the DGAT1_Vf amino acid sequence encoded by SEQ ID NO:25: MTIPETPDNSTDATTSGGAESSSDLNLSLRRRRTASNSDGAVAELASKIDELESDAGGG QVIKDPGAEMDSGTLKSNGKDCGTVKDRIENRENRGGSDVKFTYRPSVPAHRALKESP LSSDNIFKQSHAGLFNLCIVVLVAVNSRLIIENIMKYGWLIKTGFWFSSRSLRDWPLLM CCLTLPIFSLAAYLVEKLACRKYISAPTVVFLHILFSSTAVLYPVSVILSCESAVLSGVAL MLFACIVWLKLVSYAHTNFDMRAIANSVDKGDALSNASSAESSHDVSFKSLVYFMVA PTLCYQPSYPRTASIRKGWVVRQFVKLIIFTGFMGFIIEQYINPIVQNSQHPLKGDLLYAI ERVLKLSVPNLYVWLCMFYCFFHLWLNILAELLRFGDREFYKDWWNARTVEEYWRM WNMPVHKWMVRHIYFPCLRHKIPRGVALLITFFVSAVFHELCIAVPCHIFKLWAFIGIM FQIPLVGITNYLQNKFRSSMVGNMIFWFIFCILGQPMCLLLYYHDLMNRKGTTESR [0105] SEQ ID NO:27 shows an exemplary DGAT2_Vf nucleotide sequence: ATGGGCATGGTGGAGGTGAAGAACGAGGAGGAGGTGACCATCTTCAAGTCTGGCG AGATCTACCCCACAAATATCTTTCAGTCTGTGCTGGCCCTGGCAATCTGGCTGGGA AGCTTCCACTTTATCCTGTTCCTGGTGAGCTCCTCTATCTTCCTGCCTTTTTCCAAGT TCCTGCTGGTCATCGGCCTGCTGCTGTTCTTTATGGTCATCCCAATCAACGACAGGT CTAAGCTGGGCCAGTGCCTGTTCTCCTACATCAGCCGGCACGTGTGCAGCTATTTT CCTATCACACTGCACGTGGAGGACATCAATGCCTTTAGGTCCGATCGCGCCTACGT GTTCGGCTATGAGCCACACTCCGTGTTTCCCATCGGCGTGATGATCCTGTCTCTGG GCCTGATCCCCCTGCCTAACATCAAGTTCCTGGCCAGCTCCGCCGTGTTTTATACCC CCTTCCTGCGGCACATCTGGAGCTGGTGCGGACTGACCCCTGCAACAAGAAAGAA TTTCGTGAGCCTGCTGTCTAGCGGCTACTCCTGTATCCTGGTGCCCGGCGGAGTGC AGGAGACCTTCTACATGAAGCAGGACTCCGAGATCGCCTTTCTGAAGGCCCGGAG AGGCTTCATCCGGATCGCAATGCAGACCGGAACACCCCTGGTGCCCGTGTTCTGCT TTGGCCAGATGCACACCTTCAAGTGGTGGAAGCCAGATGGCGAGCTGTTTATGAA GATCGCCAGAGCCATCAAGTTTACCCCCACAATCTTCTGGGGCGTGCTGGGCACCC CACTGCCCTTCAAGAACCCTATGCACGTGGTGGTGGGCAGGCCAATCGAGGTGAA GCAGAATCCTCAGCCAACAGCAGAGGAGGTGGCAGAGGTGCAGAGGGAGTTCATC GCCAGCCTGAAGAACCTGTTTGAGCGGCATAAGGCCAGAGTGGGCTACTCCGATC TGAAGCTGGAGATCTTTTGA [0106] SEQ ID NO:28 shows the DGAT2_Vf amino acid sequence encoded by SEQ ID NO:27: MGMVEVKNEEEVTIFKSGEIYPTNIFQSVLALAIWLGSFHFILFLVSSSIFLPFSKFLLVIG LLLFFMVIPINDRSKLGQCLFSYISRHVCSYFPITLHVEDINAFRSDRAYVFGYEPHSVFP IGVMILSLGLIPLPNIKFLASSAVFYTPFLRHIWSWCGLTPATRKNFVSLLSSGYSCILVP GGVQETFYMKQDSEIAFLKARRGFIRIAMQTGTPLVPVFCFGQMHTFKWWKPDGELF MKIARAIKFTPTIFWGVLGTPLPFKNPMHVVVGRPIEVKQNPQPTAEEVAEVQREFIAS LKNLFERHKARVGYSDLKLEIF [0107] SEQ ID NO:29 shows an exemplary ELO2 nucleotide sequence: ATGCTCTCGTCAATCTCGCCCGACCTATACTCGTCCTTCTCGTTCAAAAACTCGCTC GCCGAGGCCATGCCCTCCGTGCCACACGAACTCATCAACTCAAAAACACTCTCATG GATGTACAATGCCTCTCTGGACATTCGGGTTCCTCTGACTATCGGAACCATCTACG CCGTCTCCGTGCACCTGACCAACTCATCTGAACGAATCAAGAAACGCCAGCCCATT GCCTTTGCCAAGACCGCACTCTTCAAGTGGCTCTGTGTCCTCCACAATGCAGGTCT GTGTCTCTACTCAGCATGGACCTTTGTCGGTATCCTCAACGCCGTCAAACACGCCT ACCAAATCACAGGAGACAGCTCCGCCCCCTTCTCCTTCAACACCCTCTGGGGATCG TTTTGTTCACGTGACTCCCTCTGGGTCACCGGCCTCAACTACTACGGATACTGGTTC TATCTGTCCAAATTCTACGAAGTGGTGGACACCATGATCATCCTCGCAAAGGGAAA ACCGTCCTCAATGCTCCAGACATACCACCACACCGGCGCCATGTTCTCCATGTGGG CCGGCATCCGATTCGCCTCTCCCCCCATCTGGATCTTTGTGGTTTTCAACTCCCTCA TCCACACAATCATGTACTTTTACTACACCCTCACCACCCTCAAGATCAAGGTTCCC AAGATCCTCAAGGCATCTCTGACCACCGCCCAGATCACCCAGATTGTCGGAGGTG GCATCCTGGCTGCCTCCCACGCCTTTATTTATTACAAGGACCACCAGACTGAGACC GTCTGTTCTTGTCTCACTACCCAGGGTCAGTTTTTCGCTCTCGCCGTCAATGTCATC TATCTGAGTCCTCTGGCCTATCTCTTTATTGCCTTCTGGATTCGATCTTACTTGAAG GCCAAGTCCAACTAG [0108] SEQ ID NO:30 shows the ELO2 amino acid sequence encoded by SEQ ID NO:29: MLSSISPDLYSSFSFKNSLAEAMPSVPHELINSKTLSWMYNASLDIRVPLTIGTIYAVSV HLTNSSERIKKRQPIAFAKTALFKWLCVLHNAGLCLYSAWTFVGILNAVKHAYQITGD SSAPFSFNTLWGSFCSRDSLWVTGLNYYGYWFYLSKFYEVVDTMIILAKGKPSSMLQT YHHTGAMFSMWAGIRFASPPIWIFVVFNSLIHTIMYFYYTLTTLKIKVPKILKASLTTAQ ITQIVGGGILAASHAFIYYKDHQTETVCSCLTTQGQFFALAVNVIYLSPLAYLFIAFWIR SYLKAKSN [0109] SEQ ID NO:31 shows an exemplary LPAAT_Pd nucleotide sequence: ATGGATGGATCTGGAGGAAGCTCCTTCCTGCGGGGCCGGAGACTGGAGAGCTGCT TTGAGGCATCCGTGCGGTCTAGAGGACCTGCAAAGGCCAGACCAGAGGATGCCGT GGGGCAGCCCGGCCCCAGGAGAGCCGCCGCCGCCGACTTCGTGGACGATGACCGG TGGATCACAGTGATCCTGTCCGTGGTGAGAATCGTGGTGTGCTTCCTGTCTATGGC CGTGACCACAGCCGTGTGGGCCGTGATCATGCTGCTGCTGCTGCCTTGGCCATACG CCAGGATCCGCCAGGGCAACCTGTATGGCCACGTGACCGGCAGGATGCTGATGTG GATCCTGGGCAATCCCATCACAATCGAGGGCAGCGAGTTCTCCAACACCCGCGCC ATCTTTATCTGCAATCACGCCTCCCCAATCGATATCTTCCTGGTCATGTGGCTGACC CCAACAGGAACCGTGGGAATCGCCAAGAAGGAGATCATCTGGTACCCCCTGTTTG GCCAGCTGTATATGCTGGCCAACCACCTGAGGATCGACCGCTCTAATCCTACAGCC GCCATCGAGAGCATCAAGCAGGTGGCCCGGGCCATCGTGAAGAAGAAGCTGAGCC TGATCATCTTCCCTGAGGGCACACGGTCCAAGACCGGCAGACTGCTGCCATTCAAG AAGGGCTTTGTGCACATCGCCCTGCAGACCAGGCTGCCAATCGTGCCCATGGTGCT GACAGGAACCCACCTGGCATGGCGCAAGAACAGCCTGCGGGTGAGACCAGCACCT CTGACAGTGAAGTACCTGCCCCCTATCAAGACCGATGACTGGGAGGCCGAGAAGA TCGATGACTACGTGGAGATGATCCACGCCCTGTATGTGGATCACCTGCCCGAGTCT CAGAAGCCTCTGGTGAGCGAGGGCAGGGACGTGTCCAAGCGGAGCAATAGCTGA [0110] SEQ ID NO:32 shows the LPAAT_Pd amino acid sequence encoded by SEQ ID NO:31: MDGSGGSSFLRGRRLESCFEASVRSRGPAKARPEDAVGQPGPRRAAAADFVDDDRWI TVILSVVRIVVCFLSMAVTTAVWAVIMLLLLPWPYARIRQGNLYGHVTGRMLMWILG NPITIEGSEFSNTRAIFICNHASPIDIFLVMWLTPTGTVGIAKKEIIWYPLFGQLYMLANH LRIDRSNPTAAIESIKQVARAIVKKKLSLIIFPEGTRSKTGRLLPFKKGFVHIALQTRLPIV PMVLTGTHLAWRKNSLRVRPAPLTVKYLPPIKTDDWEAEKIDDYVEMIHALYVDHLP ESQKPLVSEGRDVSKRSNS [0111] SEQ ID NO:33 shows an exemplary DGAT1A_Pd nucleotide sequence: ATGGCCATCCCATCCGATAGAGAGACCCTGGAGAGGGCACCAGAGCCTTCTCCAG CAAGCGACCTGCAGAGCTCCCTGCGGAGAAGGCTGCACTCTACCGTGGCAGCAGT GGTGGTGCCAGATTCTAGCTCCAAGACATCTAGCCCCAGCGCCGAGAACCTGACC ACAGACAGCGGAGAGGATTCCAGGGGCGACACCTCCTCTGACGCCGATACAAGGG ATAGGGTGGTGGACGGAGTGGATAGGGAGGAGGAGAACAAGACCGTGAGCGTGC TGAATGGCAGACAGTACGAGGACGGAGGCGGCAGGGGACAGGGACAGGGCACAG GCGGCGGCGTGCCCGCCAAGTTTCTGTATAGGGCATCTGCCCCTGCACACAGGAA GGTGAAGGAGAGCCCACTGAGCTCCGATGCCATCTTCAAGCAGAGCCACGCCGGC CTGCTGAACCTGTGCATCGTGGTGCTGATCGCCGTGAACTCCAGGCTGATCATCGA GAATCTGATGAAGTACGGCCTGCTGATCCGCGCCGGCTATTGGTTTTCTAGCAAGT CCCTGCGGGACTGGCCTCTGCTGATGTGCTGTCTGACCCTGCCAGCATTTCCTCTGG GAGCCTTCATGGTGGAGAAGCTGGCCCAGCACAATTTCATCTCCGAGTCTGTGGTC ATCAGCCTGCACGTGATCATCACCACAGCCGAGCTGCTGTACCCAGTGATCGTGAT CCTGAGATGCGATTCTGCCGTGCTGAGCGGCATCACACTGATGCTGTTTGCCAGCG TGGTGTGGCTGAAGCTGGTGTCCTACGCCCACACCAACTATGACATGAGGACACTG AGCAAGTCCATCGACAAGGAGGATATGTACTCCAAGTGTCCAGAGATCGATAATC TGAAGGGCGACTCCTTTAAGTCTCTGGTGTATTTCATGGTGGCCCCCACCCTGTGCT ACCAGCCAAGCTATCCAAGGACCACCTGCATCAGGAAGGGATGGGTCATCCGCCA GGTGGTGAAGCTGGTCATCTTCACCGGCCTGATGGGCTTCATCATCGAGCAGTACA TCAACCCCATCGTGCAGAATTCCCAGCACCCTCTGAAGGGCAACTTTCTGAATGCC ATCGAGCGGGTGCTGAAGCTGTCTGTGCCCACCCTGTACGTGTGGCTGTGCATGTT CTATTGTTTCTTTCACCTGTGGCTGAACATCCTGGCCGAGCTGCTGTGCTTTGGCGA TAGAGAGTTCTACAAGGACTGGTGGAACGCCAAGACAATCGAGGAGTATTGGAGG ATGTGGAATATGCCTGTGCACCGCTGGATGATCCGGCACATCTACTTCCCTTGTCT GAGAAATGGCCTGCCAAGGGCCGTGGCCATCCTGATCTCCTTTCTGGTGTCTGCCA TCTTCCACGAGATCTGCATCGCCGTGCCCTGTCACATCTTTAAGTTCTGGGCCTTTA TCGGCATCATGTTCCAGATCCCCCTGGTCATCCTGACCAAGTATCTGCAGCACAAG TTTACAAACTCCATGGTGGGCAATATGATCTTCTGGTTCTTTTTCTCTATCCTGGGC CAGCCTATGTGCGTGCTGCTGTACTATCACGACGTGATGAATAGAAAGGTGAGGA CCGAGTGA [0112] SEQ ID NO:34 shows the DGAT1A_Pd amino acid sequence encoded by SEQ ID NO:33: MAIPSDRETLERAPEPSPASDLQSSLRRRLHSTVAAVVVPDSSSKTSSPSAENLTTDSGE DSRGDTSSDADTRDRVVDGVDREEENKTVSVLNGRQYEDGGGRGQGQGTGGGVPAK FLYRASAPAHRKVKESPLSSDAIFKQSHAGLLNLCIVVLIAVNSRLIIENLMKYGLLIRA GYWFSSKSLRDWPLLMCCLTLPAFPLGAFMVEKLAQHNFISESVVISLHVIITTAELLYP VIVILRCDSAVLSGITLMLFASVVWLKLVSYAHTNYDMRTLSKSIDKEDMYSKCPEIDN LKGDSFKSLVYFMVAPTLCYQPSYPRTTCIRKGWVIRQVVKLVIFTGLMGFIIEQYINPI VQNSQHPLKGNFLNAIERVLKLSVPTLYVWLCMFYCFFHLWLNILAELLCFGDREFYK DWWNAKTIEEYWRMWNMPVHRWMIRHIYFPCLRNGLPRAVAILISFLVSAIFHEICIA VPCHIFKFWAFIGIMFQIPLVILTKYLQHKFTNSMVGNMIFWFFFSILGQPMCVLLYYH DVMNRKVRTE [0113] SEQ ID NO:35 shows an exemplary HzDST nucleotide sequence: ATGGCCCAGTCTTACCAGAGCACCACAGTGCTGTCCGAGGAGAAGGAGCTGACCC TGCAGCACCTGGTGCCACAGGCATCTCCTCGGAAGTACCAGATCGTGTATCCTAAC CTGATCACATTCGGCTATTGGCACATCGCCGGGCTGTACGGCCTGTATCTGTGCTTT ACCTCTGCCAAGTGGGCCACAATCCTGTTCAGCTACATCCTGTTTGTGCTGGCAGA GATCGGAATCACCGCAGGAGCACACAGACTGTGGGCACACAAGACATATAAGGCC AAGCTGCCACTGGAGATCCTGCTGATGGTGTTCAACTCCATCGCCTTTCAGAATTC TGCCATCGATTGGGTGCGGGACCACAGACTGCACCACAAGTACTCCGACACCGAT GCCGACCCACACAACGCCAGCAGGGGCTTCTTTTATTCCCACGTGGGATGGCTGCT GGTGCGGAAGCACCCCGAGGTGAAGAAGAGAGGCAAGGAGCTGAATATGTCTGA TATCTACAACAATCCCGTGCTGCGCTTCCAGAAGAAGTATGCCATCCCTTTCATCG GCGCCGTGTGCTTTGCCCTGCCAACCATGATCCCCGTGTACTTTTGGGGCGAGACA TGGAGCAATGCCTGGCACATCACAATGCTGAGGTATATCATGAACCTGAATGTGA CATTCCTGGTGAACTCCGCCGCCCACATCTGGGGCAATAAGCCTTACGACGCCAAG ATCCTGCCAGCACAGAACGTGGCCGTGAGCGTGGCAACCGGAGGAGAGGGCTTCC ACAATTACCACCACGTGTTTCCATGGGATTATAGGGCAGCAGAGCTGGGAAACAA TTCTCTGAACCTGACCACAAAGTTCATCGACCTGTTTGCCGCCATCGGCTGGGCCT ACGATCTGAAGACAGTGAGCGAGGACATGATCAAGCAGAGGATCAAGAGGACCG GCGATGGAACAGACCTGTGGGGACACGAGCAGAATTGCGATGAAGTGTGGGATGT GAAGGACAAGAGCTCCTGA [0114] SEQ ID NO:36 shows the HzDST amino acid sequence encoded by SEQ ID NO:35: MAQSYQSTTVLSEEKELTLQHLVPQASPRKYQIVYPNLITFGYWHIAGLYGLYLCFTSA KWATILFSYILFVLAEIGITAGAHRLWAHKTYKAKLPLEILLMVFNSIAFQNSAIDWVR DHRLHHKYSDTDADPHNASRGFFYSHVGWLLVRKHPEVKKRGKELNMSDIYNNPVL RFQKKYAIPFIGAVCFALPTMIPVYFWGETWSNAWHITMLRYIMNLNVTFLVNSAAHI WGNKPYDAKILPAQNVAVSVATGGEGFHNYHHVFPWDYRAAELGNNSLNLTTKFID LFAAIGWAYDLKTVSEDMIKQRIKRTGDGTDLWGHEQNCDEVWDVKDKSS [0115] SEQ ID NO:37 shows an exemplary DST076 nucleotide sequence: ATGCACATCGAGTCTGAGAACTGCCCCGGCAGGTTTAAGGAGGTGAACATGGCCC CTAATGCCACCGATGCCAATGGCGTGCTGTTCGAGACCGATGCCGCCACACCTGAC CTGGCCCTGCCACACGCACCTGTGCAGCAGGCCGACAACTACCCAAAGAAGTACG TGTGGCGCAATATCATCCTGTTTGCCTACCTGCACATCGCCGCCCTGTACGGCGGC TATCTGTTTCTGTTCCACGCCAAGTGGCAGACCGATATCTTCGCCTACATCCTGTAT GTGATGTCTGGACTGGGAATCACAGCAGGAGCACACAGGCTGTGGGCCCACAAGA GCTACAAGGCCAAGTGGCCTCTGAGACTGATCCTGGTCATCTTCAACACACTGGCC TTTCAGGACTCTGCCATCGATTGGAGCAGGGACCACCGCATGCACCACAAGTATTC CGAGACCGACGCCGATCCCCACAATGCCACACGGGGCTTCTTTTTCTCTCACATCG GCTGGCTGCTGGTGCGGAAGCACCCTGAGCTGAAGAGAAAGGGCAAGGGCCTGGA CCTGTCCGATCTGTATGCCGACCCAATCCTGAGATTTCAGAAGAAGTACTATCTGA TCCTGATGCCCCTGACCTGTTTCGTGCTGCCAACAGTGATCCCCGTGTACTATTGGG GCGAGACCTGGACAAACGCCTTTTTCGTGGCCGCCCTGTTTAGGTACGCCTTCATC CTGAACGTGACCTGGCTGGTGAATAGCGCCGCCCACAAGTGGGGCGATAAGCCTT ATGACCGCAACATCAAGCCATCCGAGAATATCAGCGTGTCCATGTTTGCCCTGGGC GAGGGCTTCCACAACTACCACCACACCTTCCCATGGGATTATAAGACAGCCGAGCT GGGCAACAATATGCTGAACTTCACCACAAACTTCATCAACTTCTTCGCCAAGATCG GCTGGGCCTACGATCTGAAGACCGTGTCCGACGAGATCGTGCGGTCTAGAGCAAA GAGGACAGGCGACGGAAGCCACCACCTGTGGGGATGGGGCGACAAGGATCACTC CAGGGAGGAGATGGCTGCCGCCATCCGCATCCACCCCAAGGACGAT [0116] SEQ ID NO:38 shows the DST076 amino acid sequence encoded by SEQ ID NO:37: MHIESENCPGRFKEVNMAPNATDANGVLFETDAATPDLALPHAPVQQADNYPKKYV WRNIILFAYLHIAALYGGYLFLFHAKWQTDIFAYILYVMSGLGITAGAHRLWAHKSYK AKWPLRLILVIFNTLAFQDSAIDWSRDHRMHHKYSETDADPHNATRGFFFSHIGWLLV RKHPELKRKGKGLDLSDLYADPILRFQKKYYLILMPLTCFVLPTVIPVYYWGETWTNA FFVAALFRYAFILNVTWLVNSAAHKWGDKPYDRNIKPSENISVSMFALGEGFHNYHH TFPWDYKTAELGNNMLNFTTNFINFFAKIGWAYDLKTVSDEIVRSRAKRTGDGSHHL WGWGDKDHSREEMAAAIRIHPKDD [0117] SEQ ID NO:39 shows a Lepidopteran Z9 desaturase signature motif. [0118] SEQ ID NO:40 shows an exemplary DST148 nucleotide sequence: ATGGAGGCCAAGCAGAACAATCTGGCACCCACCCTGGAGGAGGAGGCACAGTTCG AGAAGCTGATCGCCCCTCAGGCCTCCGATCGCAAGCACGAGATCATCTACGCCAA CCTGATCACCTTCGCCTATGGCCACATCTCTGCCCTGTACGGCCTGTATCTGTGCTT TAGCTCCGCCAAGTGGGCCACAATCATCATGGCCTACGTGATCCTGATCGCAGCAG AAGTGGGAGTGACCGCAGGAGCCCACAGGCTGTGGACACACCGCGCCTATAAGGC CAAGCGGCCCCTGCAGATCATCCTGATGGTCATGAACTCCTTCGCCTTTCAGAATT CTGCCATCACATGGATCAGGGACCACCGCATGCACCACAGGTACTCTGACACCGA TGCCGACCCACACAACGCCACACGCGGCTTCTTTTATAGCCACATCGGATGGCTGC TGGTGCGGAAGCACCCAGAGGTGAAGCGGAGAGGCAAGACCATCGATATGTCTGA CATCTACAGCAATCCTGTGCTGGTGTTCCAGAAGAAGTATGCCATCCCATTCATCG GCGCCGTGTGCTTTGTGATCCCAACACTGGTGCCCATCTACTTTTGGGGCGAGACC CTGACAAACGCCTGGCACATCACCCTGCTGAGATATATCATCAGCCTGCACGTGAC ATTCCTGGTGAATTCCGCCGCACACCTGTGGGGAACCAGGGCATACGATAAGAGA ATCTTTCCCGCCCAGAACCTGATCGTGTCCCTGCTGGCAGTGGGAGAGGGCTTCCA CAATTACCACCACGTGTTTCCTTGGGATTATAGGACAGCCGAGCTGGGCAACAATT ACCTGAATCTGACCACAAAGTTTATCGACTTCTTTGCCTGGCTGGGCTGGGCCTAT GATCTGAAGTCCGTGCCTGACTCTGCCGTGCAGAGCAGGGCAGCAAGAACCGGCG ACGGAACAAACAGCTGGGGATGGCCAGAGGAGGATGCCAATGAGGACATCCTGA AGCAGACCCCCCCTCTGTGA [0119] SEQ ID NO:41 shows the DST148 amino acid sequence encoded by SEQ ID NO:40: MEAKQNNLAPTLEEEAQFEKLIAPQASDRKHEIIYANLITFAYGHISALYGLYLCFSSAK WATIIMAYVILIAAEVGVTAGAHRLWTHRAYKAKRPLQIILMVMNSFAFQNSAITWIR DHRMHHRYSDTDADPHNATRGFFYSHIGWLLVRKHPEVKRRGKTIDMSDIYSNPVLV FQKKYAIPFIGAVCFVIPTLVPIYFWGETLTNAWHITLLRYIISLHVTFLVNSAAHLWGT RAYDKRIFPAQNLIVSLLAVGEGFHNYHHVFPWDYRTAELGNNYLNLTTKFIDFFAWL GWAYDLKSVPDSAVQSRAARTGDGTNSWGWPEEDANEDILKQTPPL [0120] SEQ ID NO:42 shows an exemplary DST183 nucleotide sequence: ATGGCCCCAAACATCAGCGAGGATGTGAATGGCGTGCTGTTCGAGTCCGATGCCG CCACACCAGACCTGGCCCTGTCTACCCCACCTGTGCAGAAGGCAGACAACAGGCC CAAGCAGCTGGTGTGGAGAAATATCCTGCTGTTTGCATACCTGCACCTGGCAGCAC AGTACGGAGGCTATCTGTTTCTGTTCTCTGCCAAGTGGCAGACAGATATCTTCGCC TACATCCTGTATGTGATCAGCGGACTGGGAATCACCGCAGGAGCACACCGGCTGT GGGCCCACAAGTCCTACAAGGCCAAGTGGCCTCTGAGAGTGATCCTGGTCATCTTC AACACCGTGGCCTTTCAGGACGCAGCAATGGATTGGGCAAGGGACCACAGAATGC ACCACAAGTATTCTGAGACAGACGCCGATCCTCACAATGCCACCAGGGGCTTCTTT TTCAGCCACATCGGCTGGCTGCTGGTGCGCAAGCACCCAGATCTGAAGGAGAAGG GCAAGGGCCTGGACATGAGCGATCTGCTGGCCGACCCCATCCTGAGGTTTCAGAA GAAGTACTATCTGATCCTGATGCCTCTGGCCTGCTTTGTGATGCCAACAGTGATCC CCGTGTACTTCTGGGGCGAGACATGGACCAACGCCTTTTTCGTGGCCGCCATGTTT CGCTATGCCTTCATCCTGAACGTGACCTGGCTGGTGAATTCTGCCGCCCACAAGTG GGGCGATAAGCCTTACGACAAGAGCATCAAGCCATCCGAGAACCTGTCTGTGGCC ATGTTTGCCCTGGGCGAGGGCTTCCACAATTACCACCACACATTCCCCTGGGACTA TAAGACCGCCGAGCTGGGCAACAATAAGCTGAACTTTACCACAACCTTCATCAACT TCTTCGCCAAGATCGGCTGGGCCTATGATCTGAAGACAGTGTCCGACGATATCGTG AAGAATAGGGTGAAGAGGACCGGCGACGGAAGCCACCACCTGTGGGGCTGGGGC GATGAGAACCAGTCCAAGGAGGAGATCGACGCCGCCATCCGGATCAATCCTAAGG ACGATTGA [0121] SEQ ID NO:43 shows the DST183 amino acid sequence encoded by SEQ ID NO:42: MAPNISEDVNGVLFESDAATPDLALSTPPVQKADNRPKQLVWRNILLFAYLHLAAQY GGYLFLFSAKWQTDIFAYILYVISGLGITAGAHRLWAHKSYKAKWPLRVILVIFNTVAF QDAAMDWARDHRMHHKYSETDADPHNATRGFFFSHIGWLLVRKHPDLKEKGKGLD MSDLLADPILRFQKKYYLILMPLACFVMPTVIPVYFWGETWTNAFFVAAMFRYAFILN VTWLVNSAAHKWGDKPYDKSIKPSENLSVAMFALGEGFHNYHHTFPWDYKTAELGN NKLNFTTTFINFFAKIGWAYDLKTVSDDIVKNRVKRTGDGSHHLWGWGDENQSKEEI DAAIRINPKDD [0122] SEQ ID NO:44 shows an exemplary DST189 nucleotide sequence: ATGGATTTTCTGAACGAGATCGACAATTGCCCCGAGCGGCTGAGAAAGCCAGAGA AGATGGCCCCCAACGTGACCGAGGAGAATGGCGTGCTGTTCGAGTCCGATGCAGC AACCCCAGACCTGGCCCTGGCAAGGACACCTGTGGAGCAGGCCGACGATTCTCCA AGGATCTACGTGTGGCGCAACATCATCCTGTTTGCCTATCTGCACCTGGCCGCCAT CTACGGCGGCTATCTGTTTCTGTTCTCCGCCAAGTGGCAGACCGATATCTTCGCCTA CCTGCTGTATGTGGCATCTGGACTGGGAATCACAGCAGGAGCACACAGGCTGTGG GCACACAAGAGCTACAAGGCCAAGTGGCCTCTGCGCCTGATCCTGACCATCTTTAA CACAATCGCCTTTCAGGACAGCGCCATCGATTGGGCCAGGGACCACCGCATGCAC CACAAGTATTCCGAGACCGACGCCGATCCACACAATGCCACACGGGGCTTCTTTTT CTCTCACATCGGATGGCTGCTGGTGCGGAAGCACCCAGAGCTGAAGAGAAAGGGC AAGGGCCTGGACCTGTCTGATCTGTACAGCGATCCCATCCTGAGATTTCAGAAGAA GTACTATATGATCCTGATGCCTCTGGCCTGTTTCATCCTGCCCACCGTGATCCCCGT GTATATGTGGAACGAGACATGGAGCAATGCCTTTTTCGTGGCCGCCCTGTTTAGGT ATACCTTCATCCTGAACGTGACATGGCTGGTGAATTCCGCCGCCCACAAGTGGGGC GATAAGCCTTACGACAAGTCCATCAAGCCATCTGAGAACATGAGCGTGTCCCTGTT TGCCTTCGGCGAGGGCTTTCACAATTACCACCACACCTTCCCTTGGGACTATAAGA CAGCCGAGCTGGGCAACCACCGGCTGAACTTCACCACAAAGTTCATCAACTTCTTC GCCAAGATCGGCTGGGCCTATGATATGAAGACCGTGTCTCAGGAGATCGTGCAGC AGCGGGTGAAGAGAACAGGCGACGGAAGCCACCACCTGTGGGGATGGGGCGACA AGGATCACGCACAGGAGGAGATCAACGCCGCCATCCGCATCAATCCAAAGGACGA TTGA [0123] SEQ ID NO:45 shows the DST189 amino acid sequence encoded by SEQ ID NO:44: MDFLNEIDNCPERLRKPEKMAPNVTEENGVLFESDAATPDLALARTPVEQADDSPRIY VWRNIILFAYLHLAAIYGGYLFLFSAKWQTDIFAYLLYVASGLGITAGAHRLWAHKSY KAKWPLRLILTIFNTIAFQDSAIDWARDHRMHHKYSETDADPHNATRGFFFSHIGWLL VRKHPELKRKGKGLDLSDLYSDPILRFQKKYYMILMPLACFILPTVIPVYMWNETWSN AFFVAALFRYTFILNVTWLVNSAAHKWGDKPYDKSIKPSENMSVSLFAFGEGFHNYH HTFPWDYKTAELGNHRLNFTTKFINFFAKIGWAYDMKTVSQEIVQQRVKRTGDGSHH LWGWGDKDHAQEEINAAIRINPKDD [0124] SEQ ID NO:46 shows an exemplary DST192 100G/V/I/L 245I/F nucleotide sequence: ATGGATTTTCTGAACGAGATCGACAATTGCCCCGAGCGGCTGAGAAAGCCAGAGA AGATGGCCCCCAACGTGACCGAGGAGAATGGCGTGCTGTTCGAGTCCGATGCAGC AACCCCAGACCTGGCCCTGGCAAGGACACCTGTGGAGCAGGCCGACGATTCTCCA AGGATCTACGTGTGGCGCAACATCATCCTGTTTGCCTATCTGCACCTGGCCGCCAT CTACGGCGGCTATCTGTTTCTGTTCTCCGCCAAGTGGCAGACCGATATCTTCGCCTA CCTGCTGTATGTGGCATCTXXXCTGGGAATCACAGCAGGAGCACACAGGCTGTGG GCACACAAGAGCTACAAGGCCAAGTGGCCTCTGCGCCTGATCCTGACCATCTTTAA CACAATCGCCTTTCAGGACAGCGCCATCGATTGGGCCAGGGACCACCGCATGCAC CACAAGTATTCCGAGACCGACGCCGATCCACACAATGCCACACGGGGCTTCTTTTT CTCTCACATCGGATGGCTGCTGGTGCGGAAGCACCCAGAGCTGAAGAGAAAGGGC AAGGGCCTGGACCTGTCTGATCTGTACAGCGATCCCATCCTGAGATTTCAGAAGAA GTACTATATGATCCTGATGCCTCTGGCCTGTTTCATCCTGCCCACCGTGATCCCCGT GTATATGTGGAACGAGACATGGAGCAATGCCTTTTTCGTGGCCGCCCTGTTTAGGT ATACCTTCXXXCTGAACGTGACATGGCTGGTGAATTCCGCCGCCCACAAGTGGGG CGATAAGCCTTACGACAAGTCCATCAAGCCATCTGAGAACATGAGCGTGTCCCTGT TTGCCTTCGGCGAGGGCTTTCACAATTACCACCACACCTTCCCTTGGGACTATAAG ACAGCCGAGCTGGGCAACCACCGGCTGAACTTCACCACAAAGTTCATCAACTTCTT CGCCAAGATCGGCTGGGCCTATGATATGAAGACCGTGTCTCAGGAGATCGTGCAG CAGCGGGTGAAGAGAACAGGCGACGGAAGCCACCACCTGTGGGGATGGGGCGAC AAGGATCACGCACAGGAGGAGATCAACGCCGCCATCCGCATCAATCCAAAGGACG ATTGA [0125] SEQ ID NO:47 shows the DST192 G100V/I/L 245I/F amino acid sequence encoded by SEQ ID NO:46: MDFLNEIDNCPERLRKPEKMAPNVTEENGVLFESDAATPDLALARTPVEQADDSPRIY VWRNIILFAYLHLAAIYGGYLFLFSAKWQTDIFAYLLYVASXLGITAGAHRLWAHKSY KAKWPLRLILTIFNTIAFQDSAIDWARDHRMHHKYSETDADPHNATRGFFFSHIGWLL VRKHPELKRKGKGLDLSDLYSDPILRFQKKYYMILMPLACFILPTVIPVYMWNETWSN AFFVAALFRYTFXLNVTWLVNSAAHKWGDKPYDKSIKPSENMSVSLFAFGEGFHNYH HTFPWDYKTAELGNHRLNFTTKFINFFAKIGWAYDMKTVSQEIVQQRVKRTGDGSHH LWGWGDKDHAQEEINAAIRINPKDD [0126] SEQ ID NO:48 shows an exemplary DST192100G/V/I/L nucleotide sequence: ATGGATTTTCTGAACGAGATCGACAATTGCCCCGAGCGGCTGAGAAAGCCAGAGA AGATGGCCCCCAACGTGACCGAGGAGAATGGCGTGCTGTTCGAGTCCGATGCAGC AACCCCAGACCTGGCCCTGGCAAGGACACCTGTGGAGCAGGCCGACGATTCTCCA AGGATCTACGTGTGGCGCAACATCATCCTGTTTGCCTATCTGCACCTGGCCGCCAT CTACGGCGGCTATCTGTTTCTGTTCTCCGCCAAGTGGCAGACCGATATCTTCGCCTA CCTGCTGTATGTGGCATCTXXXCTGGGAATCACAGCAGGAGCACACAGGCTGTGG GCACACAAGAGCTACAAGGCCAAGTGGCCTCTGCGCCTGATCCTGACCATCTTTAA CACAATCGCCTTTCAGGACAGCGCCATCGATTGGGCCAGGGACCACCGCATGCAC CACAAGTATTCCGAGACCGACGCCGATCCACACAATGCCACACGGGGCTTCTTTTT CTCTCACATCGGATGGCTGCTGGTGCGGAAGCACCCAGAGCTGAAGAGAAAGGGC AAGGGCCTGGACCTGTCTGATCTGTACAGCGATCCCATCCTGAGATTTCAGAAGAA GTACTATATGATCCTGATGCCTCTGGCCTGTTTCATCCTGCCCACCGTGATCCCCGT GTATATGTGGAACGAGACATGGAGCAATGCCTTTTTCGTGGCCGCCCTGTTTAGGT ATACCTTCATCCTGAACGTGACATGGCTGGTGAATTCCGCCGCCCACAAGTGGGGC GATAAGCCTTACGACAAGTCCATCAAGCCATCTGAGAACATGAGCGTGTCCCTGTT TGCCTTCGGCGAGGGCTTTCACAATTACCACCACACCTTCCCTTGGGACTATAAGA CAGCCGAGCTGGGCAACCACCGGCTGAACTTCACCACAAAGTTCATCAACTTCTTC GCCAAGATCGGCTGGGCCTATGATATGAAGACCGTGTCTCAGGAGATCGTGCAGC AGCGGGTGAAGAGAACAGGCGACGGAAGCCACCACCTGTGGGGATGGGGCGACA AGGATCACGCACAGGAGGAGATCAACGCCGCCATCCGCATCAATCCAAAGGACGA TTGA [0127] SEQ ID NO:49 shows the DST192100G/V/I/L amino acid sequence encoded by SEQ ID NO:48: MDFLNEIDNCPERLRKPEKMAPNVTEENGVLFESDAATPDLALARTPVEQADDSPRIY VWRNIILFAYLHLAAIYGGYLFLFSAKWQTDIFAYLLYVASXLGITAGAHRLWAHKSY KAKWPLRLILTIFNTIAFQDSAIDWARDHRMHHKYSETDADPHNATRGFFFSHIGWLL VRKHPELKRKGKGLDLSDLYSDPILRFQKKYYMILMPLACFILPTVIPVYMWNETWSN AFFVAALFRYTFILNVTWLVNSAAHKWGDKPYDKSIKPSENMSVSLFAFGEGFHNYH HTFPWDYKTAELGNHRLNFTTKFINFFAKIGWAYDMKTVSQEIVQQRVKRTGDGSHH LWGWGDKDHAQEEINAAIRINPKDD [0128] SEQ ID NO:50 shows an exemplary DST192245I/F nucleotide sequence: ATGGATTTTCTGAACGAGATCGACAATTGCCCCGAGCGGCTGAGAAAGCCAGAGA AGATGGCCCCCAACGTGACCGAGGAGAATGGCGTGCTGTTCGAGTCCGATGCAGC AACCCCAGACCTGGCCCTGGCAAGGACACCTGTGGAGCAGGCCGACGATTCTCCA AGGATCTACGTGTGGCGCAACATCATCCTGTTTGCCTATCTGCACCTGGCCGCCAT CTACGGCGGCTATCTGTTTCTGTTCTCCGCCAAGTGGCAGACCGATATCTTCGCCTA CCTGCTGTATGTGGCATCTGGACTGGGAATCACAGCAGGAGCACACAGGCTGTGG GCACACAAGAGCTACAAGGCCAAGTGGCCTCTGCGCCTGATCCTGACCATCTTTAA CACAATCGCCTTTCAGGACAGCGCCATCGATTGGGCCAGGGACCACCGCATGCAC CACAAGTATTCCGAGACCGACGCCGATCCACACAATGCCACACGGGGCTTCTTTTT CTCTCACATCGGATGGCTGCTGGTGCGGAAGCACCCAGAGCTGAAGAGAAAGGGC AAGGGCCTGGACCTGTCTGATCTGTACAGCGATCCCATCCTGAGATTTCAGAAGAA GTACTATATGATCCTGATGCCTCTGGCCTGTTTCATCCTGCCCACCGTGATCCCCGT GTATATGTGGAACGAGACATGGAGCAATGCCTTTTTCGTGGCCGCCCTGTTTAGGT ATACCTTCXXXCTGAACGTGACATGGCTGGTGAATTCCGCCGCCCACAAGTGGGG CGATAAGCCTTACGACAAGTCCATCAAGCCATCTGAGAACATGAGCGTGTCCCTGT TTGCCTTCGGCGAGGGCTTTCACAATTACCACCACACCTTCCCTTGGGACTATAAG ACAGCCGAGCTGGGCAACCACCGGCTGAACTTCACCACAAAGTTCATCAACTTCTT CGCCAAGATCGGCTGGGCCTATGATATGAAGACCGTGTCTCAGGAGATCGTGCAG CAGCGGGTGAAGAGAACAGGCGACGGAAGCCACCACCTGTGGGGATGGGGCGAC AAGGATCACGCACAGGAGGAGATCAACGCCGCCATCCGCATCAATCCAAAGGACG ATTGA [0129] SEQ ID NO:51 shows the DST192245I/F amino acid sequence encoded by SEQ ID NO:50: MDFLNEIDNCPERLRKPEKMAPNVTEENGVLFESDAATPDLALARTPVEQADDSPRIY VWRNIILFAYLHLAAIYGGYLFLFSAKWQTDIFAYLLYVASGLGITAGAHRLWAHKSY KAKWPLRLILTIFNTIAFQDSAIDWARDHRMHHKYSETDADPHNATRGFFFSHIGWLL VRKHPELKRKGKGLDLSDLYSDPILRFQKKYYMILMPLACFILPTVIPVYMWNETWSN AFFVAALFRYTFXLNVTWLVNSAAHKWGDKPYDKSIKPSENMSVSLFAFGEGFHNYH HTFPWDYKTAELGNHRLNFTTKFINFFAKIGWAYDMKTVSQEIVQQRVKRTGDGSHH LWGWGDKDHAQEEINAAIRINPKDD [0130] SEQ ID NO:52 shows an exemplary DST192 nucleotide sequence: ATGGATTTTCTGAACGAGATCGACAATTGCCCCGAGCGGCTGAGAAAGCCAGAGA AGATGGCCCCCAACGTGACCGAGGAGAATGGCGTGCTGTTCGAGTCCGATGCAGC AACCCCAGACCTGGCCCTGGCAAGGACACCTGTGGAGCAGGCCGACGATTCTCCA AGGATCTACGTGTGGCGCAACATCATCCTGTTTGCCTATCTGCACCTGGCCGCCAT CTACGGCGGCTATCTGTTTCTGTTCTCCGCCAAGTGGCAGACCGATATCTTCGCCTA CCTGCTGTATGTGGCATCTGGACTGGGAATCACAGCAGGAGCACACAGGCTGTGG GCACACAAGAGCTACAAGGCCAAGTGGCCTCTGCGCCTGATCCTGACCATCTTTAA CACAATCGCCTTTCAGGACAGCGCCATCGATTGGGCCAGGGACCACCGCATGCAC CACAAGTATTCCGAGACCGACGCCGATCCACACAATGCCACACGGGGCTTCTTTTT CTCTCACATCGGATGGCTGCTGGTGCGGAAGCACCCAGAGCTGAAGAGAAAGGGC AAGGGCCTGGACCTGTCTGATCTGTACAGCGATCCCATCCTGAGATTTCAGAAGAA GTACTATATGATCCTGATGCCTCTGGCCTGTTTCATCCTGCCCACCGTGATCCCCGT GTATATGTGGAACGAGACATGGAGCAATGCCTTTTTCGTGGCCGCCCTGTTTAGGT ATACCTTCATCCTGAACGTGACATGGCTGGTGAATTCCGCCGCCCACAAGTGGGGC GATAAGCCTTACGACAAGTCCATCAAGCCATCTGAGAACATGAGCGTGTCCCTGTT TGCCTTCGGCGAGGGCTTTCACAATTACCACCACACCTTCCCTTGGGACTATAAGA CAGCCGAGCTGGGCAACCACCGGCTGAACTTCACCACAAAGTTCATCAACTTCTTC GCCAAGATCGGCTGGGCCTATGATATGAAGACCGTGTCTCAGGAGATCGTGCAGC AGCGGGTGAAGAGAACAGGCGACGGAAGCCACCACCTGTGGGGATGGGGCGACA AGGATCACGCACAGGAGGAGATCAACGCCGCCATCCGCATCAATCCAAAGGACGA TTGA [0131] SEQ ID NO:53 shows the DST192 amino acid sequence encoded by SEQ ID NO:52: MDFLNEIDNCPERLRKPEKMAPNVTEENGVLFESDAATPDLALARTPVEQADDSPRIY VWRNIILFAYLHLAAIYGGYLFLFSAKWQTDIFAYLLYVASGLGITAGAHRLWAHKSY KAKWPLRLILTIFNTIAFQDSAIDWARDHRMHHKYSETDADPHNATRGFFFSHIGWLL VRKHPELKRKGKGLDLSDLYSDPILRFQKKYYMILMPLACFILPTVIPVYMWNETWSN AFFVAALFRYTFILNVTWLVNSAAHKWGDKPYDKSIKPSENMSVSLFAFGEGFHNYH HTFPWDYKTAELGNHRLNFTTKFINFFAKIGWAYDMKTVSQEIVQQRVKRTGDGSHH LWGWGDKDHAQEEINAAIRINPKDD [0132] SEQ ID NO:54 shows an exemplary DST192 G100V nucleotide sequence: ATGGATTTTCTGAACGAGATCGACAATTGCCCCGAGCGGCTGAGAAAGCCAGAGA AGATGGCCCCCAACGTGACCGAGGAGAATGGCGTGCTGTTCGAGTCCGATGCAGC AACCCCAGACCTGGCCCTGGCAAGGACACCTGTGGAGCAGGCCGACGATTCTCCA AGGATCTACGTGTGGCGCAACATCATCCTGTTTGCCTATCTGCACCTGGCCGCCAT CTACGGCGGCTATCTGTTTCTGTTCTCCGCCAAGTGGCAGACCGATATCTTCGCCTA CCTGCTGTATGTGGCATCTGTGCTGGGAATCACAGCAGGAGCACACAGGCTGTGG GCACACAAGAGCTACAAGGCCAAGTGGCCTCTGCGCCTGATCCTGACCATCTTTAA CACAATCGCCTTTCAGGACAGCGCCATCGATTGGGCCAGGGACCACCGCATGCAC CACAAGTATTCCGAGACCGACGCCGATCCACACAATGCCACACGGGGCTTCTTTTT CTCTCACATCGGATGGCTGCTGGTGCGGAAGCACCCAGAGCTGAAGAGAAAGGGC AAGGGCCTGGACCTGTCTGATCTGTACAGCGATCCCATCCTGAGATTTCAGAAGAA GTACTATATGATCCTGATGCCTCTGGCCTGTTTCATCCTGCCCACCGTGATCCCCGT GTATATGTGGAACGAGACATGGAGCAATGCCTTTTTCGTGGCCGCCCTGTTTAGGT ATACCTTCATCCTGAACGTGACATGGCTGGTGAATTCCGCCGCCCACAAGTGGGGC GATAAGCCTTACGACAAGTCCATCAAGCCATCTGAGAACATGAGCGTGTCCCTGTT TGCCTTCGGCGAGGGCTTTCACAATTACCACCACACCTTCCCTTGGGACTATAAGA CAGCCGAGCTGGGCAACCACCGGCTGAACTTCACCACAAAGTTCATCAACTTCTTC GCCAAGATCGGCTGGGCCTATGATATGAAGACCGTGTCTCAGGAGATCGTGCAGC AGCGGGTGAAGAGAACAGGCGACGGAAGCCACCACCTGTGGGGATGGGGCGACA AGGATCACGCACAGGAGGAGATCAACGCCGCCATCCGCATCAATCCAAAGGACGA TTGA [0133] SEQ ID NO:55 shows the DST192 G100V amino acid sequence encoded by SEQ ID NO:54: MDFLNEIDNCPERLRKPEKMAPNVTEENGVLFESDAATPDLALARTPVEQADDSPRIY VWRNIILFAYLHLAAIYGGYLFLFSAKWQTDIFAYLLYVASVLGITAGAHRLWAHKSY KAKWPLRLILTIFNTIAFQDSAIDWARDHRMHHKYSETDADPHNATRGFFFSHIGWLL VRKHPELKRKGKGLDLSDLYSDPILRFQKKYYMILMPLACFILPTVIPVYMWNETWSN AFFVAALFRYTFILNVTWLVNSAAHKWGDKPYDKSIKPSENMSVSLFAFGEGFHNYH HTFPWDYKTAELGNHRLNFTTKFINFFAKIGWAYDMKTVSQEIVQQRVKRTGDGSHH LWGWGDKDHAQEEINAAIRINPKDD [0134] SEQ ID NO:56 shows an exemplary DST192 I245F nucleotide sequence: ATGGATTTTCTGAACGAGATCGACAATTGCCCCGAGCGGCTGAGAAAGCCAGAGA AGATGGCCCCCAACGTGACCGAGGAGAATGGCGTGCTGTTCGAGTCCGATGCAGC AACCCCAGACCTGGCCCTGGCAAGGACACCTGTGGAGCAGGCCGACGATTCTCCA AGGATCTACGTGTGGCGCAACATCATCCTGTTTGCCTATCTGCACCTGGCCGCCAT CTACGGCGGCTATCTGTTTCTGTTCTCCGCCAAGTGGCAGACCGATATCTTCGCCTA CCTGCTGTATGTGGCATCTGGACTGGGAATCACAGCAGGAGCACACAGGCTGTGG GCACACAAGAGCTACAAGGCCAAGTGGCCTCTGCGCCTGATCCTGACCATCTTTAA CACAATCGCCTTTCAGGACAGCGCCATCGATTGGGCCAGGGACCACCGCATGCAC CACAAGTATTCCGAGACCGACGCCGATCCACACAATGCCACACGGGGCTTCTTTTT CTCTCACATCGGATGGCTGCTGGTGCGGAAGCACCCAGAGCTGAAGAGAAAGGGC AAGGGCCTGGACCTGTCTGATCTGTACAGCGATCCCATCCTGAGATTTCAGAAGAA GTACTATATGATCCTGATGCCTCTGGCCTGTTTCATCCTGCCCACCGTGATCCCCGT GTATATGTGGAACGAGACATGGAGCAATGCCTTTTTCGTGGCCGCCCTGTTTAGGT ATACCTTCTTCCTGAACGTGACATGGCTGGTGAATTCCGCCGCCCACAAGTGGGGC GATAAGCCTTACGACAAGTCCATCAAGCCATCTGAGAACATGAGCGTGTCCCTGTT TGCCTTCGGCGAGGGCTTTCACAATTACCACCACACCTTCCCTTGGGACTATAAGA CAGCCGAGCTGGGCAACCACCGGCTGAACTTCACCACAAAGTTCATCAACTTCTTC GCCAAGATCGGCTGGGCCTATGATATGAAGACCGTGTCTCAGGAGATCGTGCAGC AGCGGGTGAAGAGAACAGGCGACGGAAGCCACCACCTGTGGGGATGGGGCGACA AGGATCACGCACAGGAGGAGATCAACGCCGCCATCCGCATCAATCCAAAGGACGA TTGA [0135] SEQ ID NO:57 show the DST192 I245F amino acid sequence encoded by SEQ ID NO:56: MDFLNEIDNCPERLRKPEKMAPNVTEENGVLFESDAATPDLALARTPVEQADDSPRIY VWRNIILFAYLHLAAIYGGYLFLFSAKWQTDIFAYLLYVASGLGITAGAHRLWAHKSY KAKWPLRLILTIFNTIAFQDSAIDWARDHRMHHKYSETDADPHNATRGFFFSHIGWLL VRKHPELKRKGKGLDLSDLYSDPILRFQKKYYMILMPLACFILPTVIPVYMWNETWSN AFFVAALFRYTFFLNVTWLVNSAAHKWGDKPYDKSIKPSENMSVSLFAFGEGFHNYH HTFPWDYKTAELGNHRLNFTTKFINFFAKIGWAYDMKTVSQEIVQQRVKRTGDGSHH LWGWGDKDHAQEEINAAIRINPKDD [0136] SEQ ID NO:58 shows an exemplary MGA2act nucleotide sequence: ATGGCTAAAGACAAGGAAATCGACTTTGACTACACGGGAGAACTGGTGATGGACG ATTTCGAGTTCCCCATCGACGACATGCTCCACAACGACGGAGATGACTTTGTCAAG AAGGAAACGTGGGACGAGGGTTTTGGTTTCGGAACAAATGGCGCCGTGGGTGCGC AGATGGACGTCCAGACCAGCCCATTTAGCGACCCTGTTTTTGGCGGCGTGGGAGCA GGCCCTGACATGATGGGTCTCATGGATACAAACATGAACCACATCAACGGTAGTC ACAACATGAACAGCGTCGTCAAGCAGGAGGACTACTACACACCGTCCATGGGCAC TCCCATGAACCCCCAACAGCAACAGTCCATGACCCCTCAACAGCAGCATCACATG AACCACAACCAGCCCTCTCAGCTCCAATCTTTGCATCAACAGTCCCAGAAGGCTCA ACCACAGCAGCAACAACAACAGCCACATCAGTCGACAGGAGTCGATAGCATAATC ACAAAGGCATACACCAGGGCAGCAGGAGACCTACCGTACGGACGAAAGTACTCAC GACAACTCAACAAGTACCCCGAGGACGTGGAGTATTCATCTTTCGACCCATCGCTA TGGAGCAATTTGCTGACCAACTCGGAAACTCCGTACCAATACCAGATACATGTCCA TTCCATGCCCGGAAAATCACGTGTGGAGACCCAAATCAAATGTGCATTATCAATCT ACCCTCCGCCTCCACAGCAGTCCGTTCGACTTCCGACAGACACCATTTCGCGTCCC AAGTTCCAGCTCAAGCAGGGCCACATTCCAGACTCGTGTCTCTCCTTGGAAGTATA CATTGTGGGCGAGCAGAACCCCAGCAAGCCCGTCAATTTGTGTTCTAGATGCATCA AACGAGAACAGAAGCGAGCCTGTCGAAAGAAACTCTTTGACGAGTCGGAGGAGCT GTCGTGGGTCGAGACTCGTCAACGACGTCTGGCTGTCTTCAACTGCTCCGAGGTGC TTGAGTTCAAGGATGTGGAACGGCGAGTATACATCCCCGAGTCCGGCACTACAGTT ACCGCCAAGCAGCTGGTTCTGCCCCTGCGTCTGGCTTGCTACTGTAGACACCACGG GGAGAAAAAGGGATTTCGAATCCTCTTTTGTCTTAGAGACGAGGGAGGCCAGATT GTGGGTGTGGGCCAGAGTGGAACGACCGTCATGATCACTGACGACCACAAGGTTG TGGGAGACGCGGTTGCCATGCCGACTACAGCCACTGCTCCTGCCACCGCTGGCTCT TCACAACCCCCCACCCAGGTTCCTACCCCCGCTGCATCTTCGTCGACGAGCTATCG TCCTCGAAACTCGCTTCCTCTATCGCCTACTTCCATGGAAGACTCTTCGTCGGAGTT CACCTCGGACCATTCTCATTACTCCAACTATGGTTCTAAACGACGACGAGACGGCT CTTCCATCAGCGATTGGAGCGGCATGATGAACGTGCGAGGCATGGATAGACAGGC TTCCATTACCAGCATTCCCGAAATGGTTGGTGGCATGTCGAACATGACTGTGGCCA GTGCTTCGGGTAGCGCCACTAATCTGGCTGCTCACAACATGAACAACCCCGCAGAC GAAAACCTGCCCGTCATCAAGCGAATCATCCCCTCGCAGGGTTCCATTCGAGGCGG CATTGAAGTAACCCTGCTTGGATCTGGCTTCAAGTCCAATCTGGTGGCTGTTTTCG GTGACAACAAGGCCGTGGGCACCCACTGCTGGTCTGATTCGACCATCGTGACCCAT CTGCCGCCTTCGACCATCGTGGGTCCCGTTGTGGTGTCTTTCGAAGGTTTTGTGCTC GACAAGCCTCAGATTTTTACCTATTTTGACGACACAGACGGCCAGTTGATTGAGTT GGCGCTCCAGGTTGTGGGTCTCAAGATGAACGGACGGCTGGAAGACGCCCGAAAC ATTGCCATGCGAATCGTGGGCAAC [0137] SEQ ID NO:59 shows the MGA2act amino acid sequence encoded by SEQ ID NO:58: MAKDKEIDFDYTGELVMDDFEFPIDDMLHNDGDDFVKKETWDEGFGFGTNGAVGAQ MDVQTSPFSDPVFGGVGAGPDMMGLMDTNMNHINGSHNMNSVVKQEDYYTPSMGT PMNPQQQQSMTPQQQHHMNHNQPSQLQSLHQQSQKAQPQQQQQQPHQSTGVDSIIT KAYTRAAGDLPYGRKYSRQLNKYPEDVEYSSFDPSLWSNLLTNSETPYQYQIHVHSM PGKSRVETQIKCALSIYPPPPQQSVRLPTDTISRPKFQLKQGHIPDSCLSLEVYIVGEQNP SKPVNLCSRCIKREQKRACRKKLFDESEELSWVETRQRRLAVFNCSEVLEFKDVERRV YIPESGTTVTAKQLVLPLRLACYCRHHGEKKGFRILFCLRDEGGQIVGVGQSGTTVMIT DDHKVVGDAVAMPTTATAPATAGSSQPPTQVPTPAASSSTSYRPRNSLPLSPTSMEDSS SEFTSDHSHYSNYGSKRRRDGSSISDWSGMMNVRGMDRQASITSIPEMVGGMSNMTV ASASGSATNLAAHNMNNPADENLPVIKRIIPSQGSIRGGIEVTLLGSGFKSNLVAVFGD NKAVGTHCWSDSTIVTHLPPSTIVGPVVVSFEGFVLDKPQIFTYFDDTDGQLIELALQV VGLKMNGRLEDARNIAMRIVGN [0138] SEQ ID NO:60 shows an exemplary MGA2act G643R nucleotide sequence: ATGGCTAAAGACAAGGAAATCGACTTTGACTACACGGGAGAACTGGTGATGGACG ATTTCGAGTTCCCCATCGACGACATGCTCCACAACGACGGAGATGACTTTGTCAAG AAGGAAACGTGGGACGAGGGTTTTGGTTTCGGAACAAATGGCGCCGTGGGTGCGC AGATGGACGTCCAGACCAGCCCATTTAGCGACCCTGTTTTTGGCGGCGTGGGAGCA GGCCCTGACATGATGGGTCTCATGGATACAAACATGAACCACATCAACGGTAGTC ACAACATGAACAGCGTCGTCAAGCAGGAGGACTACTACACACCGTCCATGGGCAC TCCCATGAACCCCCAACAGCAACAGTCCATGACCCCTCAACAGCAGCATCACATG AACCACAACCAGCCCTCTCAGCTCCAATCTTTGCATCAACAGTCCCAGAAGGCTCA ACCACAGCAGCAACAACAACAGCCACATCAGTCGACAGGAGTCGATAGCATAATC ACAAAGGCATACACCAGGGCAGCAGGAGACCTACCGTACGGACGAAAGTACTCAC GACAACTCAACAAGTACCCCGAGGACGTGGAGTATTCATCTTTCGACCCATCGCTA TGGAGCAATTTGCTGACCAACTCGGAAACTCCGTACCAATACCAGATACATGTCCA TTCCATGCCCGGAAAATCACGTGTGGAGACCCAAATCAAATGTGCATTATCAATCT ACCCTCCGCCTCCACAGCAGTCCGTTCGACTTCCGACAGACACCATTTCGCGTCCC AAGTTCCAGCTCAAGCAGGGCCACATTCCAGACTCGTGTCTCTCCTTGGAAGTATA CATTGTGGGCGAGCAGAACCCCAGCAAGCCCGTCAATTTGTGTTCTAGATGCATCA AACGAGAACAGAAGCGAGCCTGTCGAAAGAAACTCTTTGACGAGTCGGAGGAGCT GTCGTGGGTCGAGACTCGTCAACGACGTCTGGCTGTCTTCAACTGCTCCGAGGTGC TTGAGTTCAAGGATGTGGAACGGCGAGTATACATCCCCGAGTCCGGCACTACAGTT ACCGCCAAGCAGCTGGTTCTGCCCCTGCGTCTGGCTTGCTACTGTAGACACCACGG GGAGAAAAAGGGATTTCGAATCCTCTTTTGTCTTAGAGACGAGGGAGGCCAGATT GTGGGTGTGGGCCAGAGTGGAACGACCGTCATGATCACTGACGACCACAAGGTTG TGGGAGACGCGGTTGCCATGCCGACTACAGCCACTGCTCCTGCCACCGCTGGCTCT TCACAACCCCCCACCCAGGTTCCTACCCCCGCTGCATCTTCGTCGACGAGCTATCG TCCTCGAAACTCGCTTCCTCTATCGCCTACTTCCATGGAAGACTCTTCGTCGGAGTT CACCTCGGACCATTCTCATTACTCCAACTATGGTTCTAAACGACGACGAGACGGCT CTTCCATCAGCGATTGGAGCGGCATGATGAACGTGCGAGGCATGGATAGACAGGC TTCCATTACCAGCATTCCCGAAATGGTTGGTGGCATGTCGAACATGACTGTGGCCA GTGCTTCGGGTAGCGCCACTAATCTGGCTGCTCACAACATGAACAACCCCGCAGAC GAAAACCTGCCCGTCATCAAGCGAATCATCCCCTCGCAGGGTTCCATTCGAGGCGG CATTGAAGTAACCCTGCTTGGATCTGGCTTCAAGTCCAATCTGGTGGCTGTTTTCG GTGACAACAAGGCCGTGGGCACCCACTGCTGGTCTGATTCGACCATCGTGACCCAT CTGCCGCCTTCGACCATCGTGGGTCCCGTTGTGGTGTCTTTCGAAGGTTTTGTGCTC GACAAGCCTCAGATTTTTACCTATTTTGACGACACAGACGGCCAGTTGATTGAGTT GGCGCTCCAGGTTGTGGGTCTCAAGATGAAC [0139] SEQ ID NO:61 shows the MGA2act G643R amino acid sequence encoded by SEQ ID NO:60: MAKDKEIDFDYTGELVMDDFEFPIDDMLHNDGDDFVKKETWDEGFGFGTNGAVGAQ MDVQTSPFSDPVFGGVGAGPDMMGLMDTNMNHINGSHNMNSVVKQEDYYTPSMGT PMNPQQQQSMTPQQQHHMNHNQPSQLQSLHQQSQKAQPQQQQQQPHQSTGVDSIIT KAYTRAAGDLPYGRKYSRQLNKYPEDVEYSSFDPSLWSNLLTNSETPYQYQIHVHSM PGKSRVETQIKCALSIYPPPPQQSVRLPTDTISRPKFQLKQGHIPDSCLSLEVYIVGEQNP SKPVNLCSRCIKREQKRACRKKLFDESEELSWVETRQRRLAVFNCSEVLEFKDVERRV YIPESGTTVTAKQLVLPLRLACYCRHHGEKKGFRILFCLRDEGGQIVGVGQSGTTVMIT DDHKVVGDAVAMPTTATAPATAGSSQPPTQVPTPAASSSTSYRPRNSLPLSPTSMEDSS SEFTSDHSHYSNYGSKRRRDGSSISDWSGMMNVRGMDRQASITSIPEMVGGMSNMTV ASASGSATNLAAHNMNNPADENLPVIKRIIPSQGSIRGGIEVTLLGSGFKSNLVAVFGD NKAVGTHCWSDSTIVTHLPPSTIVGPVVVSFEGFVLDKPQIFTYFDDTDGQLIELALQV VGLKMNRRLEDARNIAMRIVGN [0140] SEQ ID NO:62 shows an exemplary CBR1 nucleotide sequence: ATGCGCTCCTCTTCTTCACGACAACCTCAAATGCAGTCCTATTACGCCATCGCCAC CGTGTGGGCGCTCATCATTGGCGCCGCCACCTACTACTTCTTCTCCAACTCCAAGC CCAAGGCTGTCTTGCAGCGAGGAGATACGGCCTTCAAGGAGTTCCCTCTCATCCAG AAGACGGTGCTCTCTCACAATTCGGCCATCTATCGATTTGGCCTGCCCCGACCCTC CCACGTGCTGGGTCTGCCCATCGGACAGCACGTGTCGCTTTCTGCCAACATTGGAG GCAAGGAGGTGCTGCGTTCCTACACCCCCACCTCGTCCGACCTGTACGACAAGGGC TACTTCGACATTCTCATCAAGACCTACCCCCAGGGAAACATCTCCAAGTACGTGTC TGAGCTGGCCATTGGTGACACTATGAAGGTGCGGGGCCCCAAGGGCAACTTTGTCT ACAACCACGGCCTGGTCGAGTCCTTCGGCATGGTTTGTGGAGGAACCGGCATCACC CCCATGTACCAGATTCTGCGACACATTGCCGCCGATCCCGCCGACAACACCAAGGT TAACCTCGTCTACGCCAACGTGAACCACGACGACATTCTGCTCAAGAAGGAGCTG GACGCCATTGCCGCCGAGAACGACAACATCAAGATCCACTACGTGCTCAACAACG CTCCCGAGGACTGGACCGGTTCCGTGGGCTTTGTCACCAAGGAGATTCTCGAGAAG CACTGCCCCCCTCCTGGCCCCAACACCAAGCTGCTTCTGTGTGGCCCCCCTCCCAT GATCTCTGCTCTCAAGAAGGCTTCCGTCGAGCTCGGATACGAGAAGGCCCGACCTG TCAGCAAACTTGAGGATCAGGTCTTTGCCTTTTAA [0141] SEQ ID NO:63 shows the CBR1 amino acid sequence encoded by SEQ ID NO:62: MRSSSSRQPQMQSYYAIATVWALIIGAATYYFFSNSKPKAVLQRGDTAFKEFPLIQKTV LSHNSAIYRFGLPRPSHVLGLPIGQHVSLSANIGGKEVLRSYTPTSSDLYDKGYFDILIKT YPQGNISKYVSELAIGDTMKVRGPKGNFVYNHGLVESFGMVCGGTGITPMYQILRHIA ADPADNTKVNLVYANVNHDDILLKKELDAIAAENDNIKIHYVLNNAPEDWTGSVGFV TKEILEKHCPPPGPNTKLLLCGPPPMISALKKASVELGYEKARPVSKLEDQVFAF [0142] SEQ ID NO:64 shows an exemplary HAP1 nucleotide sequence: ATGACAGACCAAGAGTCTAGACGAAACGGACTGGAGCAACTGGCGGTCCGGGCCA TTGCGGAAATGAGAGAAGGACAGGGTCCCCGACCGCCAGATACCCGCGACCAGCA GCCGCTGGATCTACTACCGGATCACGAGGACAAGTCCGTGAACAACAATGGCACT CGATTGGCGCCCTACGAGCAGTGCATTCCGGACCTGTCTGCGCCCGGAGCAGGGTT CGAGGACGATTTTTCACGGGTCGTCAAGCGGCGGCGTAGAGCGCCTGTCAGCTGC CTGCTTTGTCGGAAACGCAAAGTGCGTTGCGACAAGCAGATGCCGTGCTCGGCCTG CAAGACTGCTAACGTGACGGGCTCGTGCGAATACGCCCCGCCCACTTGGGGAGGA CGAGAGGTCCGAGGAGGACAGGAGATCCAGTTCACGGTGGACGAGGCCGAGCCC ATTTCACAGCCTCCGGGCAATAACAGCTCCAACAGCGATACCCGAGGTCACCACA GTAGCAGCAGCAGTTGCTCCAACATCAGTGTGCCGCCGATGCAAAAGCCGTTGAC GCCGCCCCAGATGCTGCAGCCCATCGTCAGTCCACTGCAGCCCACCGTCAAGTCCA AGTCCTCGCACCAGCGCATGTACGGCCGGTTCCCTAATGCCGCAAACTTGCTCCGA GCGGACCTCAGCGAACGTCAACGGGCCAAGGGCGTGGAGGTGCCGCCTTCTCCGA GAAAAAAGCAGCCGCTCCACAACGACGCAAAGTGCAAAAGGGCTATTGAAACGCT GCAGGAGACCCGCAGACGCAGGTCCGACAGCCCCATCGACCCCGTCTACAAGCCG CTGCTGGACCTCTTCCCCGGCCAGCAGTACTGTGAGTTTCTCATTGACTCGTTCCTC AAACGGGTCAACACAGTGCATTACTGCGCCTGTCCAGAGGGTCTGAGAACGGATT TCAACGCCCTGTGGTCGGCCAAACAGCGTATCGAGGATGGCGAACAGTTCAATGC CCGAACATACCTCAATCTACCGTCCATAGCCGTCATGCTGTTGGTATTCCGACTCG GCAGAGGCTCATACCCGCTCAAGGACTGGAGCCCTCCAAACTACTGCATCAAGGA CGGCCACCGAGACAACACGTATCTGGGTCCCCAGGTGTTGGAGGTGGCCGAGGCA TGTCTTAACGCGTCGCATATTTTCCGACACGGTGGGCTCAAGACCATCCAGGCTCT TGTGCTCATGAAACTGGACTGCTTGTACGCTCCAGACTCCGAATACTCGCCTGATG GGGTCGATTCCGTCAACCTTACCGGTGTCATTCTTCAGCTGGCGCTCTCGCAGGGT CTGTACATGGATCCCGCCAACTTTGATACCCAGAGCACCTCAGTGTTGGCGTTCCA GGACTCGCTGTTGGTGCAGTCTCCCAAGGCCCACAGGCGGTCCATGAGCGTGTCGT CTGCGGGCTCTGCATCGCTCATCGAGCCGTCCTCGGCCAAATTGTGGCGTATACTG TACGGATGCGCACTCATTCTGGACGCCAAACGAAACCTGGAGGTTGGAATTCCGTT GTCGTTACCTCAGGGCGATTGTGATGTGTACTTCGACCACACAGACGATGGTGGAA AGACCGCCAGCAACGTCCTGGCGTTCCGTCGGGCCATGCTGCGCTTCTGTGCCCTC TCGGGCTGTGTTATGACCGAAACAAGCCGACCAGTCATGTACAGAAACGAACCTC TGATCCAACGACTGGGCCAGGACTTACACAAGTTCGGGGTGGACGAGGTGGCTTC TCTTGCCCAGCTGCGAGAAGAACTTGGCATCCCCAAGGACGACTCGTTGGCATTTT CGTCCGATGACGACCATGCCGACGAGTCTGACACCTCGTCGGTTGCGTCGTCGGCC ACCTCTGCCCTCCAGCTGATCCACAGATGCCGCATTTACTGTCTGTGGTCCAAGCT GTGTGTGCACTATGAGATGGACACGTATGACGCCTCCGAGGAGCCTCTGTACGGCC ACCGTGAGCTGTGTGTTCGACTGGGCATTGAGCAGGACCAGGCCGGGGTGCTGTC CCAGTACCGGGTCATTCTACGCATGTCCAGTCTCATTACTCTGCTGGTGTGGGTGG CCCGGCGACGCACTGCCTCTGTGGAACTTGGCGCCCATTCATGGTACATTGTGTCT CTTGTGCATTCCATGATGGGTCTTCCGTTGACCGGCGTCATCACGTGCTGGTTCCGT GTGGCTATTTTGGCCAAGGAGACGGGCTCTCTCAAGGGCACACGCTGGGACCCGG ATTGGCTGTTTCGACTCATCACCCAGGGTATTGCCGTACTTGGCGACACGGATGTC GGAGACCGCCAGATGCATGGTCTTTGCGTTTCGTTTCTCAAGAAGGCCCGTTCCGA CTTGGGGCGTTTGCTGGCCAAGATGCCTGGATCCGACCCGGCGTACCTCTCCTCTC AGTTTGATGCCATTGACAAGCTGGCAGAAGTCGAGGTGCTGAGCGAGGAGGATGA GGGCTTATGA [0143] SEQ ID NO:65 shows the HAP1 amino acid sequence encoded by SEQ ID NO:64: MTDQESRRNGLEQLAVRAIAEMREGQGPRPPDTRDQQPLDLLPDHEDKSVNNNGTRL APYEQCIPDLSAPGAGFEDDFSRVVKRRRRAPVSCLLCRKRKVRCDKQMPCSACKTA NVTGSCEYAPPTWGGREVRGGQEIQFTVDEAEPISQPPGNNSSNSDTRGHHSSSSSCSNI SVPPMQKPLTPPQMLQPIVSPLQPTVKSKSSHQRMYGRFPNAANLLRADLSERQRAKG VEVPPSPRKKQPLHNDAKCKRAIETLQETRRRRSDSPIDPVYKPLLDLFPGQQYCEFLID SFLKRVNTVHYCACPEGLRTDFNALWSAKQRIEDGEQFNARTYLNLPSIAVMLLVFRL GRGSYPLKDWSPPNYCIKDGHRDNTYLGPQVLEVAEACLNASHIFRHGGLKTIQALVL MKLDCLYAPDSEYSPDGVDSVNLTGVILQLALSQGLYMDPANFDTQSTSVLAFQDSLL VQSPKAHRRSMSVSSAGSASLIEPSSAKLWRILYGCALILDAKRNLEVGIPLSLPQGDC DVYFDHTDDGGKTASNVLAFRRAMLRFCALSGCVMTETSRPVMYRNEPLIQRLGQDL HKFGVDEVASLAQLREELGIPKDDSLAFSSDDDHADESDTSSVASSATSALQLIHRCRI YCLWSKLCVHYEMDTYDASEEPLYGHRELCVRLGIEQDQAGVLSQYRVILRMSSLITL LVWVARRRTASVELGAHSWYIVSLVHSMMGLPLTGVITCWFRVAILAKETGSLKGTR WDPDWLFRLITQGIAVLGDTDVGDRQMHGLCVSFLKKARSDLGRLLAKMPGSDPAY LSSQFDAIDKLAEVEVLSEEDEGL [0144] SEQ ID NO:66 shows an exemplary PAH1 nucleotide sequence: ATGAAGGTGGGCGATGGAGGAGAAGTCTTCTTTGTGTTCGAGACCGACGCAGACG TGCCCGAAGAGCTCCTGACATCCCCCGTCATTTCTCCCTCTTCGTCGCCATCCTGGG GCCAGGAGGAAGGCGGGGATGGTGAGCCGGACTACCTTGCTCTGAACGACTCTAA ACAGGGTGGCGACAGCAAGCACGGCAGATCGCCCTCGGAGGGCCCACCATTCAGA TCACCTTCGGCGGATCACTTACATGAGATGGGCAGCTTCGATGATGAGAATGACCC TGAGGTGAACAGAAGACAACGTGCGAGCACGGCAGCTCCAGAGCCTGTTCCTGGT TCGTTGAAACACCCAGCCACTATCTCGGAAGGCATCTCTTCGGCCTCGTTTTCCAA CAGCGATACTGATCGAACAGACACTTCTGGACCCACAGAGACAGAACCCACAGAG CTCACAGAGCCTACAGAGCCCACAGAGCCCACAGAGCCTCTGGATCTTGAGCAGA GTCTCCACCGGGCTGCCACTTCTCCCGCCCCTTCGTCCGAGGAGATTTGGGAGAAG GCCCGTGCACTGTCCAAGAAACTCACATCAGAAAACATTCAGAGTAAAATCTCCG ACAACGGAGACATTATTCTGGATATGACTGGTTACAAGTACGACCACGAGGACGT GAGTCGATCAGAGGAGCTGGTCAAGAAAATCCTCGCTGAGGAACTGGGAGAAGAC AGAGACCTGTCCCACATCCTGGTTGAAGACGAGGAGGGTAACCTTGTGATTCAGA GCGCTGGAGACAGCCACCATCACGAGCATATGAGCTCGCCCGAGTCTCTGGCCCA CTCCCCTCAGCCCCTCCCTTCTTCTAACCTTCCGTCTCAGGCCTCGGACAACAAGCA CTACGCCAAGACCATCCGTTTGACGTCTGACCAGCTCAAGTCTCTGGATCTCAAGC CCGGCAAGAACGAGGTCACCTTTGCTGTCAATAACGGCAAGACGTCGTGTTCGGC CCAGCTGTTCTACTGGAAGTACGACATTCCTGTTGTCATTTCCGACATTGATGGCA CGATCACCAAGTCCGATGCTCTGGGCCATCTGCTCACCATGATGGGCCGAGACTGG ACCCACACCGGCGTGGCCAAGCTCTTTTCCGATATCAGAGCCAACGGGTATAATAT CATGTATCTGACAGCACGATCAGTGGGACAGGCAGATGCAACCAGGGCATATCTA GGCGGTGTTGACCAGTTTGGCTTCAAGCTGCCTCCAGGACCCGTCATCTTGTCGCC TGATAGAACCCTGGCGGCTCTCAAGAGAGAGGTGATTCTTAAGAAACCTGAGGTA TTCAAGATGGCGTGTCTGCGGGACATTAAGTCGCTGTTTGGCGAGACCGAAGACG CCACCAATCCATTCTACGCTGGATTTGGCAACCGAATCACCGACGCGTTGTCGTAT AGATCTGTCGGTGTGCCGTCGTCTAGAATCTTCACAATCAACTCGAACGCCGAGGT CCATATGGAGCTGCTTGAACTGGCTGGCTACAAGTCCTCGTATGTCCACATTGCCG ATCTTGTCGACCACTTTTTCCCTCCGGAAAGCGAGTTCACGACCATTCAGGAGGAA AAATACACGGACGTCAACTACTGGCGAGATCCCATTATTGACCTGTCTGATCTGAC CGACGACGAGCTGACTGACGATGATGAGCTCTCCAAGTCGCCCAAGTCGCCCAGA TCTCCTAGAAGCCCGCGGGCCGGTTCGGCAGGCTCCAGCGCGGCTCCCTCAGGCTC GGGCGCCGACCCTGCCGGACCCTCCGAGCCGAAGGACTCCGCGAACCCGTCGAAG TTCAGCTATAAGAAGGCTCCTACGAACTCTCGATTCCAGCCCGTTTCGTACGATCT TGATCTTGACGACGGATACGAGTACGACGATGACGATGACTATGATGACGATGAG GAGTTTGTGGACGCTGAGAGCGACGCGCTGGAGGAGGATGACGACGATGATGATG ACGTCGACCTAGACAACGACTCTGACCACTCCCCTGTCAAGCCGCCCTCGCAGATG CAGCGAGTCATCAACAAGACTATTGAGGACAACAAGGGCCTGCACATGGATGAGG ATGACGTTCAAAAAGCCATGAAGGCCCTGAAGATGGAACGAGCAAGCATCAATCC TGAGTAA [0145] SEQ ID NO:67 shows the PAH1 amino acid sequence encoded by SEQ ID NO:66: letterPSEGPPFRSPSADHLHEMGSFDDENDPEVNRRQRASTAAPEPVPGSLKHPATISEGI SSASFSNSDTDRTDTSGPTETEPTELTEPTEPTEPTEPLDLEQSLHRAATSPAPSSEEIWE KARALSKKLTSENIQSKISDNGDIILDMTGYKYDHEDVSRSEELVKKILAEELGEDRDL SHILVEDEEGNLVIQSAGDSHHHEHMSSPESLAHSPQPLPSSNLPSQASDNKHYAKTIRL TSDQLKSLDLKPGKNEVTFAVNNGKTSCSAQLFYWKYDIPVVISDIDGTITKSDALGHL LTMMGRDWTHTGVAKLFSDIRANGYNIMYLTARSVGQADATRAYLGGVDQFGFKLP PGPVILSPDRTLAALKREVILKKPEVFKMACLRDIKSLFGETEDATNPFYAGFGNRITDA LSYRSVGVPSSRIFTINSNAEVHMELLELAGYKSSYVHIADLVDHFFPPESEFTTIQEEK YTDVNYWRDPIIDLSDLTDDELTDDDELSKSPKSPRSPRSPRAGSAGSSAAPSGSGADP AGPSEPKDSANPSKFSYKKAPTNSRFQPVSYDLDLDDGYEYDDDDDYDDDEEFVDAE SDALEEDDDDDDDVDLDNDSDHSPVKPPSQMQRVINKTIEDNKGLHMDEDDVQKAM KALKMERASINPE [0146] SEQ ID NO:68 shows an exemplary MPL1 amino acid sequence: MPIKDFTNPAFSNPQETSSQHTHTKMPSVADNTSNGPIEGNVLPQSKFIQHLSEYPAVA AVTGFAASFPVVKIFASNAVPLIQAIQNRGAPVAEPVVKRAAPYISQIDNAADEALNRL DKAVPSLKNTKPDEVYSRIVTQPLENVRGTVDKYADETKNTVSRVVVQPIRDVASRV QSQVVTYYDAHGKPIVHARLDPIFHPLNDRLEALINAYLPKGQEIVTDAENELARAWR LTVVAFDRARPLIEQQTSQIQEINQHTREHIQKVYDGKRSEIDDKKTVSGPVYATVATV RDLSQEGLQYAQSILNAKKPEEKADSNSGVAPVSQPHTTSAVDNSLAAPTAVHEVTAS A [0147] SEQ ID NO:69 shows an exemplary FIT2 amino acid sequence: MSKPQIVKQETITSPDGLTSTTTTVDSDGVTTTTSTCGTVSTTTVVGPDGTHTASEGDA AFESSFPNLVKIPVGQPQEANGTTPINRRGANPSSSVRNNYKHPDLHSELTAKSFGNGM FSLLDLSLIGLVFSVLILGALVGKIVDHTYFADKRNFLNILFVKNGWLWTTIAFGYIVYE TFSGSIGLGVSFGTNTSTGTRTGTTHDGVSETNSDSKANAALKLLVGISAPTPIGQLSRY IIHSLWWLLFTQWFLGIPIMDRFFVATGGKCEYQKENASPITGKTISSASCRRSGGTWIG GYDPSGHCFLLVLSTMFLVYETIPHIKRRPYKLSAKIALGTAALWTWMYFMTSLYFHT FWEKLMGVIFGLLTVQLVYVVIPYLTRPKPSVGSNN DETAILED DESCRIPTION ONE-POT BIOSYNTHESIS OF COMPLEX COMPOSITIONS OF SPECIFIC FATTY ALCOHOLS, FATTY ALDEHYDES, AND/OR FATTY ACETATES [0148] Disclosed herein are apparatuses suitable for the one-pot synthesis of complex blends comprising particular amounts of multiple fatty acid products or derivatives thereof and corresponding one-pot synthetic methods. Embodiments herein utilize at least one reaction step in an apparatus, wherein the reaction step(s) may be a biosynthetic step catalyzed by one or more enzymes of a biocatalyst (e.g., a microorganism), a chemical synthetic step, or a combination of biosynthetic step(s) and chemical synthetic step(s). Particular embodiments herein include an apparatus or method wherein multiple synthetic pathways (e.g., pathways catalyzed by different biocatalysts) are utilized in tandem (e.g., simultaneously, and non-simultaneously) to produce different fatty acid products or derivatives thereof, which fatty acid products or derivatives may be blended to produce a product blend. In some embodiments, the products of the one-pot synthesis may be blended with further fatty acids or derivatives thereof, isolated (e.g., via distillation), or otherwise subjected to downstream processing to produce a biologically active synthetic insect pheromone composition. TERMS [0149] Organism: As used herein with regard to naturally occurring and genetically modified organism biocatalysts, the term “organism” includes cells, where the term “cell,” unless it is clear to a person in the art from its particular context, includes in this and other contexts cells in suspension, adhered cell cultures, and other cultures and multicellular forms that make the catalytic activity available to substrate in the reaction volume. [0150] One-pot: As used herein, the term “one-pot” refers to a chemical synthesis or reaction whereby a reactant is subjected to successive chemical reactions a single reaction volume; e.g., the working volume of a reactor. In some embodiments, a one-pot synthesis provides advantages over conventional reactions that require separation or purification of intermediates or products during the reaction process. Unless indicated otherwise, the one-pot reactions described herein include “telescoping” reactions, whereby the synthesis is sequential, and reagents or other reaction components (e.g., genetically engineered organisms) are added during the synthesis, but without isolation and purification of the product(s) of a chemical reaction. [0151] Organic substrate: As used herein, the term “organic substrate” specifically includes sugars, glycerol, ethanol, organic acids, alkanes. Unless it is clear from its particular context, the term also includes fatty acids. Some embodiments herein utilize an organic substrate as a carbon source for the production of a saturated or unsaturated lipid moiety. In particular embodiments, the organic substrate is glucose. [0152] Exogenous: The term “exogenous,” as applied to polynucleotides and polypeptides herein, refers to one or more species that are not normally present within their specific environment or context. For example, if a host cell is transformed with a nucleic acid molecule that does not occur in the untransformed host cell in nature, then that nucleic acid molecule and polynucleotides therein are exogenous to the host cell. The term exogenous, as used herein, also refers to a polynucleotides or polypeptides that are identical in sequence to a species already present in a host cell, but that is located in a different cellular or genomic context than the species with the same sequence already present in the host cell. For example, a polynucleotide that is integrated in the genome of a host cell in a different location than a polynucleotide with the same sequence is normally integrated in the genome of the host cell is exogenous to the host cell. Furthermore, a polypeptide that is present in a subcellular compartment in a host cell is exogenous to the host cell when a polypeptide with the same sequence is only normally present in a different compartment of the host cell. [0153] Heterologous: The term “heterologous,” as applied to nucleic acids and polypeptides herein, means of different origin. For example, if a host cell is transformed with a nucleic acid molecule that does not occur in the untransformed host cell in nature, then that nucleic acid molecule and polynucleotides therein are heterologous (and exogenous) to the host cell. Furthermore, different elements (e.g., promoters, enhancers, coding sequences, and terminators) of a transforming nucleic acid molecule may be heterologous to one another and/or to the transformed host. Biosynthesis steps and Biocatalysts [0154] Apparatuses and methods of embodiments herein may therefore include biocatalysts and biosynthetic methods for producing an insect pheromone or precursor thereof. Such methods may comprise, for example, culturing a biocatalyst (e.g., a microorganism) as herein described, and feeding the culture with a saturated or unsaturated substrate. In particular embodiments, the method further comprises isolating an insect pheromone or precursor thereof produced from the substrate. For example, the method may comprise isolating the pheromone or precursor via distillation. Alternatively, isolating the pheromone or precursor may comprise membrane-based separation. In particular embodiments, a pheromone precursor is isolated, and is then converted into an active pheromone via chemical methods. [0155] In particular embodiments herein, a method for producing an insect pheromone or precursor thereof does not utilize significant amounts of organic solvents, proceeds in one step, and results in high yield of a particular product isomer, providing a significant improvement upon conventional production methods. Also disclosed herein are microorganisms, comprising at least one component of a biosynthesis pathway to produce one or more product unsaturated lipid moiety isomers. For example, a biocatalyst may be used to catalyze, for example, any combination of the following reactions, in one or more steps: synthesis of a product unsaturated lipid moiety isomer and synthesis of an intermediate selected from among a saturated lipid moiety, an unsaturated lipid moiety isomer, a fatty alcohol, a fatty aldehyde, a FFA, a FAE (e.g., a FAME), a fatty alcohol, a fatty aldehyde, or and a fatty acetate. The product unsaturated lipid moiety isomer is a stereospecific and regiospecific isomer with a specific chain length. Accordingly, an intermediate synthesized with the use of a biocatalyst herein typically is produced in a stereospecific and/or regiospecific reaction utilizing one or more enzymes of the biocatalyst. In particular product or intermediate synthesis reactions, the chain length of a saturated lipid moiety or stereospecific and regiospecific unsaturated lipid moiety isomer is modified by one or more enzymes of the biocatalyst. [0156] In some embodiments, the biocatalyst is a microorganism comprising at least one component of a biosynthesis pathway to produce one or more product unsaturated lipid moiety isomers. In particular embodiments, the microorganism is a bacterium, yeast, or a cultured cell (e.g., a plant cell or insect cell in cell culture). In some examples, the microorganism is a naturally occurring organism. In other examples, the microorganism is a genetically engineered microorganism comprising at least one exogenous polynucleotide encoding a biosynthetic enzyme that catalyzes at least one step in the biosynthesis. For example, the biocatalyst may be a genetically engineered yeast, bacterium, insect cell, or plant cell. In specific examples, the biocatalyst is a genetically engineered yeast selected from the group consisting of Saccharomyces, Scizosacchoromyces pombe, Pichia pastoris, Hansanula polymorpha, Yarrowia lipolytica, Candida albicans, Candida tropicalis, and Candida viswanathii. In further specific examples, the biocatalyst is a genetically engineered insect cell of the genus Amyelois (e.g., A. transitella). The biocatalyst is preferably suitable for large-scale culture in a bioreactor, for example, as shown in FIG.1. [0157] In some embodiments, a biocatalyst comprises at least one exogenous (e.g., heterologous) biosynthetic enzyme selected from the group consisting of desaturases (e.g., fatty acyl-CoA desaturases, and fatty acyl-ACP desaturases), acyl-CoA oxidases, acylglycerol lipases, flavoprotein pyridine nucleotide cytochrome reductases (e.g., cytochrome-b5 reductases, and NADPH-dependent cytochrome P450 reductases), elongases (e.g., ELO1, ELO2, and ELO3), thioesterases (e.g., acyl-ACP thioesterases, and acyl-CoA thioesterases), glycerol-3-phosphate acyltransferases (e.g., dual glycerol-3-phosphate O- acyltransferase/dihydroxyacetone phosphate acyltransferases), lysophosphatidic acid acyltransferases (e.g., SLC1, ALE1, and LOA1), diacylglycerol acyltransferases (e.g., acyl- CoA dependent acyltransferase and a phospholipid: diacylglycerol acyltransferase), and lipid binding proteins (e.g., sterol carrier protein 2 (SCP2)). In particular embodiments, the biosynthetic enzyme(s) is native to an organism from a genus selected from the group consisting of Saccharomyces (e.g., S. cerevisiae), Yarrowia (e.g., Y. lipolytica), Candida (e.g., C. albicans, C. tropicalis, and C. viswanathii), Helicoverpa (e.g., H. armigera, and H. zea), Thalassiosira, (e.g., T. pseudonana), Agrotis (e.g., A. segetum), Trichoplusia (e.g., T. ni), Spodoptera (e.g., S. littoralis, and S. exigua), Ostrinia (e.g., O. scapulalis, O. fumacalis, O. nubilalis, O. latipennis, and O. ovalipennis), Amyelois (e.g., A. transitella), Lobesia (e.g., L. botrana), Cydia (e.g., C. pomonella), Grapholita (e.g., G. molesta), Lampronia (e.g., L. capitella), Sesamia (e.g., S. inferens), Plodia (e.g., P. interpunctella), Bombyx (e.g., B. mori), Phoenix (e.g., P. dactylifera), Vernicia (e.g., V. fordii), Macadamia (e.g., M. tetraphylla), Rattus (e.g., R. norvegicus), Arabidopsis (e.g., A. thaliana), Plutella (e.g., P. xylostella), and Bicyclus (e.g., B. anynana). [0158] In particular embodiments, a biocatalyst comprises a heterologous desaturase capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. For example, the desaturase may be capable of utilizing a C16 or C18 fatty acyl-CoA as a substrate. In these and further embodiments, a biocatalyst may comprise a desaturase that generates a double bond at position C5, C6, C7, C8, C9, C10, C11, C12, or C13 in the fatty acid or its derivatives, such as, for example, fatty acid CoA esters. For example, the desaturase may generate a double bond at position C9, C11, or C13. In particular examples, the desaturase generates the Z isomer of an unsaturated fatty acid or fatty acid derivative. In particular examples, the desaturase is at least 90% identical to SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, or SEQ ID NO:47 (e.g., SEQ ID NOs:49, 51, 53, 55, and 57). For example, the desaturase may be at least 95% or at least 98% identical to SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, or SEQ ID NO:47. [0159] In certain embodiments, a biocatalyst comprises a heterologous Z11 desaturase that catalyzes the conversion of a fatty acyl-CoA into a monounsaturated or polyunsaturated product selected from Z11-13:Acyl-CoA, (Z,Z)-7,11-13:Acyl-CoA, Z11-14:Acyl-CoA, (E,Z)- 9,11-14:Acyl-CoA, (Z,Z)-9,11-14:Acyl-CoA, (E,Z)-9,11-15:Acyl-CoA, (Z,Z)-9,11-15:Acyl- CoA, Z11-16:Acyl-CoA, (E,Z)-6,11-16:Acyl-CoA, (E,Z)-7,11-16:Acyl-CoA, (E,Z)-8,11- 16:Acyl-CoA, (E,Z)-9,11-16:Acyl-CoA, (Z,Z)-9,11-16:Acyl-CoA, (Z,E)-11,13-16:Acyl-CoA, (Z,Z)-11,13-16:Acyl-CoA, (Z,E)-11,14-16:Acyl-CoA, (E,E,Z)-4,6,11-16:Acyl-CoA, (Z,Z,E)- 7,11,13-16:Acyl-CoA, (E,E,Z,Z)-4,6,11,13-16:Acyl-CoA, Z11-17:Acyl-CoA, (Z,Z)-8,11- 17:Acyl-CoA, Z11-18:Acyl-CoA, and (Z,Z)-11,13-18:Acyl-CoA. [0160] In certain embodiments, a biocatalyst comprises a heterologous Z9 desaturase that catalyzes the conversion of a fatty acyl-CoA into a monounsaturated or polyunsaturated product selected from Z9-11:Acyl-CoA, Z9-12:Acyl-CoA, (E,Z)-7,9-12:Acyl-CoA, (Z,Z)-7,9- 12:Acyl-CoA, Z9-13:Acyl-CoA, (E,Z)-5,9-13:Acyl-CoA, (Z,Z)-5,9-13:Acyl-CoA, Z9- 14:Acyl-CoA, (E,Z)-4,9-14:Acyl-CoA, (Z,E)-9,11-14:Acyl-CoA, (Z,Z)-9,11-14:Acyl-CoA, (Z,E)-9,12-14:Acyl-CoA, (Z,Z)-9,12-14:Acyl-CoA, Z9-15:Acyl-CoA, (Z,Z)-6,9-15:Acyl- CoA, Z9-16:Acyl-CoA, (Z,E)-9,11-16:Acyl-CoA, (Z,Z)-9,11-16:Acyl-CoA, Z9-17:Acyl- CoA, Z9-18:Acyl-CoA, (Z,Z)-9,12-18:Acyl-CoA, (Z,Z,Z)-3,6,9-18:Acyl-CoA, and (Z,Z,Z)- 9,12,15-18:Acyl-CoA. [0161] In particular embodiments, a biocatalyst comprises an exogenous acyl-CoA oxidase that catalyzes the conversion of a monounsaturated or polyunsaturated C6-C24 fatty acyl-CoA into a truncated monounsaturated or polyunsaturated fatty acyl-CoA after one or more successive cycle of acyl-CoA oxidase activity, with a given cycle producing a monounsaturated or polyunsaturated C4-C22 fatty acyl-CoA intermediate with a two carbon truncation relative to a starting monounsaturated or polyunsaturated C6-C24 fatty acyl-CoA substrate in that cycle. In particular examples, the acyl-CoA oxidase is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. For example, the acyl-CoA oxidase may be at least 95% or at least 98% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. [0162] In particular embodiments, a biocatalyst comprises at least one heterologous glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferases (LPAAT), glycerolphospholipid acyltransferase (GPLAT), or diacylglycerol acyltransferases (DGAT). In particular examples, the biocatalyst comprises a heterologous polypeptide that is at least 90% identical to a DGAT1 (e.g., SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:34) or an LPAAT (e.g., SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:32). For example, the heterologous polypeptide may be at least 95% or at least 98% identical to SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:32. [0163] In particular embodiments, a biocatalyst comprises at least one exogenous enzyme that extends the length of a fatty acid or fatty acid derivative. Two different types of fatty acid elongation takes place in different organisms. These elongation pathways use Coenzyme-A as acyl carrier rather than the acyl carrier protein (ACP) of fatty acid synthesis systems FASI and FASII. The first type, mitochondrial fatty acid elongation, is the reversal of fatty acid oxidation. This utilizes acetyl-CoA as a substrate and extends the chain length of fatty acids with two carbons. This process acts mainly on acyl-CoA shorter than C16. The second process is the elongation pathway of the endoplasmic reticulum, which is present in plants, mammals, yeast, and other lower eukaryotes. This is a four-step reaction, where the steps are catalyzed by individual enzymes; beta-ketoacyl-CoA synthase, beta-ketoacyl-CoA reductase, beta-hydroxyacyl-CoA dehydratase, and trans-2-enoyl-CoA reductase. This pathway mainly acts with acyl-CoA of chain length C16 or larger, and it is important in the generation of very long chain fatty acids. This process utilizes malonyl-CoA rather than acetyl-CoA for chain elongation. The first enzyme which leads to condensation of malonyl-CoA with acyl-CoA (beta-ketoacyl-CoA synthase) is also called elongase. [0164] Several variants of elongase exist, depending upon the host organism. In yeast, three different genes each encode an elongase (ELO1, ELO2 (FEN1), and ELO3 (FEN12)), and they have different substrate specificities. ELO1 prefers shorter saturated fatty acids (C14-C16), ELO2 prefers longer saturated and monounsaturated fatty acids, and ELO3 prefers monounsaturated and polyunsaturated fatty acids. In particular examples, the biocatalyst comprises an exogenous elongase that is at least 90% identical to an ELO1, ELO2 (e.g., SEQ ID NO:30), or ELO3. For example, the exogenous elongase may be at least 95% or at least 98% identical to SEQ ID NO:30. [0165] In particular embodiments, a biocatalyst comprises at least one exogenous lipid regulator. In particular examples, the biocatalyst comprises a lipid regulator that is at least 90% identical to a truncated transcriptional activator fragment (e.g., MGA2act (e.g., SEQ ID NO:59 and SEQ ID NO:61)), a cytochrome B5 reductase (e.g., SEQ ID NO:63), a heme-responsive zinc finger transcription factor (e.g., SEQ ID NO:65), or a diacylglycerol phosphate phosphatase (e.g., SEQ ID NO:67). For example, the exogenous lipid regulator may be at least 95% or at least 98% identical to SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, or SEQ ID NO:67. [0166] Fatty acid products and derivatives thereof produced by the foregoing biocatalysts and biosynthetic methods are modified by non-biological means such as chemical synthesis in the one-pot synthesis in some embodiments herein to yield final products. [0167] Further details regarding microorganisms suitable for use as biocatalysts in embodiments herein, and regarding methods for utilizing such microorganisms to produce chemicals in a biosynthetic reaction, may be found in PCT International Patent Publication No. WO 2018/213554 A1, the contents of which are incorporated herein by this reference in their entirety. Chemical reaction steps and catalysts [0168] Apparatuses and methods of embodiments herein may also include chemical synthetic methods. Chemical synthesis is the preparation of a compound by performing various chemical reactions to change the molecular structure of a starting material by reactions with other chemicals. The starting materials for a chemical synthesis may be simple compounds removed from oil and natural gas, or more complex chemicals isolated in from biological sources or biosynthetic fermentation reactions. In some embodiments, the one-pot synthesis includes the chemical synthesis of at least one free fatty acid (FFA), fatty acid alkyl ester (FAAE), fatty alcohol, or fatty aldehyde from an unsaturated lipid (e.g., monounsaturated fatty acids, polyunsaturated fatty acids, branched unsaturated fatty acids, unsaturated triacylglycerol, and unsaturated triglyceride). In particular embodiments, the unsaturated lipid is produced by a biocatalyst in the one-pot synthesis. [0169] In particular embodiments, an unsaturated lipid is chemically converted to at least one FFA and/or at least one FAAEs. In these and further embodiments, an unsaturated lipid, FFA and/or FAAE is reduced to a fatty alcohol and/or a fatty aldehyde by contacting the unsaturated lipid, FFA, and/or FAAE with at least one stoichiometric reducing agent (e.g., sodium bis(2-methoxyethoxy)aluminum hydride (Vitride™, Red-Al, SMEAH), and diisobutylaluminum hydride (DIBAL)), and/or at least one transition metal catalyst (e.g., a group VIII catalyst). In some examples, a bulky cyclic nitrogen Lewis base is used to modify the reducing agent to allow for selectivity of the reducing step to produce one or more fatty aldehydes. [0170] In particular embodiments, a fatty alcohol is oxidized to a fatty aldehyde by an oxidation step, which oxidation step may be, for example, a partial oxidation method such as NaOCl/TEMPO, TEMPO-bleach, TEMPO-PhI(OAc)2, TEMPO-copper-air, copper-catalyzed aerobic oxidation, Swern oxidation, and noble metal-air oxidation. In some examples, a fatty alcohol may be converted into a fatty acetate using chemical methods, e.g., via chemical catalysis utilizing a chemical agent; for example, acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride, and propionic anhydride. [0171] The following EXAMPLES are provided to illustrate certain particular features and/or embodiments. The EXAMPLES should not be construed to limit the disclosure to the particular features or embodiments exemplified. EXAMPLES Example 1: Selective Biocatalysis of Z9-16 and Z11-16 from Substrate [0172] Z11 desaturase from Helicoverpa zea was identified as particularly active on a 16Acid substrate from among a library of Z11 specific insect desaturases (data not shown). The Gen1 strain SPV458 (H. zea desaturase integrated at XPR2 locus) was selected for further engineering. FIG.2. 22 plasmid constructs were integrated into SPV739, the marker rescued descendant of SPV458 to create Gen2 strains. FIG.2A. Strains harboring multiple copies of the H. zea desaturase produced increased Z11-16Acid titers. Four of those strains were evaluated for improved performance in both a small-scale assay and in large-scale fermentors. [0173] Two Gen2 strains, SPV968 and SPV969, were marker rescued, and additional DNA cassettes were integrated to create a small library of Gen3 strains with 2 or 3 copies of Z11 desaturase, and deletions of key lipid metabolism genes. FIG.2A. Increasing desaturase copy number was found to increase both Z11-16Acid productivity and selectivity. Data from Gen1 to Gen3 strains indicated that total fatty acid content made up 50-60% of dry cell weight. Further increases in productivity may be made by expressing recombinant acyltransferases. Integrating acyltransferases was found to further increase Z11-16Acid titer and selectivity. [0174] Both Z9-16Ald and Z11-16Ald are recognized as active ingredients in the pheromone for Helicoverpa zea and H. armigera. To produce a Z11-16Acid/Z9-16Acid synthetic pheromone for Helicoverpa, it is desired to produce Z11-16Acid at ≥97% of the unsaturated C16 fatty acid composition from the microbial bioprocess, for example, so that a blend of 97% Z11-16Acid, 3% Z9-16Acid can be synthesized, or a small amount of metathesis- derived Z9-16Ald can be doped into the final mixture. Towards this end, the Gen4 strain expressing P. dactylifera DGAT1A (SPV1629) was selected for further engineering (96% Z11- 16Acid/Z9-16Acid selectivity at 1 L scale). FIG.2A. [0175] Two distinct strategies were employed to achieve this goal. First, the native Z9 desaturase encoded by the OLE1 gene was replaced with recombinant desaturases that could either reduce Z9-16 desaturase activity while maintaining Z9-18 desaturase activity or replace Z9 desaturase activity completely with Z11-18 desaturase activity (Example 1.1; FIG. 2B). Second, incremental gains in selectivity were made by increasing the H. zea desaturase copy number to four and combined expression of both P. dactylifera DGAT1A and LPAAT in the same strain (Example 1.2; FIG.2A (Gen5)). Example 1.1: Replacing OLE1 with Alternate Z9-18CoA and Z11-18CoA Desaturases [0176] The native Z9 desaturase (OLE1) was replaced with alternative Z9-18CoA and Z11-18CoA desaturases, improving Z11-16Acid/Z9-16Acid selectivity. A Y. lipoltyica strain devoid of Z9 desaturase activity was engineered that can be used for a Z11-18Acid/Z13- 18Acid blend strain to produce the Z11-18Ald/Z13-18Ald pheromone blend of the rice pest Cnaphalocrocis medinalis. [0177] Three lepidopteran desaturases and one fungal desaturase were integrated into the marker rescued descendant of SPV1994 (xpr2::HzDST, fat1::HzDST, fao1::HzDST, axp::DGAT1A_Pd, and pox5::HzDST) at the OLE1 locus. The resulting strains had a deletion of OLE1, and the expression of the recombinant desaturase was driven by either the native OLE1 promoter or the TEF promoter. Table 1. [0178] Table 1. OLE1 knockout desaturase constructs. Desaturase Source species Activity Promoter
Figure imgf000063_0002
[0179] The resulting strains were screened in both a 24-well plate bioconversion assay using methyl palmitate as substrate, and a 48-well Biolector® assay to assess growth rate. The fatty acid composition of all cultures was measured using GC-FAME analysis. Strains were evaluated by examining the overall lipid profile and by assessing the following seven metrics: (1) Z11-16Acid titer; (2) Z11-16Acid/Z9-16Acid selectivity:
Figure imgf000063_0001
(4) Z11 total selectivity:
Figure imgf000064_0001
(5) Z11-16Acid/products selectivity:
Figure imgf000064_0002
and (7) Growth rate. [0180] When transformed in the SPV1994 background, four constructs produced viable strains with deletion of the native OLE1 gene. Strains using the TEF promoter to drive DST301 (R. irregularis) expression produced equivalent titers of Z11-16Acid (4.1 ± 0.3 g/L) and improved Z11-16Acid/Z9-16Acid selectivity (98%) when compared with the SPV1994 control. Growth rates were equivalent in semi-defined FERM1 medium and 65% of SPV1629 in Solulys®-based medium. Strains using the OLE1 promoter to drive DST301 expression produced 2.76 ± 0.26 g/L Z11-16Acid with a Z11-16Acid/Z9-16Acid selectivity of >99%. Titers of Z9-16Acid (<10 mg/L) and Z9-18Acid (0.11 ± 0.01 g/L) were greatly reduced relative to the control. Growth rates were significantly reduced. [0181] DST148 (B. mori, Z11-18 desaturase) expressing strains were also isolated. The lipid profiles were devoid of detectable Z9 unsaturated species, resulting in a Z11-16Acid/Z9- 16Acid selectivity of 100%. Z11-16Acid titers were 1.36 ± 0.12 g/L with Z11-18Acid titers of 1.62 ± 0.14 g/L. A C18 diene was produced at 0.30 ± 0.02 g/L. Growth was significantly reduced (29% of SPV1629 growth rate in FERM1, and undetected in Solulys®-based medium). [0182] Table 2. Performance of OLE1 knockout strains.
Figure imgf000064_0003
Figure imgf000065_0001
[0183] Four previously screened desaturases were selected for replacement of the native Y. lipolytica Z9 desaturase (OLE1). Three desaturases were Z9 selective and had displayed increased selectivity for an 18CoA over a 16CoA substrate. Two Z9 desaturases (DST199 and DST200) were lepidopteran variants from the butterfly P. xuthus. The third Z9 desaturase was from the fungus R. irregularis. A fourth desaturase variant (DST148, B. mori) as a Z11 desaturase that prefers an 18CoA substrate was discovered. A high expression level may be preferable for the desaturase making the non-native Z11-18Acid. [0184] Each desaturase was subcloned from an existing expression vector into a plasmid that targets the OLE1 locus. This OLE1 construct uses the native OLE1 promoter and terminator sequences as homology arms, so that upon integration the entire coding sequence of the native Z9 desaturase is removed. The recombinant Z9 desaturases were cloned with two different expression approaches. In the first approach, the coding sequence of the recombinant Z9 desaturase was cloned directly downstream of the native OLE1 promoter sequence, which also served as the homology arm; the resulting constructs used the native OLE1 promoter to drive expression of the desaturase. The second strategy added a TEF promoter sequence downstream of the OLE1 promoter/homology arm sequence; this second set of constructs used the TEF promoter to drive expression. [0185] Positive integrants were screened in a 24-well plate assay in a semi-defined medium used previously for 24-well plate and Biolector® assays. [0186] Fatty Acid Profiling of OLE1 Replacement Strains [0187] Relative to the control, SPV2073 (pTEF-DST301, R. irregularis) produced less Z9-16Acid (60%), statistically equivalent Z11-16Acid, more 18Acid (130%), and similar unsaturated C18 fatty acid titers. FIG. 3; FIG. 4. The result was a strain with a similar C18 fatty acid profile to the control, but with increased Z11-16Acid/Z9-16Acid selectivity (98% vs. 96%). SPV2072 (pOLE1-DST200, P. xuthus) displayed similar trends relative to the control, but had a lower average Z11-16Acid titer. FIG.3. These lower titers correlated with a lower cell density. FIG. 5. SPV2074 (pOLE1-DST301), displayed a dramatically different phenotype: Z9 unsaturated fatty acid titers were greatly reduced, while Z11-16Acid and Z13- 18Acid were reduced by ~40%. FIG. 3. The Z9-16Acid titers, 10 mg/L, were near the detection limit of the assay. Interestingly, 18Acid and Z11-18Acid were increased by ~5-fold and ~3.5-fold, respectively. FIG. 3; FIG. 4. While Z11-18Acid can be synthesized through elongation of Z9-16Acid, the complete fatty acid profile suggests an alternative mechanism; it is likely that 18CoA concentrations increased due to lower Z9 desaturase activity, and at these high concentrations of 18CoA, the H. zea Z11 desaturase had activity on 18CoA, leading to an increase in Z11-18Acid. The Z11-16Acid/Z9-16Acid selectivity for SPV2074 was >99%. [0188] Like SPV2074, SPV2075 and SPV2076 (pTEF-DST148, B. mori Z11 desaturase) displayed a unique fatty acid profile. All three native Z9 fatty acids were below the detection limit in these strains. FIG. 3A. The dominant fatty acid products were Z11- 16Acid (1.2-1.6 g/L) and Z11-18Acid (1.4-1.9 g/L). FIG.3B. Relative titers were consistent between the two isolates; absolute titers correlated with final cell density measurements. Final cell densities were 25% (SPV2075) and 40% (SPV2076) lower than the SPV1994 control. FIG.5. [0189] Growth of OLE1 Replacement Strains [0190] Growth rates for OLE1 replacement strains were independently measured through online cell density monitoring using a Biolector®. Growth rates were compared to both the current production strain SPV1629 and the direct parent SPV1994 in two different media; the small-scale bioconversion medium FERM1, and a large-scale bioprocess medium using an initial batch phase with glycerol as the primary carbon source along with corn-steep liquor (Solulys® 95) to supply both nitrogen and vitamins. Growth rates for all strains and conditions are shown in FIG. 6. In general, a greater diversity of growth rates was observed in the bioprocess medium. Strains devoid of Z9 desaturase activity (SPV2075 and SPV2076) had a greatly reduced growth rate in FERM1, and no growth was detected in the bioprocess medium. This lower growth rate may partially result from low initial seeding density, because the initial YPD cultures for these two strains also grew poorly. Improved growth rates are achieved by supplementing media with Z9-18Acid or by fine-tuning Z9-18Acid activity. [0191] Replacing the Y. lipolytica OLE1 with lepidopteran and fungal desaturases was successful. OLE1 was replaced with both Z9 and Z11-18 desaturases. Three of the resulting strains displayed increased Z11-16Acid/Z9-16Acid selectivity. When OLE1 was replaced by a B. mori Z11-18 desaturase (DST148), only Z11 or Z11-derived fatty acids were observed. Growth rates for strains producing low titers of the key membrane component Z9-18Acid were significantly reduced compared to strain SPV1629. Growth of the DST148 strain with methyl oleate supplementation shows that growth is recovered with Z9-18Acid supplementation. [0192] Materials & Methods [0193] Integration Vector Construction: Desaturase sequences were codon optimized (Homo sapiens expression organism) and cloned into pPV266 (XPR2 locus integration vector with TEF promoter and terminator) using SpeI/NotI restriction digestion. The XPR2 construct was used as template for desaturase coding sequence subcloning into OLE1 locus integration vector pPV235. Desaturases were cloned to either remove the TEF promoter from the construct, resulting in integration directly downstream of the native OLE1 promoter, or cloned with the intact TEF promoter for integration at the same OLE1 insertion site. The constructs simultaneously remove the native OLE1 gene. The resulting constructs as listed in Table 3. [0194] Table 3. Plasmid Descriptions.
Figure imgf000067_0001
Figure imgf000068_0001
[0195] Strain Construction: Integration constructs were linearized using PmeI restriction enzyme and transformed into the host strain. Transformants were verified for correct integration by colony PCR of the 5’ and 3’ integration junctions using primers outside of the integration junctions and within the pTEF promoter (5’ junction) or the tURA3 terminator (3’ junction). After each step of construction, all engineered loci were re-verified for correct integration and insert size using genomic DNA as template. [0196] Plasmid digestion: 1-2 μg plasmid was digested with PmeI in a 15 μL digestion reaction (12 μL DNA + 1.5 μL 10x CutSmart Buffer + 1.5 μL PmeI restriction enzyme). The reaction was incubated in the PCR machine for 1.5 hours at 37 ºC, and heat inactivated at 65 ºC for 30 minutes. [0197] Transformation: Competent cells were grown by inoculating a 2 mL YPD culture for 16 hrs at 28 ºC. A 50 mL YPD culture was inoculated at OD600 = 0.03 in a 500 mL baffled flask and grown for 6 hours before harvesting. Cells were harvested at 800 xg, and washed with 0.25x volume Solution 1 from the Zymo® Frozen-EZ™ Transformation II Kit for Yeast. Cells were resuspended 200 μL Solution 2 concentrated to OD600 = 10, and slowly frozen at -80 ºC while insulated in a styrofoam container. 50 μL cells were first mixed with the 12 μL digestion reaction (no cleanup), and then with 500 μL Solution 3. Transformations were incubated for 3 hours at 28 ºC without shaking, after which the full transformation mixture was plated to appropriate selective agar media. Petri dishes were incubated for 2-3 days before the appearance of colonies. [0198] Check PCR: Individual transformants were picked to 9 μL water in a PCR plate. Then, 3 μL cells were drop-plated by multichannel to selective omni trays and grown overnight. The remaining 6 μL cells were divided into two PCR plates for 5’ and 3’ junction PCRs. PCR plates were microwaved for 2 minutes before directly adding 15 μL PCR master mix. All DST192 constructs were verified using a desaturase-specific primer. [0199] Colony patching: Positive clones were re-streaked onto YPD petri dishes from selective media drop plates and grown overnight at 28 ºC. Colonies grown on YPD were used to inoculate bioassay cultures. [0200] 24-well plate bioconversion assay: Both growth and bioconversion phases were cultured in 17 mm diameter crimp-top glass vials inserted into 24-well plates. Y. lipolytica strains were inoculated from YPD agar patches and grown in 1 mL YPD seed cultures for 24 hours at 28 °C and 1000 rpm (Infors™ plate incubator) to an OD600 ≈ 15-20. Seed cultures were pelleted by centrifugation at 800 xg for 5 min. Supernatant was removed by pipetting before 1 mL bioconversion medium (FERM1) was added. Cells were resuspended before returning to the plate incubator (28 °C, 1000 rpm) for six hours. After the six-hour incubation, 25 μL warmed methyl palmitate was added to each vial. Cultures were then incubated under the same conditions for an additional 66 hours before sampling. Wells were mixed by pipetting the culture volume before 100 μL was transferred to a crimp-top GC vial. Sample vials were frozen and stored at -80 °C. Cell density was measured with a Tecan® M200™ Pro plate reader at sampling. [0201] Biolector® growth rate Assay: Y. lipolytica strains were inoculated from YPD agar patches and grown in 2 mL YPD in 24-well plate seed cultures for 24 hours at 28 °C and 1000 rpm (Infors™ plate incubator). Most cultures grew to OD600 ≈ 15-20, but some of the slower growing strains grew to OD600 = 10. SPV2075 and SPV2076 only grew to an OD600 = 0.25 after 24 hours. 20 μL culture was inoculated into 780 μL growth media in a 48-well flower plate. Cultures were incubated at 32 °C and 1500 rpm with online monitoring of cell density. [0202] GC sample processing of lyophilized samples: 250 μL cultures were freeze- dried in glass crimp top vials overnight using Labconco® Triad tray lyophilizer. 500 μL methanol was added to the vials, followed by the addition of 35 μL 10M KOH. Vials were sealed with a crimp cap, arrayed in a V54 rack, and mixed for 10 minutes at 2000 rpm using a MixMate® plate shaker. Following mixing, vials were transferred to 108-well aluminum racks, and placed in a convection oven for 40 minutes at 60 °C. After heating, the vials were decapped, and 29 μL 24N sulfuric acid was added. Vials were then re-sealed, arrayed in a V54 rack, and mixed again for 2 minutes at 2000 rpm using a MixMate® plate shaker. Following this mixing, the vials were transferred to 108-well aluminum racks, and again placed in a convection oven for 40 minutes at 60 °C. After this heating, the vials were decapped, and 1 mL n-hexane was added. Then, the vials were re-sealed, arrayed on V54 rack, and mixed for 10 minutes at 2000 rpm using a MixMate® plate shaker. [0203] Samples were run on GC in a one-pot format using the parameters listed in Table 4 (for both front and back detectors). The GC needle height was adjusted from 3.0 mm to 14.5 mm (sample is drawn from the very top of vials when run in “one-pot” format. [0204] Table 4. GC Parameters.
Figure imgf000070_0001
Example 1.2: Expression of DGAT1A and LPAAT with Increased HzDST Copy Number [0205] To increase the Z11-16Acid/Z9-16Acid selectivity of engineered strains from 96%/4% to at or above 97%/3%, while maintaining high titer, and productivity, two additional strategies were employed. [0206] Increases in Z11-16Acid fraction with additional copies of the Z11 desaturase were observed; the addition of a second copy produced an increase in Z11/Z9-16Acid selectivity from 83% to 96%, and a third copy increased the Z11/Z9-16Acid selectivity to 97%. P. dactylifera DGAT1A expression in a 3-copy strain further increased Z11-16Acid titer but led to a Z11/Z9-16Acid selectivity of 96-97%. Therefore, the first strategy was to express four copies of the H. zea Z11 desaturase in this background. The addition of a fourth copy could increase the Z11/Z9-16Acid selectivity above 97%. [0207] The second strategy was to combine expression of the P. dactylifera DGAT1A and LPAAT. Expression of the LPAAT alone had led to a small increase in the Z11/Z9-16Acid selectivity, while also reducing the C18 acid content of the strain. An increase in Z11-16Acid titer was also observed, but the increase was smaller than that with DGAT1A expression alone. Combining expression of these two acyltransferases can lead to improved selectivity. [0208] Strains were screened in a Biolector® 48-well plate bioconversion assay using methyl palmitate as substrate. The fatty acid composition of all cultures was measured using GC-FAME analysis. Selectivity of strains were evaluated with the following four metrics:
Figure imgf000071_0001
[0209] Characterization of 4-copy H. zea Desaturase Strains [0210] A fourth copy of the codon-optimized H. zea Z11 desaturase was integrated into the previously deleted POX5 locus in the marker-rescued descendant of SPV1629 (See Materials & Methods). Two unique isolates were screened against an SPV1629 control in the small-scale Biolector® assay (See Materials & Methods). Both clones showed similar performance, with marginally better selectivity observed for clone SPV1994. FIG.7. [0211] The fatty acid profiles of SPV1629 and SPV1994 were similar except for a small decrease in 16Acid and Z9-16Acid titers. FIG. 8E. The decrease in Z9-16Acid titer from 140 mg/L to 90 mg/L led to an increase in Z11-16/Z9-16Acid selectivity from 96.8% in SPV1629 to 97.8% in SPV1994. FIG. 8A. Based on the observed reduction in 16Acid titer for SPV1994, C16 selectivity (SC16) was reduced relative to SPV1629, and Z11-16Acid total selectivity (SZ11-16_tot) was increased (FIG. 8B and FIG. 8D). The Z11-16Acid/products selectivity (SZ11-16_prod), which excludes the 16Acid substrate, was equivalent for both strains. [0212] Table 5. Two independent replicates showing 4-copy H. zea desaturase strain improved Z11-16Acid/Z9-16Acid selectivity (SZ11/Z9-16) by 1% from 96.6% to 97.7% across two independent Biolector® experiments, relative to SPV1629 (3xHzDST + DGAT1A_Pd). The selectivity improvement resulted from a reduction in Z9-16Acid titer.
Figure imgf000072_0001
[0213] Characterization of P. dactylifera LPAAT Strains [0214] A codon-optimized copy of the P. dactylifera LPAAT was integrated into the previously deleted POX4 locus in the marker rescued descendant of SPV1629 (See Materials & Methods). Four unique isolates were screened against an SPV1629 control in the small- scale Biolector® assay (See Materials & Methods). All four clones showed similar performance, with a relative reduction in Z9-16Acid titer and C18 fatty acid titers. FIG. 9. Clone SPV1995 was selected for further screening based on Z11-16Acid titer and selectivity criteria. [0215] SPV1995 produced increased Z11-16Acid titer while producing less Z9- 16Acid and less C18 fatty acids. The observed increase in Z11-16Acid titer and decrease in Z9-16Acid titer resulted in a Z11-16Acid/Z9-16Acid selectivity increase from 97.4% in SPV1629 to 98.4% in SPV1995. FIG. 10A. The reduction in all C18 fatty acid titers led to increases in C16 selectivity (SC16), Z11-16Acid/products selectivity (SZ11-16_prod), and Z11- 16Acid total selectivity (SZ11-16_tot). FIG. 10A-C. 16Acid titers were statistically equivalent for both strains. [0216] Comparison of SPV1994 and SPV1995 [0217] SPV1994 (4-copy HzDST) and SPV1995 (P. dactylifera LPAAT and DGAT1A) were rescreened against an SPV1629 control in an independent experiment using the same small-scale assay. Selectivity metrics and Z11-16Acid titer from SPV1629 showed that SPV1629 produces a profile with SZ11/Z9-16 ≈ 96.5-97.5%, SC16 ≈ 80%, SZ11-16_prod ≈ 70%, SZ11-16_tot ≈ 50%. [0218] As observed in independent experiments, SPV1994 and SPV1995 displayed higher Z11-16Acid/Z9-16Acid selectivity (SZ11/Z9-16). FIG. 11A. The rank order of strains determined by SZ11/Z9-16 was maintained, with SPV1994 producing the highest selectivity at 97.7%. Similar consistency was observed for C16 selectivity (SC16), Z11-16Acid/products selectivity (SZ11-16_prod), and Z11-16Acid total selectivity (SZ11-16_tot), with the highest selectivities observed for SPV1995 at 85%, 76%, and 54%, respectively. FIG.11B-D. [0219] Table 6. Two independent replicates showing P. dactylifera LPAAT-expression led to a similar ~0.75% improvement in Z11-16Acid/Z9-16Acid selectivity and a ~5% increase in C16 selectivity (SC16), relative to SPV1629 (3xHzDST + DGAT1A_Pd). All C18 titers were reduced, and average Z11-16Acid titers were increased over the control in both experiments. 2
Figure imgf000073_0001
Figure imgf000074_0001
[0220] SPV1994 and SPV1995 Performance [0221] As described above, SPV1994 and SPV1995 were constructed based on previous observations on the effect of additional H. zea Z11 desaturase copy-number and the expression of P. dactylifera acyl transferases. By examining the performance of all SPV458 lineage strains (FIG. 12B), the improved performance of SPV1994 and SPV1995 was confirmed. [0222] When a second copy of the H. zea desaturase was introduced at the FAT1 locus (SPV968), an increase in Z11-16Acid titer from 2.50 to 3.17 g/L, and a large jump in Z11- 16Acid/Z9-16Acid selectivity (SZ11/Z9-16) from 83% to 96%, were observed. FIG. 12. This selectivity increase resulted from both an increase in Z11-16Acid titer, and a decrease in Z9- 16Acid titer. FIG.13. Addition of a third copy of the H. zea desaturase in SPV1056 led to a further increase in Z11-16Acid titer, and a smaller reduction in Z9-16Acid titer. The result was a small increase in SZ11/Z9-16 from 96% to 97%. FIG.12C. [0223] At this point, P. dactylifera DGAT1A was introduced to increase Z11-16Acid storage without greatly increasing storage of C18 fatty acids. The expression of P. dactylifera DGAT1A in SPV1629 led to an increase in Z11-16Acid and Z9-16Acid titers. FIG.12; FIG. 13. As a result, SZ11/Z9-16 remained constant at 97%. The expression of P. dactylifera LPAAT (SPV1796) led to a smaller increase in Z11-16Acid titer, with a significant reduction in Z9- 18Acid titer. FIG.12B. Titers of Z9-16Acid were also lower in the LPAAT-expressing strain when compared to the DGAT expressing strain (SPV1629). FIG.13. The result was a higher SZ11/Z9-16 at nearly 98%. FIG. 12C. [0224] Based on this evidence, both addition of a fourth copy of the H. zea desaturase and combining P. dactylifera DGAT1A and LPAAT expression were predicted to result in increases in SZ11/Z9-16. Data suggested that there is an upper limit to the benefit of additional desaturase copies on product titer (not shown). Before constructing SPV1994, it was unclear whether a fourth copy of the desaturase in the Z11-16Acid strain would result in increased titers. The fourth copy did reduce Z9-16Acid titer, leading to improved SZ11/Z9-16 (97.8%), but Z11-16Acid titers were lower on average than the SPV1629 parent. FIG. 12; FIG. 13. The combination of P. dactylifera acyltransferases boosted Z11-16Acid titer and improved selectivity in SPV1995, which exhibited a 10% increase in Z11-16Acid titer and a SZ11/Z9-16 of 97.9%. FIG.12. The increase in SZ11/Z9-16 resulted from both the increase in Z11-16Acid titer and the reduction in Z9-16Acid titer relative to SPV1629. FIG.13. As described above, the C18 fatty acid content was also reduced, and the resulting Z11-16Acid/Z9-18Acid ratio was between that of SPV1629 (DGAT1A only) and SPV1796 (LPAAT only). [0225] Addition of a 4th copy of the H. zea desaturase and expression of the P. dactylifera LPAAT led to increases in Z11-16Acid/Z9-16Acid selectivity (SZ11/Z9-16), such that these strains have a Z11-16Acid/Z9-16Acid selectivity of at least 97%. Expression of the P. dactylifera LPAAT reduced C18 titers and increased Z11-16Acid titers relative to the parent SPV1629. Combining expression of the H. zea desaturase from 4 gene copies and LPAAT expression shows these improvements are additive. [0226] We developed synthetic insect pheromone blend strains by adding a 4th HzDST copy and a copy of the P. dactylifera LPAAT to a Z11-16Acid production platform. . This engineered strain harbors increased Z11-16Acid/Z9-16Acid selectivity and increased Z11- 16Acid titers relative to the parent SPV1629., Strains from this strategy were engineeredto target a Z11-16Acid, Z9-16Acid blend that matches a biologically active ratio in Scirpophaga incertulas (for which the natural pheromone contains 75% Z11-16Ald, and 25% Z9-16Ald) (Example 2), and to target a Z9-16Ald, Z11-16Ald, and Z13-18Ald blend that matches a biologically active ratio in Chilo suppressalis (Example 3). [0227] Materials & Methods [0228] Integration Vector Construction: The H. zea desaturase sequence was previously cloned into a POX5 targeting vector (pPV0261, See Report: MetEng 170324 Z11- 16Acid strain screening assay). The P. dactylifera LPAAT vector was constructed from two plasmids: 1) pPV0548, an AXP targeting vector containing the codon optimized P. dactylifera LPAAT (See Report: MetEng 171024 acyltransferase screening-v1) and 2) pPV1015, a POX4 targeting vector containing an LPAAT from Cuphea avigera. The P. dactylifera LPAAT gene and a portion of the TEF promoter were subcloned from pPV0548 into the pPV1015 backbone replacing the C. avigera LPAAT with the P. dactylifera LPAAT. DNA constructs were transformed into E. coli EPI400 to propagate the plasmid. E. coli EPI400 strains were grown overnight in LB at 37 ºC and 250 rpm to an OD600 ≈ 2. Cells were back-diluted 10x into LB with copy-cutter induction solution and grown 24 hours at 37 ºC and 250 rpm before miniprepping. [0229] Strain Construction: Strain construction, including plasmid digestion, transformation, check PCR, and colony patching was performed essentially as described in Example 1.1. [0230] Biolector® Assay: Y. lipolytica strains were inoculated from YPD agar patches and grown in 2 mL YPD seed cultures for 16 hours at 28 ºC and 1000 rpm (Infors™ plate incubator) to an OD600 ≈ 15-20. Seed cultures were pelleted and concentrated to OD600 ≈ 50 in fresh YPD. A 1.5-2.5% inoculum was used to seed 700 μL FERM1 medium in a M2Plabs® 48-well flower plate. After growing for 7 hours at 32 ºC and 1500 rpm in the Biolector®, the plate was removed, 8.4 μL methyl palmitate at 37 ºC was added to each well as a liquid, and the plate was resealed and returned to the Biolector®. At 23 hours post-inoculation, an additional 8.4 μL methyl palmitate at 37 ºC was added to each well. The flower plate seal was replaced, and the plate was incubated an additional 25 hours (48-hour timepoint). Wells were mixed by pipetting the culture volume before 250 μL was transferred to a crimp-top GC vial. Sample vials were frozen and stored at -80 ºC. Cell density was measured with a Tecan® M200™ Pro plate reader at sampling. [0231] In all experiments, dissolved oxygen was monitored using oxygen sensitive optodes on the Biolector® plate. Only data from replicates with sustained oxygen transfer were used in the analysis. [0232] GC Processing of Lyophilized Samples: First, 250 μL culture was lyophilized in open glass crimp top vials for at least 3 hours. Next, 500 μL trimethylsulfonium hydroxide (TMSH) was added to the vials and sealed with a crimp cap. These vials were arrayed in racks, which were placed in a 37 ºC plate shaker for 2 hours at 250 rpm. After mixing, these dried cells with the derivatizing agent, the vials were incubated for 1 hour at 50 ºC to lyse the cell membranes. Finally, the liquid portion of the methylated sample was transferred to a clean GC vial with glass insert to prevent solid debris from clogging the column during GC analysis. Samples were run on GC-FID using the parameters (for both front and back detectors) listed in Table 4 of Example 1.1, above. Example 2: Z11-16 / Z9-16 Blend Production [0233] To engineer a strain that targets a Z11-16Acid, Z9-16Acid blend that matches a biologically active ratio in S. incertulas (70%-75% Z11-16), expression of a variety of Z9-16 desaturases were combined with expression of the H. zea Z11 desaturase to produce strains that produce a variety of Z11/Z9 ratios. The selection of Z9 desaturases was based on results of a screen of all publicly available lepidopteran desaturases (data not shown). The resulting strains produced a Z9-16Acid/Z11-16Acid blend as a precursor to Scirpophaga incertulas synthetic pheromone (Z9-16Ald/Z11-16Ald) at performances suitable for commercial scale deployment. [0234] Combinations of insect Z9-16Acid desaturases were integrated into the marker rescued descendant of a Z11-16Acid production strain (SPV1629: xpr2::HzDST, fat1::HzDST, fao1::HzDST, axp::DGAT1A_Pd) that was validated at 150L-scale to modify its profile of 97% Z11-16Acid, 3% Z9-16Acid. Z9 desaturases were integrated at either the previously deleted POX5 locus, or replaced an existing copy of the H. zea Z11 desaturase at the XPR2 locus. Nine Z9 desaturases were selected based on varying activity observed from screening 300 Lepidopteran desaturases in Y. lipolytica (data not shown). Desaturases comprising the Z9 DST motif (SEQ ID NO:39) are listed in Table 7 below. The activity level indicated is based on the increase in Z9-16Acid titer over the control strain in the desaturase screen. [0235] Table 7. Desaturases used to develop blend strains. The listed desaturases all contain the Lepidopteran Z9 DST motif (SEQ ID NO:39), a structural region within lepidopteran desaturases that correlates with enzyme activities.
Figure imgf000077_0001
Figure imgf000078_0005
*Activity levels represent the g/L increase in Z9-16Acid titer over the control strain in the desaturase library screen. [0236] The resulting strains were screened in both 24 well plate (1 mL scale) and Biolector® 48-well plate (700 μL scale) bioconversion assays using methyl palmitate as substrate. The fatty acid composition of all cultures was measured using GC-FAME analysis. [0237] Strains were evaluated, relative to SPV1629, with the following four metrics: (1) Z11-16Acid/Z9-16Acid selectivity:
Figure imgf000078_0001
(2) Combined titer =
Figure imgf000078_0002
Figure imgf000078_0003
(3) Total unsaturated C16 selectivity: ; and
Figure imgf000078_0004
(4) Growth rate. [0238] Gen1 Strains and Selection of Gen2 Parent [0239] Two sets of Gen1 blend strains were constructed to produce a library of 12 different strains (See Materials & Methods). Both sets used SPV1736, the marker rescued descendant of SPV1629, as the direct parent. See FIG.2C. The first set (Set 1) replaced one of the three copies of the H. zea Z11 desaturase (XPR2 locus) with a single Z9-16 desaturase selected from each of the three activity level groups shown in Table 7 (DST181, DST183, and DST192 (SEQ ID NO:53)). The second set (Set 2) added a single Z9-16 desaturase at the POX5 locus, producing strains with two copies of the H. zea Z11 desaturase and one copy of a Z9-16 desaturase. All nine desaturases shown in Table 7 were transformed for this set. [0240] Multiple clones of completed strains were initially screened in a Biolector® assay, and Z11-16Acid/Z9-16Acid selectivities from 68%-96% were observed. FIG.14. Most strains were above the target selectivity of 75%. Three isolates of the Set 1 DST192 (SEQ ID NO:53) construct (2xHzDST, 1xDST192) produced identical Z11-16Acid/Z9-16Acid selectivities (82%), while a fourth isolate produced a lower selectivity (68%). One of the three identical isolates (SPV2045) and the distinct fourth isolate (SPV2044) were selected. Check PCR on SPV2044 reconfirmed correct homologous recombination at both the 5’ and 3’ junctions, and the presence of the intended Z9 desaturase at the XPR2 locus. Two strains from Set 2 displayed lower selectivity than the others: SPV2025 (DST076, 88%) and SPV2029 (DST189, 87%). FIG.14B. These two strains were selected as parents for Gen 2 blend strains. [0241] Screening of Select Gen1 and Gen2 Strains [0242] Strain SPV2025 was marker rescued, and three desaturases were transformed into the marker rescued descendant SPV2043, replacing the H. zea desaturase at XPR2, and creating Gen2 blend strains with two copies of the H. zea Z11 desaturase, and two copies of Z9-16 desaturases. One medium activity desaturase (DST183), and two high activity desaturases (DST189 and DST192) were selected based on Gen1 blend results. [0243] The resulting Gen2 strains were screened in a Biolector® assay (See Materials & Methods) with the best Gen1 candidates, SPV2044 and SPV2045 (2xHzDST, 1xDST192). FIG.15; FIG.16A. The Biolector® assay was modified by reducing the initial seeding density and adding substrate later (18h) as a single bolus. These changes were made to facilitate a large-scale bioprocess protocol, and to help reduce the frequency of plate seal-clogging, and to improve the growth environment. These changes led to increases in Z11-16Acid/Z11-16Acid selectivity for SPV2045 (84%) and SPV2044 (74%). The control, SPV1629, produced a selectivity of 95%, similar to what had been observed previously (96%-97%). Gen2 strains, SPV2069 (2xHzDST, 1xDST076, 1xDST183), SPV2070 (2xHzDST, 1xDST076, 1xDST189), and SPV2071 (2xHzDST, 1xDST076, 1xDST192) produced selectivities of 79%, 74%, and 73%, respectively. All blend strains had marginally higher unsaturated C16 titer, and marginally lower unsaturated C16 selectivity (SC16) than the control SPV1629. FIG.15. [0244] A second 24-well plate assay was completed on a select panel of the best Gen1 and Gen2 strains to test the sensitivity of Z11-16Acid/Z9-16Acid selectivity to high seeding density, and alternative vessel format (See Materials & Methods). Under the 24-well plate assay format, the panel of strains produced Z11-16Acid/Z9-16Acid selectivities from 53%- 87%, while the control, SPV1629, had a selectivity of 96%. FIG. 17A. In general, strains displayed lower Z11-16Acid/Z9-16Acid selectivities (more Z9-16Acid) in this assay. Unsaturated C16 selectivity (SC16) for SPV1629 was 14% lower than in the Biolector® assay and was nearly identical for all strains. FIG. 17B. These 24-well plate results support the hypothesis that, in addition to strain modifications, changes to growth/seeding conditions can be used to adjust the selectivities of blend strains. [0245] Strain Growth Rates [0246] Final cell densities in all bioconversion assays and qualitative observations of phenotype suggested that blend strains were healthy and grew as well as the parent, SPV1629. More rigorous quantification of growth rate was completed for a select set of Gen1 and Gen2 blend strains. FIG.18. Two different media were tested; FERM1 (small-scale assay medium that primarily uses glucose, ammonium sulfate, yeast nitrogen base, and yeast extract to supply carbon, nitrogen, and vitamins), and Solulys® (batch version of a bioprocess medium that uses glycerol, ammonium sulfate, and Solulys® 95 to supply carbon, nitrogen and vitamins). Growth rates for all strains in both media were within 10% of the control SPV1629. [0247] Screening 14 strains across two small-scale assay conditions produced Z11- 16Acid/Z9-16Acid selectivities (SZ11/Z9-16) from 53% to 96%. In the Biolector® bioconversion assay, four stains displayed a Z11-16Acid/Z9-16Acid selectivity near 75%. Table 8. All tested strains had marginally higher combined Z11-16Acid/Z9-16Acid titers (TZ11/Z9-16) than SPV1629. In the 24-well plate assay with higher initial seeding density, strains SPV2025, SPV2029, and SPV2045 produced Z11-16Acid/Z9-16Acid selectivities from 73%-75%. Table 9. All tested strains had higher combined Z11-16Acid/Z9-16Acid titers (TZ11/Z9-16) than SPV1629. [0248] Table 8. Z11-16Acid/Z9-16Acid selectivity of blend strains containing lepidopteran desaturases.
Figure imgf000080_0001
[0249] Table 9. Z11-16Acid/Z9-16Acid selectivity of further blend strains containing lepidopteran desaturases.
Figure imgf000081_0001
[0250] Under both assay conditions, total unsaturated C16 selectivity (Su16) was similar to SPV1629 (72% Biolector®, 62% 24-well). In the Biolector® assay, some blend strains had reduced SC16 (67%), due to small increases in Z9-18Acid and Z11-18Acid titers. All strains grew well across media, and growth rates for select strains were found to be within 10% of SPV1629 (0.25 h-1) when cultured in a batch version of a bioprocess medium. [0251] A library of strains with varying Z11-16Acid/Z9-16Acid selectivity was constructed by combining the expression of lepidopteran desaturases with Z11 and Z9 desaturase activity on palmitoyl-CoA. Multiple strains display selectivity within 5% of a target Z11-16Acid/Z9-16Acid selectivity of 70%-75%. Growth rates and overall selectivity metrics match the performance of SPV1629, showing blend strains can be dropped into the Z11-16Acid bioprocess design. [0252] That activity of the strains with a Z11-16Acid/Z9-16Acid selectivity between 70% and 75% complements the existing Z11-16Acid strain profile (97% Z11-16Acid, 3% Z9- 16Acid) to produce a blend that matches a biologically active ratio in S. incertulas. Table 10 shows the fraction of the 97/3 blend required for different 75/25 blend strain/process performances. [0253] Table 10: Blend of strains for 75/25 Z11-16Ald/Z9-16Ald pheromone using 97:3 blend strain for Z11-16Ald enrichment.
Figure imgf000081_0002
Figure imgf000082_0001
[0254] Materials & Methods [0255] Integration vector construction: Desaturase sequences were codon optimized (Homo sapiens expression organism) and cloned into an integration vector using restriction digestion. [0256] Strain construction: Strain construction, including plasmid digestion, transformation, check PCR, and colony patching was performed essentially as described in Example 1.1. For checking PCR products from transformants with XPR2 constructs integrated into a background with a preexisting desaturase at the XPR2 locus, a desaturase-specific primer was used to confirm that the correct sequence had been integrated. [0257] 24-well plate bioconversion assay: The 24-well plate bioconversion assay was performed essentially as described in Example 1.1. [0258] Biolector® bioconversion assays: The Biolector® bioconversion assays with Gen1 and Gen2 strains were performed essentially as described in Example 1.2. For assays including Gen1 strains, 8.4 μL liquid 37 °C methyl palmitate was added to each well after growing for 7 hours, and again after growing for 23 hours. For assays including Gen2 strains, 21 μL liquid 37 °C methyl palmitate was added to each well after growing for 18 hours. [0259] Biolector® growth rate assay: The Biolector® growth rate assay was performed essentially as described in Example 1.1. [0260] GC processing of lyophilized samples: [0261] The Biolector® growth rate assay was performed essentially as described in Example 1.1. Samples were run on the GC in a one-pot format using the parameters (for both front and back detectors) listed in Table 4 of Example 1.1, above. The GC needle height was adjusted from 3.0 mm to 14.5 mm, as the sample is drawn from the very top of vials when run in “one-pot” format. Example 3: Z9-16 / Z11-16 / Z13-18 Blend Production [0262] To engineer a one-pot synthesis for a Z9-16, Z11-16, and Z13-18 blend, such as matches a biologically active ratio in C. suppressalis, production strains that are devoid of Z9 desaturase activity were developed to produce a high purity Z11-16 source (Example 3.1). Also developed were production strains that produce Z9-16, Z11-16, and Z13-18 in appropriate ratios (Example 3.2). Example 3.1: Improved Production of Z11-16 [0263] In Z11-16 production strain SPV2076 (pTEF-DST148, B. mori Z11 desaturase), the native Z9-18/16 desaturase (OLE1) was replaced with a Z11-18 desaturase from B. mori in the SPV1629 background (Example 1.1). We further characterized SPV2076, and selected SPV2076-derived isolates with improved growth rate (FIG.2D). The resulting strains serve as a platform for high purity Z11-16 production, and as a platform for C. suppressalis pheromone blend production without Z9-18 byproduct. [0264] SPV2076 is selective for Z11-16Acid and Z11-18Acid, and its growth phenotype could be improved for use as a Z11 selective production strain. Two parallel approaches were used to improve the growth phenotype of SPV2076, based on the hypothesis that the absence of Z9-18CoA (oleoyl-CoA) dysregulates lipid metabolism: First, the growth of SPV2076 with methyl oleate (Z9-18ME) supplementation was improved. Therefore, controlled oleate supplementation is useful to support the growth phase of blend bioconversions, limiting Z9-18Acid production. Second, descendants of SPV2076 were generated with an improved growth rate without oleate (Z9-18Acid) supplementation via adaptive evolution and mutagenesis. [0265] SPV2076 was propagated in rich media (YPD) before plating the resulting population and isolating colonies with increased growth rate (colony size). SPV2076 culture was randomly mutagenized using ethyl methanesulfonate (EMS), and faster growing isolates were identified by plating. Growth rates of resulting strains were measured in two different semi-defined media with online monitoring in a Biolector® assay. Bioconversion phenotypes were assayed in a 24-well plate assay with methyl-palmitate (16ME) supplementation. The fatty acid composition of all cultures was measured using GC-FAME analysis. [0266] Strains were evaluated, relative to SPV1629, with the following four metrics: (1) Exponential and linear phase growth rates; (2) Z11-16Acid/Z9-16Acid selectivity:
Figure imgf000083_0001
(3) Z11-16Acid total selectivity: ; and
Figure imgf000084_0001
(4) Z11-16Acid/products selectivity:
Figure imgf000084_0002
[0267] Methyl Oleate Supplementation [0268] It was hypothesized that the absence of oleoyl-CoA (Z9-18CoA) in SPV2076 led dysregulated lipid metabolism and created a slower growth rate than parent strains still containing the Ole1p Z9 desaturase. To test this hypothesis, SPV2076 was cultured in FERM1 medium with supplementation of varying amounts of methyl oleate (See Materials & Methods). Y. lipolytica will readily hydrolyze methyl oleate, and import the resulting free acid. [0269] When grown from an initial OD600 = 0.1, SPV2076 grew more slowly, and to a lower final cell density than the strain SPV1629. FIG.19A. Supplementation of either 0.9 or 4.4 g/L methyl oleate led to an increase in growth rate and final cell density by 90 hours. FIG. 19A. While the exponential growth phase was slower than for SPV1629, the following linear growth phase was similar. Therefore, methyl oleate supplementation to SPV2076 at either 1 g/L or 5 g/L improved exponential growth rate (10%) and linear growth rate (40%). [0270] Derivative Strains with Improved Growth Phenotypes [0271] We also screened for isolates with improved growth rates based on the hypothesis that mutations in the host genome could overcome dysregulation caused by the absence of Z9-18CoA. Two approaches, adaptive evolution and random mutagenesis, were taken to isolate faster growing strains. First, SPV2076 was cultured in YPD medium, and the resulting stationary phase culture was struck out on YPD agar to identify faster growing colonies. This approach led to the isolation of faster growing isolate SPV2135. Second, SPV2076 was treated with ethyl methanesulfonate (EMS) to increase the natural mutation rate (See Materials & Methods). EMS treatment led to the isolation of five fast growing colonies, from which strain SPV2148 was selected. [0272] A Biolector® growth rate assay with two different media was used to evaluate SPV2076, SPV2135 (growth selection), SPV2148 (EMS treatment), and strain SPV1629 (See Materials & Methods). The first medium, FERM1, uses glucose, glycerol, ammonium sulfate, yeast extract, and YNB as major components. The second medium, here referred to as Solulys®, mimics a batch phase medium. Growth rates for both SPV2135 and SPV2148 were significantly higher than the parent SPV2076 in both media. FIG.20; Table 11. Growth rates of SPV2135 and SPV2148 were most similar in the FERM1 medium, but SPV2148 was able to achieve similar final cell density to SPV1629 in the Solulys® medium with a delay of ~5 hours. Fatty acid titers for each strain were determined by GC-FAME analysis of whole culture samples. Selectivities were calculated from fatty acid titers as described in the Approach section above. [0273] Table 11. Bioconversion performance of SPV2076 lineage strains. Uncertainty in selectivity was less than 0.01 unless indicated.
Figure imgf000085_0001
[0274] Despite a marginally slower growth rate and lower final cell density, SPV2135 produced more lipid than any of the other strains in both media. FIG. 21. Cultures were incubated for 70 hours before sampling to profile fatty acid content, and SPV2135 produced >4 g/L of Z11-16Acid without the addition of methyl palmitate. These results showed that growth rate could be improved, and that SPV2135 displayed an increased lipid capacity relative to other strains. [0275] Derivative Strain Methyl Palmitate Bioconversion [0276] After identifying strains with improved growth rate, bioconversion performance was tested using a standard 24-well bioconversion assay (See Materials & Methods). The assay used FERM1 medium, and methyl palmitate was added in excess (~22 g/L). SPV2135 continued to display an improved lipid accumulation phenotype with >10 g/L of Z11-16Acid produced. FIG. 22. This titer was 50% greater than that observed with SPV1629. Cell densities measured by optical density were marginally lower for SPV2135 compared to SPV1629. The SPV2076 control also produced lower fatty acid titers and lower final cell density. SPV2148 produced significantly less Z11-16Acid, while producing more Z11-18Acid than SPV2148. [0277] Selectivity metrics were equivalent or improved for SPV2135; Z11/Z9-16 selectivity was nearly 1.0 for SPV2135. FIG.24A. Total Z11-16 selectivity was marginally improved over SPV1629, and Z11-16 products selectivity was equivalent to SPV1629. FIG. 24B; FIG. 24C. Total and products selectivities were lower for SPV2148, due to the lower Z11-16Acid titer and higher Z11-18Acid titer. [0278] The growth rates of SPV2076 lineage strains (adaptive evolution, mutagenesis) were improved relative to SPV2076; growth rates were ~80% of SPV1629 in FERM1, and ~50- 60% of SPV1629 in Solulys®. The growth rate rank order of the strains was generally consistent between both media. Table 12. Growth rates were measured with online optical density detection using the Biolector®. All strains exhibited two growth phases, with a true exponential growth period followed by a linear growth phase. [0279] Table 12. Growth rates of SPV1629 and SPV2076 lineage strains. Relative Linear phase Exponential exponential growth rate Relative linear Medium Strain growth rate (h-1) growth rate (OD/h) growth rate
Figure imgf000086_0001
[0280] Maximum cell densities were comparable for all strains in FERM1 medium, except for SPV2135, which generated a higher cell density when the culture was grown for 70 hours. Maximum cell density in Solulys® medium, as measured by optical density, was reduced for SPV2076 (21% lower) and SPV2135 (12%-28% lower) relative to SPV1629 and SPV2148. SPV2135 produced higher Z11-16Acid titers (2- to 3-fold) than SPV1629 in both media under growth assay conditions (no addition of methyl palmitate). [0281] In the 24-well bioconversion assay (Table 11), SPV2135 produced 50% higher Z11-16Acid titer (~10 g/L) than SPV1629 (~7 g/L), while only producing saturated acids (16Acid, 18Acid) and Z11 acids (Z11-16Acid, Z11-18Acid, Z13-18Acid, and Z11Z14- 18Acid). As observed in the growth assays, the measured cell density for SPV2135 was lower than that observed for SPV1629. [0282] This Example shows that adaptive evolution and chemical mutagenesis of SPV2076 produced strains with an improved growth rate phenotype (SPV2135 and SPV2148), and additionally an increased lipid accumulation phenotype and high Z11-16Acid selectivity (SPV2135). Deletion of FAD2 (Δ12) desaturase in SPV2135, combined with overexpression of elongases, and replacement of the B. mori Z11-18 desaturase with Manduca sexta Z13-18 desaturase (DST143), generates a strain that produces only Z11-16Acid and Z13-18Acid. Similarly, the product profile of FAD2-deleted SPV2135, when coupled to Z-selective metathesis, leads to the production of Z11-16FAME and Z13-18FAME when using a 1-hexene substrate. In all cases, the resulting Z11-16 and Z13-18 products, when blended with a Z11- 16/Z9-16 blend (See Example 6) and/or Z9-16FAME, produce a Z9-16, Z11-16, and Z13-18 blend. FIG.25. [0283] Materials & Methods [0284] Growth selection strain isolation: Strain SPV2076 was inoculated from a YPD streak plate into 2 mL YPD medium in a 24-well culture plate. The culture was grown for 48 hours to reach a saturated cell density. After 48 hours, a loop was used to streak out culture on YPD agar plates. SPV2135 was an isolate from these plates. An improved growth rate of SPV2135 was readily observed on YPD agar when compared to SPV2076. [0285] Chemical mutagenesis strain isolation: Strain SPV2076 was inoculated from a YPD streak plate into mL YPD medium in a 14 mL round-bottom culture tube. The culture was incubated overnight at 28 ºC and 250 rpm in an Infors™ flask shaker. Cells were harvested after 28 hours at OD600 = 3. The 1 mL culture was transferred to a sterile microcentrifuge tube and pelleted before washing twice with 100 mM phosphate buffer (pH 7). After the second wash, the cell pellet was resuspended in 1.5 mL 100 mM phosphate buffer (pH 7), and 700 μL was transferred to a 14 mL round-bottom culture tube. 25 μL ethyl methanesulfonate was added to the 700 μL resuspended culture, and the tube was returned to the incubator for 35 minutes at 28 ºC. Zero, 10-fold, 100-fold, and 1,000-fold dilutions of the treated cells were made using phosphate buffer, and 100 μL aliquots were plated on YPD agar plates. Multiple growth rates were observed based on visual inspection of colony size. Five of the fastest growing colonies were further screened for growth rate in the Biolector® growth assay and one of those isolates was banked as SPV2148. [0286] 24-well plate methyl oleate supplementation assay: Y. lipolytica strains were inoculated from YPD streak plates and grown in 2 mL YPD seed cultures in 24-well plates for 24 hours at 28 ºC and 1000 rpm (Infors™ plate shaker/incubator). Seed cultures at OD600 between 5 and 8 were added to 1 mL YE-glucose medium to an initial OD600 of ~1. Zero, 1, or 5 μL methyl oleate was added to triplicate cultures. Strains were incubated at 28 ºC and 1000 rpm for the duration of the experiment (90 hours), with samples taken for OD600 cell density measurements at intervals (Tecan® plate reader). [0287] 24-well plate bioconversion assay: The 24-well plate bioconversion assay was performed essentially as described in Example 1.1. [0288] Biolector® growth rate assay: The Biolector® growth rate assay was also performed essentially as described in Example 1.1. [0289] GC sample processing of lyophilized samples: GC sample processing of lyophilized samples was performed essentially as described in Example 1.1. Samples were run on the GC in a one-pot format, using the parameters (for both front and back detectors) listed in Table 4 of Example 1.1. The GC needle height was adjusted from 3.0 mm to 14.5 mm. Example 3.2: Production of Z13-18 in a Z11-16 / Z9-16 Background [0290] To develop strains that produce blends of unsaturated fatty acids that can be processed to a synthetic Chilo suppressalis pheromone from our Z11-16 platform, the Gen1 blend strain expressing the recombinant Z9 desaturase DST183 from Helicoverpa assulta (SPV2028) was selected for further development. A target composition was selected for an active C. suppressalis pheromone a blend containing 81% Z11-16Ald : 9% Z9-16Ald : 10% Z13-18Ald after distillation. For production, we aimed to generate a strain that produces a Z9- 16Acid/Z11-16Acid ratio of 0.091 - 0.12, and a Z13-18Acid/Z11-16Acid ratio of >0.14. We aimed to produce an excess of Z13-18Acid over the target, because we anticipated a loss of Z13-18Acid during distillation. [0291] The native Y. lipolytica fatty acid synthase predominantly produces 18CoA (stearoyl-Coenzyme A), and the native Z9 desaturase (OLE1) has a selective preference for 18CoA, converting it to the unsaturated product Z9-18CoA. Z9-18Acid (oleic acid), one of our byproducts, is required for rapid cell growth, because it is a key regulator of native lipid metabolism and is a major cellular membrane component. A strain devoid of desaturase activity that can act on 18CoA leads to a build-up of saturated 18Acid, which reduces membrane fluidity and renders the strain inviable. [0292] To develop a strain devoid of Z9 desaturase activity, OLE1 was replaced with a Lepidopteran Z11-18 desaturase (DST148) from Bombyx mori in our Z11-16Acid producing strain, SPV1994. Example 1.1. The resulting strain had a significantly reduced growth rate, which was improved through adaptive evolution (Example 3.1). We hypothesized that Z11- 18Acid can serve as a substitute for Z9-18Acid and provide some degree of membrane fluidity, but ultimately lacks recognition in the regulatory pathway, which hampers the efficient regulation of cell growth. Subsequently, the native Z12 desaturase FAD2 was deleted to eliminate Z9Z12-18Acid synthesis. [0293] The native elongase, ELO2, was overexpressed in SPV2028 to test for increased Z13-18Acid production from Z11-16Acid (SPV2366). The resulting marker rescued strain (SPV2416) served as a clean background for screening Z9 desaturase selectivity. [0294] SPV2028, generated in the Scirpophaga incertulas blend strain library, was selected as a parent for the C. suppressalis blend production strain, because it produced a Z9- 16/Z11-16 ratio of 0.15, close to our target range of 0.091-0.12. FIG.26. To increase the Z13- 18 content, a 14-strain combinatorial library of CoA elongation overexpression constructs in the SPV2028 background was tested. To tune the final Z9-16 content, the lepidopteran Z9 desaturase DST192 (SEQ ID NO:47) was engineered for improved selectivity to enable independent control of Z9-16 desaturase and Z9-18 desaturase activities. DST192 variants were incorporated into the lead strain to reduce the Z9-18Acid byproduct titer. [0295] All strains were screened in a 1 mL 24-well plate assay format, and all growth rates were measured through a Biolector® assay. Fatty acid composition was measured using GC- FAME analysis. [0296] Elongase Overexpression in a FAD2 Deletion Background [0297] To increase the Z13-18Acid content, a library of DNA constructs overexpressing key genes of the native CoA elongation pathway (ELO2, IFA38, and TSC13) were integrated into the SPV2028 background. Multiple elongase copies were stacked at different loci, resulting in a library with various combinations of ELO2, IFA38, and TSC13 overexpression, as well as the deletion of the Z12 desaturase FAD2 (See Materials & Methods for constructs). Results from our 1 mL bioconversion assay showed that increased overexpression of ELO2 led to an increase in elongation of both Z11-16Acid to Z13-18Acid, and elongation of 16Acid substrate to 18Acid, which was subsequently converted to Z9- 18Acid. Expression of IFA38 did not have a detectable effect on the elongation rate, while TSC13 overexpression tended to reduce Z13-18Acid titers. FIG. 27. The reduction due to TSC13 overexpression can be observed when comparing performance to SPV2322, SPV2166, and SPV2324. A second copy of TSC13 in SPV2327 also reduced the Z13-18Acid titer relative to SPV2325. FIG.27C. [0298] The overexpression of ELO2 in the SPV2028 background increased elongation of Z11-16Acid to Z13-18Acid, and 16Acid substrate to 18Acid, which was subsequently converted to Z9-18Acid. SPV2165 was selected as Gen1 C. suppressalis blend production strain. [0299] OLE1 Deletion [0300] In a 1L bioconversion, SPV2165 produces Z9-16Acid/Z11-16Acid and Z13- 18Acid/Z11-16Acid titer ratios close to the targets of 0.091-12 and >0.14, respectively, but produces an excess of Z9-18Acid that is nearly three-fold higher than our target of 2.0 g/L. To reduce Z9-18 activity, we hypothesized that the native Z9 desaturase OLE1 could be removed if sufficient Z9-18 desaturase activity could be provided by our recombinant Z9 desaturase DST183. DST183 was tested in Z11 strain SPV2416, and it was found to generate sufficient flux towards Z9-18Acid for cell growth. OLE1 was therefore deleted in the SPV2165 background to reduce the Z9-18Acid content. The resulting strain (SPV2480) utilizes DST183 as the sole producer of Z9-18Acid. Though the deletion of OLE1 did reduce Z9-18Acid production, DST183 still produced excess Z9-18Acid, suggesting strong activity on 18CoA. FIG.28. To further reduce Z9-18Acid production, DST183 was replaced with a more selective Z9 desaturase. [0301] In a DST library screen, a range of Z9-16Acid/Z9-18Acid ratios were observed for Lepidopteran desaturases. DST192 produced a similar Z9-18Acid titer to DST183, while producing higher Z9-16Acid titers, which suggests a higher selectivity for the 16CoA substrate. FIG.29C. It is also possible that the higher activity of DST192 draws more flux of 16CoA away from elongation by producing Z9-16CoA, which lowers the local concentration of 18CoA. We hypothesized that its high wild type activity and potential higher selectivity made DST192 the best choice for protein engineering. [0302] DST192 Engineering [0303] A simplified but accurate binding pocket model of DST192 was created using structural information from mammalian Z9-18 desaturases, and sequence-function data for Lepidopteran desaturases. FIG.29(A-B). The ω-tail of the fatty acyl-CoA substrates sits in a pocket between two transmembrane helices of the desaturase that were labeled as Transmembrane Helix 2 (TM2) and Transmembrane Helix 4 (TM4), based on the order they appear in the primary sequence of the enzyme. A correlation was identified between predictions from the binding pocket model and our data observed in the DST screen; Z9 desaturases producing higher relative titers of Z9-18Acid compared to Z9-16Acid had smaller amino acids at positions toward the bottom of the binding pocket (FIG.29(B-C)). The smaller side chains likely reduce the binding constant Km for the longer substrate 18CoA. [0304] Accordingly, a library of point mutants at 8 positions split across both TM2 and TM4 was created to improve substrate selectivity for 16CoA. Three positions (F244, I245, and Y96) at the bottom of the binding pocket were mutated to bulkier amino acids to create steric hindrance against the binding of an 18CoA substrate. Additionally, DBK degenerative codon libraries were created for six-positions (Y96, V97, A98, S99, G100, and I101) along the center of the binding pocket to test if bulkier amino acids at positions in the center of the pocket would also increase selectivity for 16CoA (See Materials & Methods for constructs). [0305] All mutants were screened in Z11-only background strain, SPV2416, so that the only Z9 activity observed would come from the recombinant mutants. One point mutant, I245F (SEQ ID NO:57), improved in vivo 16/18 selectivity to 1:1, compared with a wild type ratio of 0.4:1. FIG. 30A. Data from the DBK libraries identified the G100 position of TM2 as a key determinant of substrate selectivity. Mutations at G100 produced a trend in substrate selectivity that correlated well with amino acid size, indicating that bulkier substitutions hinder longer chain length substrates from entering and accessing the binding pocket (FIG.30A; FIG. 30C). A G100V mutation produced a highly C16-selective desaturase (SEQ ID NO:55), whereas smaller amino acids like alanine (A) and serine (S) were less selective for 16CoA. Larger side chains like phenylalanine (F) and arginine (R) eliminated Z9 activity on both 16CoA and 18CoA (FIG.30A; FIG.30C). [0306] From the DST192 mutant data, a double mutant library was generated, combining the I245F mutation with four different G100 mutations. DST192 double mutants were also screened in SPV2416. Results from our 1 mL bioconversion assay showed that I245F coupled with G100V displayed no difference in selectivity or activity when compared with a single G100V mutant. FIG.30B. This result supports the hypothesis that mutations at the G100 position alone are sufficient to control selectivity, and the effect is independent of the bulkiness of residues at the bottom of the pocket. On the contrary, combining G100A and I245F mutations led to 16/18 selectivity that was higher than either single mutation by itself. As with the single mutants, G100I and G100L side chains were too large, eliminating Z9 activity. In total, the results support a model where the G100 position is a structural determinant of substrate specificity, whereby bulky residues limit the binding of 18CoA without drastically impacting 16CoA binding. A residue that is too large at position 100 on TM2 will sterically hinder even a 16CoA substrate (FIG.30C). Assaying these mutants with a 14Acid substrate would elucidate whether mutations of G100 to amino acids bulkier than valine retain activity on shorter chain length substrates. [0307] The DST192_I245F point mutant improved the 16/18 selectivity to 1:1 in vivo. The DST192_G100V point mutant resulted in a highly C16 selective Z9 desaturase (>16:1 in vivo). Replacing the native Z9 desaturase OLE1 with a low-expression wild type (SPV2665) and an engineered point mutant (DST192 I245F, SPV2667) reduced the titer of the byproduct Z9- 18Acid by 52% and 70%, respectively, in the 1 mL assay. Growth rates (SPV2665: 38%; SPV2667: 38%) and fatty acid productivity (SPV2665: 22%; SPV2667: 30%) of the resulting strains were reduced relative to the parent. [0308] Reduced Z9-18 Desaturase Activity Strains [0309] Both wild type DST192 (SEQ ID NO:53) and the I245F mutant (SEQ ID NO:55) were selected for expression to provide levels of Z9-18 desaturase activity that would be sufficient to support growth while reducing Z9-18Acid titer. Weak promoters pPXA1 and pAMO2 were selected to drive expression in view of desaturase activity data generated in a previous screen of Y. lipolytica promoters (data not shown), because previous strains using the strong pTEF promoter had produced high titers of Z9-18Acid. DST192 and DST192_I245F were cloned behind pPXA1 and pAMO2 at the POX5 locus, and resulting plasmids were transformed into the SPV2588 background (1xELO2, 1xIFA38, Δole1, 1xDST192-I245F), replacing the previously integrated DST183. Resulting strains expressing two copies of DST192 variants reduced Z9-18 titers by 52% (SPV2665) and 70% (SPV2667). FIG. 31C. While these strains were successful in restricting Z9-18Acid titer, the overall fatty acid productivity and growth rate were reduced. FIG. 31A; FIG. 32. Further tuning of Z9-18 desaturase activity through the expression of additional copies of DST192_G100V achieves a higher growth rate while reducing Z9-18Acid titer and increasing Z9-16Acid content. [0310] Single-copy overexpression of the native elongase ELO2 increased Z11-16Acid elongation to Z13-18Acid to an acceptable level for a C. suppressalis blend application. Lepidopteran Z9 desaturases were engineered according to our binding pocket model to yield high C16 substrate selectivity. Engineered desaturases were used to balance the Z9-16Acid and Z11- 16Acid content in blend production strains. Z9-18 desaturase activity was reduced by using weak promoters and a Z9 desaturase mutant for the purpose of maintaining higher growth rate and productivity. [0311] Table 13. Ratios of C. suppressalis pheromone blend precursors and Z9-18 in a 1 mL bioconversion assay.
Figure imgf000093_0001
[0312] Accordingly, the DST192_G100V point mutant (high C16 selectivity) increases the Z9-16Acid content in the lead strain, and Y. lipolytica fatty acid synthase (FAS) mutants engineered in the ketosynthase domain bias de novo fatty acid synthesis to 16CoA instead of 18CoA, so that the accumulation of saturated 18Acid is reduced during the growth phase. [0313] Materials & Methods [0314] Strain construction: Strain construction, including plasmid digestion, transformation, check PCR, and colony patching was performed essentially as described in Example 1.1. [0315] 24-well bioconversion assay: The 24-well bioconversion assay was performed essentially as described in Example 1.1. [0316] Biolector® growth rate assay: The Biolector® growth rate assay was performed essentially as described in Example 1.1. [0317] GC Sample processing of lyophilized samples: [0318] GC and GC sample processing of lyophilized samples was performed essentially as described in Example 1.1, with the parameters (for both front and back detectors) listed in Table 4. Example 4: Selective Biocatalysis of Z9-14 from Substrate [0319] To demonstrate how the Lepidopteran desaturase active site structural determinants identified in Example 3 that confer substrate and regioselectivity can be used to rationally engineer desaturases at will, a Z9-14 specific desaturase was engineered as a tool for increasing the Z9-14 fraction in production strains. Z9-14 is an active ingredient or valuable intermediate in insect pheromones; for example, Spodoptera frugiperda (83% Z9-14Ac and 13% Z11-16Ac). However, a Z9-14-specific desaturase has not been previously reported. [0320] Preliminary evidence suggested that some POX variants (e.g., pPV642; BaACX1) preferentially chain shorten saturated 16CoA (data not shown). See FIG.33 (POX catalyzing conversion of 16CoA to 14CoA). A desaturase operating on 14CoA was believed to drive flux toward Z9-14CoA in a pathway circuit even when 16ME (the substrate of choice for many biocatalysts and blend strains) is fed. FIG. 33. A Z9-16 specific mutant desaturase (DST192 G100V) was engineered by modeling the active site of Z9-16/Z9-18 desaturase DST192. See Example 3.2. Therefore, a DST192 G100DBK library was screened in SPV2416 (a mutant strain that does not produce Z9-unsaturated fatty acids and produces only Z11-unsaturated fatty acids) against 14CoA substrate. [0321] A DST192 G100(DBK) library (SEQ ID NO:49) was used to engineer a Z9- 16-specific desaturase, wherein the degenerate DBK codon encodes the following set of canonical amino acids: alanine (A), arginine (R), cysteine (C), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), serine (S), threonine (T), tryptophan (W), and valine (V). From the result that DST192 G100V accepted 16CoA but not 18CoA (See Example 3.2), even conservative changes are useful to rationally tune substrate preferences. When screening this library against 14CoA, mutant DST192 G100V was shown to accept both 14CoA and 16CoA. FIG. 34. However, DST192 G100I and DST192 G100L both retain activity against 14:0 substrate (fed), but not 16CoA or 18CoA (either extension products of 14CoA or produced de novo). FIG.34. [0322] Mutants DST192 G100F and DST192 G100R, with bulky substitutions, were inactive against all three monitored chain lengths. FIG.34. G100S retained almost the same ratio of activity as wildtype against all three substrates, but had a lower overall activity. FIG. 34. ore mutants were not screened, as two variants (DST192 G100I and DST192 G100L) with the desired Z9-14 selectivity were discovered. [0323] From homology models of WT DST192 and Z9-14 selective mutants DST192 G100I and DST192 G100L (with 14CoA, 16CoA, and 18CoA bound), G100I and G100L were found to not impede the binding of 14CoA (FIG. 35). By contrast, the models presented in FIG.36 and FIG.37 show that the G100I and G100L occlude the active site, and prevent the binding of 16CoA and 18CoA, respectively. Taken together, the models support the hypothesis that these mutations do not impede binding of 14CoA, but fully abrogate 16CoA and 18CoA binding. Notably, these models are consistent with GC-FID data showing that the G100I and G100L mutants harbor little to no activity against 16CoA and 18CoA, but retain full WT level activity against 14CoA (FIG.38; FIG.39; FIG.40). [0324] Models of the bulkier mutants DST192 G100F and DST192 G100R show at least two reasons why these enzymes completely lose activity against 14CoA, 16CoA, and 18CoA. FIG. 41 shows that the phenol abuts the CoA substrate in at least one hypothetical rotamer of the G100F mutant. Separately, the sidechain of G100R abuts the opposing helix lining the binding pocket to prevent proper folding. [0325] Materials & Methods [0326] Strain construction: Strain construction, including plasmid digestion, transformation, check PCR, and colony patching was performed essentially as described in Example 2.1. Integration constructs are listed in Table 14. [0327] Table 14. Integration vector constructs.
Figure imgf000095_0001
Example 5: Increased Lipid Content During Selective Biocatalysis [0328] To determine whether further increases in Z11-16 productivity could be achieved in our production platform strains, both expression of additional recombinant acyltransferases and the overexpression of native lipid metabolism genes were investigated, with the goal of increasing the lipid content of our strains while maintaining product selectivity. Such strains are preferable for production of a synthetic Helicoverpa sp. pheromone, where ≥97% of the unsaturated C16 fatty acid composition is Z11-16 (Both Z9-16Ald (≤3%) and Z11-16Ald (≥97%) are recognized as active ingredients in the pheromone for H. zea and H. armigera). [0329] Six different acyl transferases were expressed in the SPV1629 strain background that already expresses P. dactylifera DGAT1A: Macadamia tetraphylla DGAT1 (SEQ ID NO:18); Thalassiosira pseudonana DGAT1 (SEQ ID NO:20); Vernicia fordii DGAT1 (SEQ ID NO:26); V. fordii DGAT2 (SEQ ID NO:28); T. pseudonana LPAAT1 (SEQ ID NO:22); and T. pseudonana LPAAT2 (SEQ ID NO:24). Overexpressing native Y. lipolytica lipid regulators MGA2act (SEQ ID NO:59) (a truncated transcriptional activator fragment), MGA2act G643R (SEQ ID NO:61) (enhanced activity mutant), HAP1 (SEQ ID NO:65) (Heme-responsive zinc finger transcription factor), the diacylglycerol phosphate (PA) phosphatase PAH1 (SEQ ID NO:67), and the cytochrome B5 reductase CBR1 (SEQ ID NO:63) were also screened in SPV1629. [0330] PAH1 (phosphatidate phosphatase) catalyzes the first committed step in TAG synthesis by removing the sn-3 phosphate, exposing the final hydroxyl group which forms an ester with a third acyl group in the final TAG. Post-translational phosphorylation of PAH1 in S. cerevisiae has been implicated in down-regulation of PAH1 activity. Since many phosphorylation sites are not conserved in the Y. lipolytica variant, we set out to test whether simple overexpression would be sufficient. However, overexpressing Y. lipolytica PAH1 was not expected to to enhance activity. See Choi et al. (2011) J. Biol. Chem. 286:1486-98; Choi et al. (2012) J. Biol. Chem. 287:11290-301; Karanasios et al. (2010) Proc. Natl. Acad. Sci. USA 107(41):17539-44. [0331] To determine whether microsomal cytochrome B5 reductase (CBR1) is involved in the transfer of electrons to enzymes of lipid synthesis, utilizing an NADH cofactor, in the Y. lipolytica variant, the effects of CBR1 overexpression on lipid content and fatty acid profile of our Z11-16 strains were tested. Bioconversion phenotypes were assayed in a 24-well plate assay with methylpalmitate (16ME) supplementation. The fatty acid composition of all cultures was measured using GC-FAME analysis. Strains were evaluated, relative to SPV1629, with the following metrics: (1) Exponential and linear phase growth rates; (2) Z11-16Acid titer and productivity; (3) Z11-16Acid/Z9-16Acid selectivity:
Figure imgf000096_0001
(4) Z11-16Acid total selectivity:
Figure imgf000096_0002
; and (5) Z11-16Acid/products selectivity: .
Figure imgf000097_0001
[0332] Growth rates of promising strains based on lipid profile in the bioconversion assay were measured in two different semi-defined media with online monitoring in a Biolector® assay. Select promising strains were screened at 1 L in fermentors. [0333] Bioconversions with Methyl Palmitate Supplementation [0334] Multiple (generally 4) clonal isolates of each construct were screened using a 1 mL assay in 24-well plate format with glass vial inserts (See Materials & Methods). Briefly, individual clones were confirmed by colony PCR, struck out for single colonies, and inoculated into 1 mL YPD seed cultures. Methyl palmitate was added as the bioconversion substrate after a period of adjustment to the bioconversion medium (6h). [0335] Twenty-four replicates of the parental control strain SPV1629 were used across three assay plates to accurately capture the titer distribution. Including outliers, Z11- 16Acid titers for SPV1629 ranged from 5.4 to 10.7 g/L. FIG.42; FIG.54. The average value was 9.6 g/L with 16 of 24 replicates producing titers from 9.2 - 10.1 g/L. Five samples were low outliers, which were verified to be the result of variations in substrate loading (data not shown). [0336] Surprisingly, different lipid phenotypes were observed for strains expressing a second recombinant acyl transferase. Clones expressing the two T. pseudonana LPAAT variants produced a wider range of Z11-16Acid titer that spanned the range of the non-outlier SPV1629 titers. FIG.42. Both DGAT variants from V. fordi produced a split distribution of Z11-16Acid titer with two higher and two lower replicates. The higher values exceeded the control distribution, with the lower values at the upper end of the control distribution. The titers of byproduct fatty acids relative to the Z11-16Acid titer were marginally lower than those observed for SPV1629, indicating a potential increase in selectivity. FIG. 42. The clearest phenotypes were observed for M. tetraphylla and T. pseudonana DGAT1 overexpression. Three of four replicates produced Z11-16Acid titers above the highest observed values for SPV1629, with the fourth replicate titer equivalent to the highest observed titers for SPV1629. FIG. 42. Additionally, byproduct titers also increased relative to Z11-16Acid titer. FIG. 42; FIG.44. The byproduct increase resulted mostly from Z9-18Acid and 18Acid titer increases. For DGAT1_Mt, byproducts increased by 23%, with the Z9-18Acid and 18Acid titers accounting for 64% and 23% of that increase, respectively. DGAT1_Tp byproducts were 64% higher with Z9-18Acid and 18Acid titers, accounting for 73% and 13% of the increase, respectively. [0337] Strains overexpressing native lipid metabolism regulators and enzymes also generated distinct phenotypes, although none of the strains produced higher Z11-16Acid titers than the SPV1629 control. Overexpression of the full-length MGA2 gene led to a reduction in all fatty acid titers, including Z11-16Acid. FIG. 43. The Z11-16 product selectivity was comparable to the SPV1629 control. Overexpressing the activator domain of MGA2 (MGA2act, MGA2act G643R) led to increases in byproduct fatty acids, while lowering the observed Z11-16Acid titer. FIG.43. Nearly 70% of this increase was due to increase in Z9- 18Acid titer. Similarly, PAH1 overexpression led to a less selective strain because of a reduced Z11-16Acid titer relative to the control. HAP1 overexpression preserved selectivity, but reduced productivity. Finally, CBR1 overexpression led to equivalent Z11-16Acid titers with an increase in byproducts (predominantly Z9-18) leading to a reduced selectivity. [0338] Bioconversions without Methyl Palmitate Supplementation [0339] All acyl transferase strains and a subset of native lipid metabolism overexpression strains (CBR1, MGA2act, MGA2act G643R, PAH1) were rescreened following the same assay protocol, with the exclusion of methyl palmitate substrate addition. This assay tests the effect of each genetic modification on de novo lipid synthesis and storage. [0340] Without substrate addition, less noise in the data was expected, because inconsistency in substrate addition adds a significant amount of variation. Because less variance was expected, eight replicates of SPV1629 were used. The range was smaller than observed with methyl palmitate addition; 2.37 - 2.57 g/L Z11-16Acid. Variation in byproduct titer was also low, with total byproducts between 2.57 and 2.80 g/L. A coefficient of variance of 2-3% was observed for each fatty acid titer. A linear correlation between Z11-16Acid and byproduct titers was observed, suggesting the variation in titer was due to variation in cell density. [0341] Two of the acyltransferase constructs, DGAT1_Mt and DGAT1_Tp, produced higher Z11-16Acid and byproduct fatty acid titers. FIG. 45. DGAT1_Mt overexpression led to proportional increases in Z11-16Acid and byproduct fatty acid titers with the same Z11- 16Acid total selectivity as SPV1629 (SZ11-16_Tot = 48%). Total fatty acid titer increased by 23%. FIG. 46. DGAT1_Tp overexpression increased Z11-16Acid titer, but produced a higher increase in byproduct fatty acids, leading to a lower Z11-16Acid total selectivity, (SZ11-16_Tot = 43%). Total fatty acid titer was increased by 51%. FIG.46. [0342] A distinct phenotype was observed for all four native lipid metabolism overexpression constructs. Overexpressing CBR1, MGA2act, and MGA2act G643R all produced higher byproduct fatty acid titers. FIG.47. CBR1 overexpression reduced the Z11- 16Acid titer, so that the total fatty acid titer was equivalent to that of SPV1629. FIG.48. The activator domains MGA2act and MGA2act G643R produced higher total fatty acid titers, with MGA2act G643R also producing higher Z11-16Acid titers. FIG. 47; FIG. 48. This result confirmed both the mutant MGA2 and wildtype MGA2 activator domains as activators of de novo lipid synthesis and storage. [0343] Large-scale Bioconversions [0344] The most productive strains identified during the 1 mL scale screening (SPV2473 (DGAT1_Mt), SPV2474 (DGAT1_Tp), and SPV2477 (DGAT1_Vf)), and the strain SPV2479 from the native lipid metabolism group (CBR1), were screened at 1 L scale. Strain performance at 1 L mirrored that of the 1 mL results. The rank order of strains based on Z11- 16Acid productivity was maintained: SPV2474 > SPV2473 > SPV2477 > SPV1629 > SPV2479. Table 15; FIG. 49; FIG. 50. The relative increases in multiple fatty acid species also closely mirrored the small-scale results. Table 15; Table 17; FIG.49. In total, the data shows a strong correlation between 1 mL and 1 L strain performance across multiple phenotypes. [0345] Table 15. Performance of select strains in 1 L bioconversion process at 40 hours.
Figure imgf000099_0001
[0346] Table 16. Relative fatty acid titers for acyl transferase strains screened at 1 mL and 1 L scales. Percent increases are relative to the SPV1629 control. Data taken from ~40- hour timepoint to best differentiate between productivity of strains.
Figure imgf000100_0001
[0347] Table 17. Increase in fatty acid titers with overexpression of select native lipid metabolism genes. Percent increases are relative to the SPV1629 control. 1 L data taken from ~40-hour timepoint to best differentiate between productivity of strains.
Figure imgf000100_0002
[0348] With the 1 L results, distinct phenotypes were confirmed for each strain relative to SPV1629. SPV2473 (DGAT1_Mt) has an increased lipid accumulation phenotype, while maintaining similar selectivity to SPV1629. SPV2473 has 13% higher peak Z11-16Acid productivity, equivalent SZ11/Z9-16, and marginally lower SZ11-16_Prod, due to an increase in 18Acid and Z9-18Acid titer. Table 15; Table 18; FIG.49. SPV2474 (DGAT1_Tp) has the highest lipid accumulation phenotype, while sacrificing selectivity for Z11-16Acid. SPV2474 has a 30% higher peak Z11-16Acid productivity, ~5% lower SZ11/Z9-16, and 20-25% lower SZ11-16_Prod, due to increases in C18 fatty acid titers, especially Z9-18Acid. Table 15; Table 18; FIG.49. SPV2477 (DGAT1_Vf) produced a lipid profile nearly identical to that of SPV1629, with marginally higher titers early in the fermentation. FIG. 49; FIG. 50. SPV2477 did maintain a linear increase in lipid content through the later stages of the bioconversion, leading to a final lipid fraction equivalent to SPV2473. Table 18; FIG.50. The SPV2477 data suggests either lower expression or slower kinetics of DGAT1_Vf in the Y. lipolytica background, relative to DGAT1_Mt and DGAT1_Tp. For SPV2479 (CBR1), a higher Z9-18Acid fraction was observed at 1 L confirming the phenotype observed at 1 mL scale. FIG.49. [0349] Table 18. Performance of select strains in 1 L bioconversion process at 70 hours.
Figure imgf000101_0001
[0350] Tuning the methyl palmitate feeding schedule for SPV2473 or SPV2474 resulted in increases in titer. The feeding schedule of the bioconversion process was tuned for the lipid accumulation rate of SPV1629, and both SPV2473 and SPV2474 display an increased rate of both total lipid and product lipid (all fatty acids excluding 16Acid substrate) accumulation during peak productivity between 20 and 40 hours (FIG. 51; FIG. 52). The product lipid accumulation rate does not exceed substrate loading for SPV2473, but the rate is higher than SPV1629. Towards the end of peak productivity, the product lipid accumulation rate is higher than substrate loading for SPV2474. Some of the increased lipid accumulation may be due to increased de novo synthesis which does not rely on methyl palmitate as substrate. However, increased supply of methyl palmitate increases the C16 selectivity if the storage rate of Z11-16Acid (and not the desaturation rate of 16Acid to Z11-16Acid) is limiting. Improved C16 selectivity results from increased and decreased rates of methyl palmitate loading for SPV2473 and SPV2474. [0351] SPV2473 was selected for process improvement. While the increase in Z11- 16Acid productivity is less than that of SPV2474, the decrease in SZ11/Z9-16 means the product blend for SPV2474 is further from the desired target of 97% Z11-16Ald : 3% Z9-16Ald. SPV2473 produces a 96:4 ratio of Z11-16Acid:Z9-16Acid, which is equivalent to SPV1629 selectivity. Increased lipid productivity and lipid content was achieved through increased expression of DGAT activity in these two strains. Identifying DGATs or other acyl transferases that are selective for Z11-16Acid and Z9-16Acid further improves the performance of Helicoverpa sp. blend producing strains. [0352] In summary, growth rates for the best performing strains were at least 95% of the SPV1629 growth rate in both media. Table 19. All strains exhibited two growth phases in FERM1 base medium with a true exponential growth period followed by a linear growth phase. In Solulys® base medium, all strains exhibited exponential growth. [0353] Table 19. Growth rates of strains expressing multiple acyltransferases or overexpressing native lipid metabolism genes.
Figure imgf000102_0001
Figure imgf000103_0001
[0354] Strains expressing a second recombinant diacylglycerol acyl transferase produced higher Z11-16Acid and total fatty acid titers. Two strains, SPV2473 (DGAT1_Mt) and SPV2474 (DGAT1_Tp), produced significant increases in titers, with and without methyl palmitate supplementation. Table 16. In 1 L bioconversion processes, SPV2474 had the highest Z11-16Acid and total fatty acid productivity, with lower selectivity due to increases in Z9-18Acid. Table 16. SPV2473 had improved Z11-16Acid productivity, with a selectivity equivalent to SPV1629. Table 16. Three strains overexpressing native lipid metabolism genes produced higher titers of at least one fatty acid, altering the observed fatty acid profile. Table 17. At 1 mL scale, increased titers were generally more pronounced for native C18 fatty acids. SPV2479 was selected for 1 L scale fermentation. Lower biomass was observed compared to acyl transferase and SPV1629, and relatively higher titers of C18 fatty acids were observed. [0355] Table 20 and Table 21 show complete titer and selectivity summaries for all strains (uncertainty in the selectivity measurements less than 0.01). Selectivities were equivalent or better than SPV1629 except for SZ11-16_Tot and SZ11-16_Prod of SPV2474, which were lower due to an increase in Z9-18Acid titer. Table 20; Table 21. [0356] Table 20. Bioconversion performance with 16ME supplementation.
Figure imgf000104_0001
[0357] Table 21. Bioconversion performance without 16ME substrate (glucose only).
Figure imgf000105_0001
[0358] The combined expression of date palm DGAT1A_Pd with additional recombinant DGATs leads to increases in Z11-16Acid and total fatty acids in all bioconversion processes. The two best strains resulted from stacking DGAT1_Mt or DGAT1_Tp expression on top of DGAT1A_Pd expression. The expression of DGAT1_Mt increased productivity while maintaining selectivity. Expressing DGAT1_Tp generated a less selective strain with larger productivity increases. The effect is most pronounced when methyl palmitate is not supplemented into the medium. Under this condition, the additional DGAT activity serves as a sink for fatty acid synthesis drawing flux towards lipid storage. Alternative methyl palmitate feeding schedules further improve Z11-16Acid productivity increases for SPV2473 and SPV2474. [0359] Our modifications of native lipid metabolism to achieve increased lipid accumulation successfully increased C18 fatty acid synthesis. Overexpressing the activator domain of the native lipid metabolism regulator MGA2 increased fatty acid accumulation with and without methyl palmitate supplementation. Deleting the native Y. lipolytica DGATs in (SPV2473 and SPV2474) improves product selectivity. [0360] Materials and Methods [0361] Integration vector construction: Homo sapiens codon-optimized genes of the DGAT1_Mt and DGAT1_Tp acyl transferases were subcloned from existing vectors pPV0539 and pPV1001, respectively. All other acyl transferase genes were synthesized with H. sapiens codon optimization. All acyl transferase genes were cloned into existing expression plasmid pPV1130 using SpeI and Not I sites. The resulting vectors use a TEF promoter and target the TGL1 locus. Native Y. lipolytica genes were cloned from genomic DNA. A FAD2 targeting expression vector was created by cloning homology arms from existing vector pPV1132 into expression vector pPV0601. Y. lipolytica genes were then cloned into the resulting vector, pPV1295, using AvrII and NotI cloning sites. DNA constructs were transformed into E. coli EPI400 to propagate the plasmid. E. coli EPI400 strains were grown overnight in LB at 37 °C and 250 rpm to an OD600 ≈ 2. Cells were back diluted 10x into LB with copy-cutter induction solution and grown 24 hours at 37 °C and 250 rpm before miniprepping. [0362] Table 22. Construct summary.
Figure imgf000106_0001
Figure imgf000107_0001
[0363] Strain construction: Strain construction, including plasmid digestion, transformation, check PCR, and colony patching was performed essentially as described in Example 1.1. [0364] 24-well bioconversion assay: The 24-well bioconversion assay was performed essentially as described in Example 1.1. [0365] Biolector® growth rate assay: The Biolector® growth rate assay was performed essentially as described in Example 1.1. [0366] GC processing of lyophilized samples: One-pot format GC assays and lyophilized sample processing was performed essentially as described in Example 2.1. Example 6. Increasing Z9-16, Z11-16, Z9-18, Z13-18 content while maintaining the ratio [0367] We sought to increase the productivity of our Chilo suppressalis biocatalyst. This strain produces Z9-16, Z11-16, Z9-18, and Z13-18 at a desirable ratio. We tested whether overexpressing lipid body factors would increase the total lipid content without altering the ratio. Specifically, we overexpressed MPL1 (seq ID 1001) at the POX4 locus and FIT2 (seq ID 1002) at the Alk2 locus in a background (i.e., SPV1629) that produces a desirable ratio of Z19-16, Z11-16, Z9-18, and Z13-18. The resulting strains were named SPV3315 and SPV3316, respectively (Table 23). We monitored for increased lipid content but similar precursor ratios (Table 23). Each modification increases the total lipid capacity by >20% relative to the control; the ratio of the four components remains relatively unchanged. [0368] Table 23: Overexpressing lipid body factors improves overall lipid titers by >20% while retaining nominal ratios.
Figure imgf000108_0001
[0369] Materials and methods: [0370] Strain construction: Strain construction, including plasmid digestion, transformation, check PCR, and colony patching was performed essentially as described in Example 1.1. [0371] Assay: Strains were inoculated from YPD agar plates into 2mL liquid YPD and grown at 28C and 1000rpm for 24 hours in the Infors plate shaker. Then, YPD seed culture was inoculated at OD=0.2 in 1.5mL bioprocess media in 17mm glass vials wedged in a 24-well plate. These cultures were grown up for 21 hours at 28C and 1000rpm in the Infors plate shaker. Then, 1mL from each vial was transferred to substrate plates with 17mm vials that were preloaded with 12 mg methyl palmitate, for a feeding concentration of 12 g16ME/L. OD600 measurements taken after growth phase using the Tecan reader. The substrate plates were pre-loaded by adding a methyl palmitate solution in ethanol and letting the ethanol evaporate overnight. The co-substrate plates were kept in liquid form by incubating at 37 ^C until feeding. Cultures were sampled after 48 hours of bioconversion. OD600 measurements taken. Example 7. Developing a Spodoptera frugiperda blend [0372] We sought to build a biocatalyst that makes Spodoptera frugiperda blend precursors Z9-14 and Z11-16. We overexpressed DST192G100L, a Z9-14-specific desaturase, in Yarrowia lipolytica background SPV298. The one-copy variant (SPV3212) produced greater than 3 g/L Z9-14 and approximately 0.4 g/L of the elongation product Z11-16. The two-copy variant (SPV3217) produced almost 5 g/L Z9-14 and approximately 0.8 g/L Z11-16. Notably, this biocatalyst produces these precursors in a ratio close to the optimal blend of 87:13 (Z9- 14: Z11-16) (Figure 53 and Table 24). Table 24: Table of DST192G100L-dependent analytes. Values are shown in grams per liter.
Figure imgf000109_0001
[0373] Strain construction: Strain construction, including plasmid digestion, transformation, check PCR, and colony patching was performed essentially as described in Example 1.1. [0374] Assay: Strains were inoculated from YPD agar plates into 2mL liquid YPD and grown at 28C and 1000rpm for 24 hours in the Infors plate shaker. Then, YPD seed culture was inoculated at OD=0.2 in 1.5mL bioprocess media in 17mm glass vials wedged in a 24- well plate. These cultures were grown up for 21 hours at 28C and 1000rpm in the Infors plate shaker. Methyl myristate (14ME) was added to a final concentration of 20 g/L. Example 8: Large-scale Production of Biologically Active Pheromone Blends [0375] Engineered Y. lipolytica strain SPV2777 were produced as an example of a biocatalyst for the bioconversion of methyl palmitate (MP) to mixtures of Z11-16FAME/TAG (Z11-16:1), Z9-16 FAME/TAG (Z9-16:1) and Z13-18 FAME/TAG (Z13-18:1) that serve as precursors for ingredients in a biologically active Chilo suppressalis pheromone blend. This bioconversion process was successfully used at increased scale (≥150 L). Downstream processing (DSP) of the resulting broth to dried biomass was carried out with centrifugation (Alfa Laval Clara20™), followed by spray drying (GEA Niro™ FSD 4.0). [0376] Biodesaturation of methyl palmitate with SPV2777 for blend production was performed in two 150 L Frings® reactors. The culture broth was processed to dry cell pellet, and OD600 cell-dry-weight and broth metabolites were quantified via HPLC. The lipid profile was quantified via GC-FID. [0377] Bioconversion Reactions [0378] 2 mL SPV2777 glycerol stocks were used to inoculate multiple 200 mL YPD30 (10 g/L yeast extract, 20 g/L peptone, and 30 g/L glucose) cultures in 1-L baffled shake flasks. The cultures were incubated at 28 °C and 250 rpm with 2.5 cm throw. After a 24-hour incubation, the cultures reached average OD600 values of 78 – 88, and cell dry weight values of ~20 g/L. A 1.1 L inoculum was collected from the shake flasks and used to inoculate 150 L reactors loaded with 65 L initial media. [0379] Reactions were performed with an initial temperature of 32 °C, an initial pH of 3.5, and an air flow of 36 L/min. The pH was under base-only control using 6 M NaOH. Stirring was initially set to 130 rpm with 0.35 bar overpressure. Growth under these setpoints was performed for the first 6 hours post inoculation. After 6 hours, gas flow was increased to 72 L/min, stirring was increased to 345 rpm, and the overpressure was increased to 1 bar. [0380] The first methyl palmitate addition was performed at 22 h using a peristaltic pump. Additional substrate shots were added at ~26 h, 30 h, 34 h, and 38 h. Table 25. After each substrate addition, 10 g AF204 was added for foam suppression. Additional shots of AF204 were added during the reactions as needed for foam suppression. FIG.54 shows the major fermentation parameters, DO, temperature, pH, oxygen utilization rate (OUR), feed addition and fermentor weight of these two reactions. [0381] Table 25. Methyl palmitate and AF204 additions during bioconversion.
Figure imgf000110_0001
[0382] A dissolved oxygen (DO) spike due to ammonium sulfate exhaustion occurred at 21.9 h during the reaction. The oxygen utilization rate (OUR) of these reactions reached a peak value of 129 mmol/L/h at the DO spike, and again at ~54 hours. Similar bioconversion profiles were seen across reactions for all major fermentation parameters. Foaming was persistent during the process, with 310 g and 240 g of additional AF204 added during the reaction. The majority of the additional AF204 was added after 50 hours of reaction. Foam contact of the level probe resulted in a valve closure of the line to the off-gas sensor between 38 to 43 h. This closure resulted in the apparent drop in the measured OUR. The additions of glucose, base, and ammonium sulfate were performed with the intended feed rates, resulting in addition totals of 30 kg, 5 kg, and 1.9 kg, respectively. Across both Reaction 1 and Reaction 2, an overall fermentation volume/weight increase of 1.4-fold was observed. [0383] FIG.55 shows the bioconversion profile (biomass, Z11-16:1, Z9-16:1, Z13-18:1 titers, and Z9-18:1, Z11-18:1 titers) of Reaction 1, Reaction 2 (65 L initial volume), and Reaction 3 (0.9 L initial volume). Overall, the profiles of these three reactions are very similar, with the exception of Z9-16:1 and Z9-18:1. For these two compounds, produced by the ^9-desaturase, lower accumulation is observed with the 65 L reactions compared to the lab scale reaction. [0384] The biomass accumulation of these three reactions shows the same profile reaching final biomass values of 70, 71, and 77 for Reaction 1, Reaction 2, and Reaction 3, respectively. The difference in final biomass between the lab scale reaction to the 65 L reactions is mostly due to a difference in volume increase of 23% in the lab scale reaction compared to 41% in the pilot reactions. Correcting for the volume increase, the normalized biomass (on the initial volume basis) would be 98, 99, and 95 for Reaction 1, Reaction 2, and Reaction 3, respectively. [0385] The accumulation of Z11-16:1, Z9-16:1, and Z13-18:1 across these three reactions exhibited only a difference with Z9-16:1. The identical accumulation of Z11-16 / Z9- 16 / Z13-18 between Reaction 1 and Reaction 2, which differed in MP loading (45 g/L vs.48 g/L) indicates that the additional MP was not utilized for product formation. [0386] The product produced by the action of a desaturase (Z9-16:1, Z11-16:1, and Z9- 18:1) all show an inflection point at ~38 hours between a highly productive phase (22 - 38 h) and a low product accumulation phase (38 – 60 h) For Z9-16:1 and Z11-16:1, production accumulation stalls at 48 hours, whereas Z9-18:1 continues to accumulate. This difference is likely the result of the elongase acting on Z9-16:1 and Z11-16:1, and not acting on Z9-18:1. [0387] The two compounds produced by the action of the elongase (Z11-18:1 from Z9- 16:1, and Z13-18:1 from Z11-16:1) both accumulated linearly across the bioconversion phase. The STY across the bioconversion phase for these two compounds were 0.013 g/L/h and 0.046 g/L/h. [0388] At the end of the reaction (60 h), both Reaction 1 and Reaction 2 were heat inactivated with a 1-hour hold at 70 ºC. The heating to 70 ºC took ~2 hours, and the harvest operation was completed without incident. From Reaction 1 and Reaction 2, 96.7 kg and 92.2 kg of broth was collected, respectively. The broths were combined and stirred in a jacketed (40 ºC) holding tank as feed for the centrifuge. The combined feed had a dry matter (DM) content of 12.6 wt%, a total lipid content of 3.1 wt%, and Z11-16 / Z9-16 / Z13-18 content of 1.8 wt%. An Alfa Laval Clara 20™ centrifuge was used for biomass recovery at a feed rate of 200-220 L/h and 2 min discharge cycle. [0389] The loss of biomass as indicated by the pack cell volume (PCV) of the supernatant stream at the start and end of the discharge cycle was constant at a value of ~5 % (2 mL pellet from a 40 mL sample). In total, 155 kg supernatant (7.2 wt% DM, 0.5 wt% total lipid, 0.2 wt% Z11-16 / Z9-16 / Z13-18) was collected along with 40 kg sludge (30.6 wt% DM, 8.2 wt% total lipid, 5.0 wt% Z11-16 / Z9-16 / Z13-18). This represents 103% mass balance. The higher than 100% mass balance is likely due to the additional water introduced during the discharge phase to open the bowl. [0390] Spray drying of the sludge was performed with a GEA Niro™ FSD 4.0. The inlet air had a flowrate of 360 L/h, and a temperature of 220 ºC. The air at the outlet was set to a target value of 80 ºC. The feed rate of the sludge was under feedback control to maintain the outlet temperature setpoint. An average feed rate of 21.7 kg/h was maintained over the 1.7 hours of spray drying operation. A total of 10.7 kg spray dried powder (98.6 wt% DM, 37.2 wt% total lipid, 22.5 wt% Z11-16 / Z9-16 / Z13-18) was produced with an average production rate of 6.3 kg/h. Based on the sludge feed rate, an average evaporation rate of 15 kg/h was maintained during spray drying operation. [0391] Table 26 shows the DM and lipid composition of the major DPS streams, inactivated broth, sludge, and dried biomass. The 10.7 kg final dried product with 22.5 wt% Z11- 16 / Z9-16 / Z13-18 contain 2.4 kg crude Z11-16 / Z9-16 / Z13-18. This represent 70.5% recovery of the 3.4 kg crude Z11-16 / Z9-16 / Z13-18 present in the 189 kg of inactivated broth. Based on the Z11-16 / Z9-16 / Z13-18 content of the supernatant stream, only 11% of the product loss could be attributed to centrifugation. The remaining 18.5% of product loss during DSP is likely the result of handling at pilot scale. For example, only 37 kg of the 40 kg sludge produced from centrifugation was processed through spray drying. This loss of 3 kg during handling represents an 8% product loss. [0392] Table 27. Composition of DSP materials from Reaction 1 and Reaction 2 DSP.
Figure imgf000113_0001
[0393] Table 28. Summary of overall performance of bioconversion reactions carried out in 150 L reactors.
Figure imgf000113_0002
*MP loading was varied between Reaction 1 (45 g/L) and Reaction 2 (48 g/L) [0394] Reactions performed in 150 L reactors produced 18.0 g/L and 18.3 g/L Z11- 16 / Z9-16 / Z13-18, with Z11-16:Z9-16:Z13-18 ratios of 82:8:10 and 81:9:10. While the obtained titers of the reactions performed at 150 L scale are comparable to the control reactions, the ratio of the Z11-16, Z9-16, and Z13-18 components is essentially on target (81:9:10), and the reaction time was shortened from 70 h to 60 h. [0395] Table 29. Mass balance for spray dried biomass produced from Reaction 1 and Reaction 2.
Figure imgf000113_0003
Figure imgf000114_0001
[0396] From a total of 189 kg broth, 10.7 kg spray dried biomass was produced containing 2.4 kg crude C. suppressalis blend precursors (TAG/FAME). During DSP, the largest yield loss occurred during centrifugation. Based on the Z11-16 / Z9-16 / Z13-18 content of the supernatant (0.21 wt%), a 11% loss occurred during centrifugation, which accounts for 0.3 kg of the 1.0 kg overall loss. This 11% loss is consistent with previous centrifugation trials performed with a Z11-16FAME/TAG process. The other 0.7 kg product loss was attributed to processing and handling at this scale, since the mass balance for dry matter content of the centrifugation and drying steps are 98 and 95%, respectively. Additional scaling of reactions in a 15 m3 reactor is also productive for Z11-16 / Z9-16 / Z13-18 synthesis.

Claims

CLAIMS 1. A method for producing an active blend of insect pheromones and/or precursors thereof from organic substrate, the method comprising: introducing the organic substrate into a single reaction volume, thereby catalyzing the production of the insect pheromones in an active ratio, or the precursors thereof in corresponding amounts, wherein the insect pheromones and/or precursors thereof comprised in the blend are selected from: (A) at least one compound selected from the group consisting of Z11-16Acid, Z11- 16OH, Z11-16Ald, Z11-16CoA, Z11-16Ac; (B) at least one compound selected from the group consisting of Z9-16Acid, Z9- 16OH, Z9-16Ald, Z9-16CoA, and Z9-16Ac; (C) at least one compound selected from the group consisting of Z13-18Acid, Z13- 18OH, Z13-18Ald, Z13-18CoA, and Z13-18Ac; (D) at least one compound selected from the group consisting of Z11-18Acid, Z11- 18OH, Z11-18Ald, Z11-18CoA, and Z11-18Ac; and (E) at least one compound selected from the group consisting of Z9-18Acid, Z9- 18OH, Z9-18Ald, Z9-18CoA, and Z9-18Ac, preferably wherein the pheromones and/or precursors thereof comprised in the blend are selected from: (1) A and B, (2) A, B, and C, (3) A, B, C and E, (4) A, B and E, (5) A, B, C, D, and E, and (6) C and D.
2. The method according to claim 1, wherein the active blend is active against an insect selected from the group consisting of Helicoverpa zea, Helicoverpa armigera, Scirpophaga incertulas, Chilo suppressalis, and Cnaphalocrocis medinalis. 3. The method according to claim 1 or claim 2, wherein the active blend comprises 90-99.5% Z11-16Ald and 0.5-10% Z9-16Ald; 60-90% Z11-16Ald and 10-40% Z9-16Ald; 40- 90% Z11-16Ald, 5-40% Z9-16Ald and 1-20% Z9-18Ald; 70-94% Z11-16Ald,
3-15% Z9- 16Ald, and 3-15% Z13-18Ald; 40-97% Z11-16Ald, 1-20% Z9-16Ald, 1-20% Z13-18Ald, and 1-20% Z9-18Ald; 70-99% Z13-18Ald and 1-30% Z11-18Ald; or Z11-16Ald, Z9-16Ald, Z13- 18Ald, Z9-18Ald, and Z11-18Ald, preferably wherein the active blend comprises 96-99.8% Z11-16Ald and 1-4% Z9-16Ald; 66-85% Z11-16Ald and 15-33% Z9-16Ald; 60-80% Z11-16Ald, 15-25% Z9-16Ald and 5-15% Z9-18Ald; 75-90% Z11-16Ald, 5-12% Z9-16Ald, and 5-12% Z13-18Ald; 70-80% Z11-16Ald, 5-15% Z9-16Ald, 5-15% Z13-18Ald, and 5-15% Z9-18Ald; or 85-95% Z13-18Ald and 5-15% Z11-18Ald, or more preferably wherein the active blend consists essentially of 97% Z11-16Ald and 3% Z9-16Ald; 75% Z11-16Ald and 25% Z9-16Ald; 74% Z11-16Ald, 19% Z9-16Ald and 7% Z9- 18Ald; 81% Z11-16Ald, 9% Z9-16Ald, and 10% Z13-18Ald; 74% Z11-16Ald, 7% Z9-16Ald, 9% Z13-18Ald, and 10% Z9-18Ald; or 90% Z13-18Ald and 10% Z11-18Ald.
4. The method according to any of claims 1-3, wherein the reaction volume is a bioreactor.
5. The method according to claim 4, wherein the bioreactor comprises a genetically modified microorganism comprising at least one heterologous biosynthetic enzyme selected from the group consisting of desaturases, acyl-CoA oxidases, fatty acyl-CoA reductases, enoyl-CoA hydratases, 3-hydroxyacyl-CoA dehydrogenases, conjugases, elongases, thiolases, beta-oxidation enzymes, and acyltransferases, preferably wherein the genetically modified microorganism comprises at least one polynucleotide encoding a heterologous desaturase, optionally further comprising at least one polynucleotide encoding a heterologous acyltransferase, more preferably wherein the genetically modified microorganism comprises at least two HzDST desaturase polynucleotides and at least one further polynucleotide encoding a desaturase or lysophosphatidic acid acyltransferase, even more preferably wherein the genetically modified microorganism comprises at least three HzDST polynucleotides and at least one polynucleotide encoding a DST076 desaturase, a DST183 desaturase, a DST189 desaturase, a DST192 desaturase, or a lysophosphatidic acid acyltransferase, most preferably wherein the genetically modified microorganism comprises four HzDST polynucleotides and at least one polynucleotide encoding a DST076 desaturase, a DST183 desaturase, a DST189 desaturase, a DST192 desaturase, or a lysophosphatidic acid acyltransferase.
6. The method according to claim 5, wherein the genetically modified microorganism further comprises a heterologous diacylglycerol O-acyltransferase gene, preferably wherein the heterologous diacylglycerol O-acyltransferase gene is DGAT1A.
7. The method according to claim 5 or claim 6, wherein the genetically modified microorganism is a eukaryote, preferably wherein the genetically modified microorganism is a yeast, plant cell, or insect cell, more preferably wherein the genetically modified microorganism is Yarrowia lipolytica or Saccharomyces cerevisiae.
8. The method according to any of claims 1-7, wherein the organic substrate comprises glucose.
9. The method according to any of claims 1-8, wherein the organic substrate comprises fatty acids or alkyl esters, preferably wherein the fatty acids or fatty alkyl esters are C14 or C16 fatty acids or fatty alkyl esters, preferably wherein the fatty acids or alkyl esters comprise saturated molecules.
10. The method according to any of claims 1-9, further comprising isolating the active insect pheromones and/or precursors thereof from the reaction volume.
11. A active blend of insect pheromones and/or precursors thereof produced by the method according to any of claims 1-10, (I) wherein the blend comprises: at least one Z9-16 fatty acid or fatty alkyl ester, and at least one Z11-16 fatty acid or fatty alkyl ester, preferably further comprising at least one Z13-18 fatty acid or fatty alkyl ester, or at least one Z13-18 fatty acid or fatty alkyl ester, and at least one Z9-18 fatty acid or fatty alkyl ester or at least one Z11-18 fatty acid or fatty alkyl ester; (II) wherein the blend comprises Z9-14 fatty acid or fatty alkyl ester; and/or (III) wherein the insect pheromones are produced in a active ratio through a series of reaction including the conversion of E9-14 fatty acid or fatty alkyl ester to an intermediate fatty acid or fatty alkyl ester, optionally wherein the blend comprises Z9-18 fatty acid or fatty alkyl ester, and optionally including a chain shortening product.
12. The active blend of insect pheromones and/or precursors thereof of claim 11, wherein the blend comprises 40-97% Z11-16Ald, 1-20% Z9-16Ald, 1-20% Z13-18Ald, and 1- 20% Z9-18Ald, preferably wherein the blend comprises 72-97% Z11-16Ald, 5-20% Z9-16Ald, 6-20% Z13-18Ald, and 5-20% Z9-18Ald, more preferably wherein the blend comprises 72- 77% Z11-16Ald, 5-10% Z9-16Ald, 6-12% Z13-18Ald, and 5-12% Z9-18Ald, and most preferably wherein the blend comprises 74-75% Z11-16Ald, 7-8% Z9-16Ald, 8-10% Z13- 18Ald, and 7-11% Z9-18Ald.
13. A genetically modified microorganism comprising at least one heterologous biosynthetic enzyme selected from the group consisting of desaturases, acyl-CoA oxidases, fatty acyl-CoA reductases, enoyl-CoA hydratases, 3-hydroxyacyl-CoA dehydrogenases, conjugases, elongases, thiolases, beta-oxidation enzymes, and acyltransferases, preferably wherein the genetically modified microorganism comprises at least one polynucleotide encoding a heterologous desaturase, optionally further comprising at least one polynucleotide encoding a heterologous acyltransferase, more preferably wherein the genetically modified microorganism comprises at least two HzDST desaturase polynucleotides and at least one further polynucleotide encoding a desaturase or lysophosphatidic acid acyltransferase, even more preferably wherein the genetically modified microorganism comprises at least three HzDST polynucleotides and at least one polynucleotide encoding a DST076 desaturase, a DST183 desaturase, a DST189 desaturase, a DST192 desaturase, or a lysophosphatidic acid acyltransferase, most preferably wherein the genetically modified microorganism comprises four HzDST polynucleotides and at least one polynucleotide encoding a DST076 desaturase, a DST183 desaturase, a DST189 desaturase, a DST192 desaturase, or a lysophosphatidic acid acyltransferase.
14. The genetically modified microorganism of claim 13, wherein the genetically modified microorganism overexpresses ELO2.
15. A fermentation culture comprising the genetically modified microorganism of claim 13 or claim 14.
16. A chemical synthesis apparatus for performing the method of any of claims 1-10.
17. A chemical synthesis apparatus comprising the fermentation culture of claim 15.
PCT/US2021/038775 2020-06-23 2021-06-23 Synthesis of complex insect pheromone blends and reaction apparatuses for the same WO2021262906A1 (en)

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