Production of omega-hydroxy fatty acids and derivatives thereof using engineered cells

Abstract

Methods for producing macrocyclic molecules, in particular omega-hydroxy fatty acids, are described. The methods involve the use of engineered cells having modified biochemical pathways that allow the cells to efficiently produce omega-hydroxy fatty acids from a C16 precursor. The resulting omega-hydroxy fatty acids are useful in the production of perfumes and other fragrant consumer products.

Description

Technical Field

[0001] Methods for producing macrocyclic molecules, particularly ω-hydroxy fatty acids, are described. These methods involve the use of engineered cells with modified biochemical pathways that allow them to efficiently produce ω-hydroxy fatty acids from C16 precursors. The resulting ω-hydroxy fatty acids can be used to produce fragrances and other flavored consumer products. Background Art

[0002] Ampelopsis lactone, one or more isomalopsis lactones, hexadecanoic acid, and numerous related molecules are macrocyclic molecules with excellent diffusion properties and superior musk characteristics. These fragrance molecules can be produced from C16 ω-hydroxylated (ω-hydroxy) fatty acids. Currently, most ω-hydroxy fatty acid derivatives are chemically prepared from petroleum-based starting materials or through the bioconversion of paraffin waxes. The chemical processes required to produce these compounds involve the use of hazardous organic reagents, which are energy-intensive and environmentally costly.

[0003] There is a need for safer and more environmentally friendly methods for producing macrocyclic fragrance molecules that ideally use renewable feedstocks and are more cost-effective than conventional chemical methods. Summary of the Invention

[0004] Methods for producing macrocyclic molecules, particularly ω-hydroxy (ω-hydroxy) fatty acids, using engineered cells with modified biochemical pathways are described. The resulting (ω-hydroxy) fatty acids can be used to produce fragrances and other flavored consumer products. Aspects and embodiments of these methods are described in the following independently numbered paragraphs.

[0005] 1. In one aspect, provided are genetically engineered cells having reduced fatty alcohol oxidase activity and reduced fatty acid β-oxidation compared to an otherwise identical parent cell, wherein the genetically engineered cells are engineered to reduce or eliminate the expression or activity of peroxisomal β-oxidation multifunctional enzyme type 2 (MFE2) and to reduce or eliminate the expression or activity of endogenous fatty alcohol oxidase.

[0006] 2. In some embodiments of the genetically engineered cell of paragraph 1, the fatty alcohol oxidase is fatty alcohol oxidase 1 (FAO1).

[0007] 3. In some embodiments of the genetically engineered cells of paragraphs 1 or 2, the cells are further engineered to reduce or eliminate the expression or activity of one or more diacylglycerol acyltransferases.

[0008] 4. In some embodiments of the genetically engineered cell of paragraph 3, the diacylglycerol acyltransferase is acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and / or acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) or phospholipid:diacylglycerol acyltransferase (PDAT), which is functionally and / or structurally similar to the protein or homolog or has at least 60% of the amino acid sequence.

[0009] 5. In some embodiments of the genetically engineered cells of paragraphs 1-4, the cells are further engineered to reduce or eliminate the expression or activity of peroxisomal membrane proteins.

[0010] 6. In some embodiments of the genetically engineered cells of any of paragraphs 1-5, the cells are further engineered to increase the expression or activity of one or more endogenous cytochrome P450 proteins, or to overexpress one or more exogenous cytochrome P450 proteins.

[0011] 7. In some embodiments of the genetically engineered cells of paragraphs 1-6, the strain is a Yarrowia strain.

[0012] 8. In some embodiments of the genetically engineered cell of paragraph 7, the strain is a Yarrowia lipolytica strain.

[0013] 9. In another aspect, a method for producing ω-hydroxy fatty acids from a hydrophobic substrate is provided, comprising: (a) providing a genetically engineered cell as described in any one of paragraphs 1-7; (b) culturing the strain in a suitable culture medium; and (c) contacting the strain with the hydrophobic substrate to form one or more ω-hydroxy fatty acids.

[0014] 10. In some embodiments, the method of paragraph 9 further comprises (d) isolating the one or more β-hydroxy fatty acids.

[0015] These and other aspects and embodiments of the variant molecules and methods are described below with reference to any incorporated figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The β- and ω-oxidation pathways in Yarrowia are elucidated.

[0017] Figure 2 Physical and functional maps of plasmid pYRH213 are shown.

[0018] Figure 3 Shown are the physical and functional maps of the AscI / SphI DNA fragment of plasmid pYRH213.

[0019] Figure 4 The production process of isoamyl lactone from palmitic acid is described.

[0020] Figure 5 The process of producing ambrinolide from palmitic acid is described. DETAILED DESCRIPTION

[0021] 1. Definitions and Abbreviations

[0022] Before describing these variations and methods in detail, the following terms are defined. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise. This document is organized into several sections for ease of reading; however, the reader will appreciate that statements made in one section may apply to other sections. In this manner, the headings used for the different sections of this disclosure should not be construed as limiting.

[0023] 1.1. Definition

[0024] As used herein, the term "ambroxan" refers to (3aR,5aS,9aS,9bR)-dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan, which is commercially available under the names AMBROX (Firmenich), Ambroxan (Henkel), (Givaudan), (Quest), Laevo (Firmenich), (International Flavors & Fragrances), and and / or Norambrenolide Ether (Pacific). The desirable sensory benefits of Ambroxan come from the (-) stereoisomer, not the (+) enantiomer. The (-) stereoisomer has been described as musky, woody, warm, or ambery, while the (+) enantiomer has a relatively weaker note.

[0025] As used herein, the term "activity" means the ability of an enzyme to react with a substrate to provide a target product. Activity can be determined in a so-called activity assay by an increase in target product as a function of time, a decrease in substrate (or starting material), or a combination of these parameters.

[0026] As used herein, the term "nucleic acid molecule" refers to a polynucleotide of the present disclosure, which can be DNA, cDNA, genomic DNA, synthetic DNA, or RNA, and can be double-stranded or single-stranded, sense strand and / or antisense strand.

[0027] As used herein, an "expression vector" includes a recombinant nucleic acid molecule encoding a polypeptide, including the necessary regulatory regions suitable for expression of the polypeptide.

[0028] As used herein, the terms "polypeptide" and "protein" (and their respective plural forms) are used interchangeably to refer to polymers of any length comprising amino acid residues linked by peptide bonds. Conventional one-letter or three-letter codes for amino acid residues are used herein, and all sequences are presented in the N-terminal to C-terminal direction. A polymer may contain modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that are modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a labeling component. Also included within the definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art.

[0029] As used herein, "functionally and / or structurally similar proteins" are considered "related proteins" or "homologs". Such proteins can be derived from organisms of different genera and / or species, or organisms of different classes (e.g., bacteria and fungi), or be artificially designed proteins. Related proteins also encompass homologs determined by primary sequence analysis, by secondary or tertiary structure analysis, or by immunological cross-reactivity, or by their function.

[0030] As used herein, the term "homologous protein" refers to a protein having similar activity and / or structure to a reference protein. This is not intended to imply that homologs are necessarily evolutionarily related. Thus, the term is intended to encompass one or more enzymes that are identical, similar, or corresponding (i.e., in terms of structure and function) obtained from different organisms. In some embodiments, it is desirable to identify homologs having similar quaternary, tertiary, and / or primary structures to a reference protein. In some embodiments, a homologous protein induces one or more similar immune responses as a reference protein. In some embodiments, a homologous protein is engineered to produce an enzyme having one or more desired activities.

[0031] The degree of homology between sequences can be determined using any suitable method known in the art (see, e.g., Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol., 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI); and Devereux et al. (1984) Nucleic Acids Res. 12:387-95).

[0032] For example, PILEUP is a useful program for determining sequence homology levels. PILEUP uses progressive, pairwise comparisons to create a multiple sequence alignment from a group of related sequences. It can also draw a tree showing the clustering relationship used to create this comparison. PILEUP uses a simplified version of Feng and Doolittle's progressive alignment method (Feng and Doolittle (1987) J.Mol.Evol. [Journal of Molecular Evolution] 35:351-60). This method is similar to the method described by Higgins and Sharp ((1989) CABIOS [Computer Applications in Biological Sciences] 5:151-53). Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and a weighted end gap. Another example of a useful algorithm is the BLAST algorithm, described by Altschul et al. ((1990) J. Mol. Biol. 215:403-10) and Karlin et al. ((1993) Proc. Natl. Acad. Sci. USA 90:5873-87). A particularly useful BLAST program is the WU-BLAST-2 program (see, e.g., Altschul et al. (1996) Meth. Enzymol. 266:460-80). The parameters "W", "T", and "X" determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915), an alignment (B) of 50, an expectation (E) of 10, M' 5, N' -4, and a comparison of both strands.

[0033] As used herein, the phrases "substantially similar" and "substantially identical" in the context of at least two nucleic acids or polypeptides typically mean that the polynucleotides or polypeptides described or referenced in this patent application comprise sequences that are at least about 60% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or even at least about 99% identical, or more identical, compared to a reference (i.e., wild-type) sequence.

[0034] The sequence identity percentages were calculated using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22: 4673-4680. The default parameters for the CLUSTAL W algorithm were:

[0035]

[0036] Another indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide. Typically, polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions (e.g., within a range of moderate to high stringency).

[0037] As used herein, the term "gene" is synonymous with the term "allele" and refers to a nucleic acid that encodes and directs the expression of a protein or RNA.

[0038] As used herein, the term "expressing a polypeptide" and similar terms refer to the cellular process by which the polypeptide is produced using the cell's translation machinery (eg, ribosomes).

[0039] As used herein, "overexpressing a polypeptide," "increasing expression of a polypeptide," and similar terms refer to expressing a polypeptide at a level greater than normal compared to that observed in a parental or "wild-type" cell that does not include the specified genetic modification.

[0040] As used herein, "expression cassette" refers to a DNA fragment that includes a promoter, an amino acid coding region, and a terminator (i.e., promoter::amino acid coding region::terminator), as well as other nucleic acid sequences required to allow production of the encoded polypeptide in a cell. The expression cassette can be exogenous (i.e., introduced into the cell) or endogenous (i.e., present in the cell).

[0041] As used herein, the terms "wild type" and "native" are used interchangeably and refer to a gene, protein or strain as found in nature, or which has not been intentionally modified for advantage in the present cell / strain.

[0042] As used herein, "destruction of a gene" generally refers to any genetic or chemical operation (i.e., mutation) that substantially prevents a cell from producing a functional gene product (e.g., protein) in a host cell. Exemplary destruction methods include complete or partial deletion or mutagenesis of any part of a gene (including a polypeptide coding sequence, a promoter, an enhancer, or another regulatory element), wherein mutagenesis encompasses substitutions, insertions, deletions, inversions, and combinations and variations thereof, any of which substantially prevents the generation of a functional gene product. CRISPR, RNAi, antisense, or any other method for eliminating gene expression can also be used to destroy a gene. A gene can be destroyed by the deletion or genetic manipulation of non-adjacent control elements. As used herein, "gene deletion" refers to removing the gene from the host cell genome. When a gene includes a control element (e.g., an enhancer element) that is not immediately adjacent to a gene coding sequence, the deletion of a gene refers to the deletion of a coding sequence and optionally adjacent enhancer elements (e.g., including but not limited to promoter and / or terminator sequences), but the deletion of non-adjacent control elements is not required. Gene deletion also refers to the deletion of a portion of a coding sequence, or a portion of a promoter that may or may not be immediately adjacent to a coding sequence, wherein the functional activity of the gene of interest is absent in the engineered cell.

[0043] As used herein, the terms "genetic manipulation," "genetic alteration," "genetic engineering," and similar terms are used interchangeably and refer to changes in a nucleic acid sequence. Changes may include, but are not limited to, substitutions, deletions, insertions, or chemical modifications of at least one nucleic acid in a nucleic acid sequence.

[0044] As used herein, a "functional polypeptide / protein" is a protein that has activity (e.g., enzymatic activity, binding activity, surface active properties, etc.) and has not been mutagenized, truncated, or otherwise modified to eliminate or reduce this activity. As noted, a functional polypeptide can be thermostable or thermolabile.

[0045] As used herein, a "functional gene" is a gene that can be used by cellular components to produce an active gene product (typically a protein). Functional genes are the opposite of disrupted genes that have been modified so that they cannot be used by cellular components to produce an active gene product, or have a reduced ability to be used by cellular components to produce an active gene product.

[0046] As used herein, yeast cells have been "modified to prevent the production of a specified protein" if they have been genetically or chemically altered to prevent the production of a functional protein / polypeptide exhibiting an activity characteristic of the wild-type protein. Such modifications include, but are not limited to, deletion or disruption of a gene encoding a protein (as described herein), modification of a gene such that the encoded polypeptide lacks the aforementioned activity, modification of a gene that affects post-translational processing or stability, alteration of signaling to turn on gene transcription or translation within the cell, and combinations thereof.

[0047] As used herein, the term "transformed" refers to the introduction of exogenous or heterologous DNA into a cell. The transforming DNA may or may not be integrated, ie, covalently linked, into the genome of the cell.

[0048] As used herein, the phrases "engineered cells," "modified yeast cells," or similar phrases refer to cells that include the genetic modifications and features described herein. Engineered / modified yeast does not include naturally occurring yeast.

[0049] 1.2 Abbreviations and Acronyms

[0050] Unless otherwise stated, the following abbreviations / acronyms have the following meanings:

[0051] ℃ degrees Celsius

[0052] C16 refers to a chain with a length of 16 carbon atoms

[0053] dH2O or DI deionized water

[0054] FA fatty acids

[0055]

[0056] 2. Introduction: Methods for producing C-16-derived molecules in cells

[0057] The compositions and methods of the present invention relate to genetically engineered cells that produce fragrance precursor molecules from C16 saturated or unsaturated fatty acids. These molecules can be used to produce, for example, hexadecanolide, malvaceae lactone, isomaltone, and / or other fragrance molecules. The use of engineered cells allows for the production of these precursor molecules using cost-effective fermentation processes.

[0058] The theory behind the compositions and methods is that it is necessary to engineer cells with pathways capable of producing pro-fragrance molecules to reduce or eliminate lipid storage, reduce or eliminate beta-oxidation, and reduce or eliminate conversion of ω-hydroxy fatty acids to diacids in order to effectively and efficiently produce ω-hydroxy fatty acids. The enzymatic reactions in the potential pathways are as follows Figure 1 shown.

[0059] 3. Reduce beta-oxidation in cells to increase production

[0060] Many cells produce peroxisomal β-oxidation multifunctional enzymes (MFEs), particularly MFE type 2 (MFE2), which participate in the fatty acid β-oxidation pathway, which is part of normal lipid metabolism. One aspect of the present method involves eliminating or reducing such activity to reduce fatty acid β-oxidation and increase the production of ω-hydroxy fatty acids ( Figure 1 Such engineered cells are capable of producing more C16ω-hydroxy fatty acids than otherwise identical parental cells. As shown, Yarrowia expresses six acyl-CoA oxidases (POX1 to POX6) that carry out the first step of β-oxidation, and these enzymes are substrate-specific. MFE2 is the only enzyme responsible for the second and third steps of β-oxidation.

[0061] As described in USPN 10,093,950, elimination or reduction of MFE2 activity can be combined with elimination or reduction of fatty alcohol oxidase 1 (FAO1) activity, which reduces the conversion of ω-hydroxy fatty acids to dicarboxylic acids, resulting in further increased production of ω-hydroxy fatty acids, which are precursors to more valuable flavor molecules. Alternatively, elimination or reduction of MFE2 activity is performed without eliminating or reducing fatty alcohol oxidase 1 (FAO1) activity.

[0062] 4. Mutations to Reduce Lipid Biosynthesis and Accumulation

[0063] The peroxisomal membrane protein (PMP) Pex3 and its interacting partner, cytosolic Pex19, are involved in peroxisomal membrane biogenesis. Their mechanistic mode of action remains unclear. Pex3 can also recruit other proteins to the peroxisomal membrane to influence autophagy and organelle retention. Reducing or eliminating the activity of Pex3 or functionally and / or structurally similar proteins can lead to the elimination of functional peroxisomes. Reducing or eliminating the activity of Pex5 and Pex10 is expected to produce similar results.

[0064] Eukaryotes typically express up to three different classes of diacylglycerol (DAG) acyltransferases: acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2), and phospholipid:diacylglycerol acyltransferase (PDAT). Yarrowia contains at least one homolog of each molecule. Disruption of these genes, or genes encoding functionally and / or structurally similar proteins, can significantly reduce oil biosynthesis and accumulation compared to otherwise identical parental cells.

[0065] 5. Further increase in the production of C16:0ω-hydroxy fatty acids

[0066] Further increases in ω-hydroxy fatty acid production can be obtained by overexpressing endogenous cytochrome P450 (CYP) enzymes, or by expressing more efficient exogenous CYP enzymes in the cells, which will direct the conversion of C16 fatty acids to ω-hydroxy fatty acids ( Figure 1 For this purpose, two expression cassettes are introduced into the cells, e.g. Figure 3 These cassettes incorporate slightly different codon-optimized sequences encoding CYP enzymes derived from Vicia sativa (ie, VsCYP94A1; GenBank Accession No. AAD10204; and VsCPR; GenBank Accession No. Z26252).

[0067] 6. Cells for the production of C16:1(Δ9)ω-hydroxy fatty acids

[0068] Cells with reduced or eliminated lipid storage, β-oxidation, and conversion of ω-hydroxy fatty acids to diacids can be used to efficiently produce C16:1 (Δ9) ω-fatty acids, such as by expressing or overexpressing an ω-hydroxylase complex with a Δ9 desaturase (stearoyl-CoA 9-desaturase) that converts palmitic acid to palmitoleic acid. Figure 4 ), which fatty acids can be produced from vegetable oils, vegetable oil-derived fatty acids, or fatty acid esters.

[0069] Useful Δ9 desaturases are those derived from yeast and plants. Particularly useful Δ9 desaturases are from Yarrowia (GenBank Accession No. YALI0C05951), Saccharomyces (GenBank Accession No. NP_011460), or a Δ9 desaturase having 80% amino acid sequence identity to any of these enzymes.

[0070] 7. Cells for producing C16:1(Δ7)ω-hydroxy fatty acids

[0071] Cells with reduced or abolished lipid storage, β-oxidation, and conversion of ω-hydroxy fatty acids to diacids can also be used to efficiently produce C16:1(Δ7)ω-fatty acids by co-expressing an ω hydroxylase complex with a Δ7 desaturase (i.e., palmitoyl-monogalactosyldiacylglycerol desaturase) that converts palmitic acid to (Z)-7-hexadecenoic acid. Figure 5 ), which fatty acids can be produced from vegetable oils, vegetable oil-derived fatty acids, or fatty acid esters. Useful Δ7 desaturases are those that can be efficiently expressed and are functional. Particularly useful enzymes are those derived from plants and / or algae.

[0072] Like Δ7 desaturase, monogalactosyldiacylglycerol (MGDG) synthase is present in the chloroplasts of plants and eukaryotic green algae. This enzyme catalyzes the formation of MGDG, the major structural and functional lipid in chloroplasts. Useful MGDG synthases for this application will be those that can be efficiently expressed and are functional. Particularly useful enzymes are those from plants and / or algae that produce C16:1 (Δ7) fatty acids.

[0073] 8. Additional mutations controlling cell growth

[0074] The mating type (MATa) locus of the cell includes genes responsible for inducing sporulation in diploid B / B cells, inhibiting the cell's ability to mate. Genes in the MATa locus can be disrupted to prevent sporulation.

[0075] URA3 is a conventional selectable marker used in yeast cells because it enables selection of auxotrophic transformants via 5'-FOA resistance and counterselection potential for marker removal.

[0076] 9. Suitable microorganisms for pathway engineering

[0077] Any fungal cell can be used according to the present method, in particular oleaginous yeast, or yeast engineered to be oleaginous yeast. Such yeast include, but are not limited to, Yarrowia, Candida, and Saccharomyces.

[0078] 10. Recovery of flavor molecules and precursor molecules from cells

[0079] The ω-hydroxy fatty acids and their derivatives produced by the present method can be collected, for example, by steam extraction / distillation or organic solvent extraction using a water-immiscible solvent (to separate the reaction products and unreacted substrate from the biocatalyst remaining in the aqueous phase), followed by subsequent solvent evaporation to obtain the crude reaction product, as determined by gas chromatography (GC) analysis.

[0080] The molecule can be further selectively crystallized to remove unreacted substrate from the final product. In some embodiments, the isolated crystalline material contains only the desired enantiomer. In other embodiments, the isolated crystalline material contains other isomers, wherein the isomer is only present in an olfactory acceptable amount.

[0081] Examples of suitable water-miscible and water-immiscible organic solvents suitable for the extraction and / or selective crystallization of ω-hydroxy fatty acids and their derivatives include, but are not limited to, aliphatic hydrocarbons, preferably aliphatic hydrocarbons having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane; halogenated aliphatic hydrocarbons, preferably aliphatic hydrocarbons having one or two carbon atoms, such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane or tetrachloroethane; aromatic hydrocarbons, such as benzene, toluene, xylene, chlorobenzene or dichlorobenzene; aliphatic acyclic and cyclic ethers or alcohols, preferably an ether or alcohol having 4 to 8 carbon atoms, such as ethanol, isopropanol, diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, tetrahydrofuran; or an ester, such as ethyl acetate or n-butyl acetate, or a ketone, such as methyl isobutyl ketone or dioxane or a mixture of these. The solvent that is particularly preferably used is above-mentioned heptane, methyl tert-butyl ether (also referred to as MTBE, tert-butyl methyl ether and iBME), diisopropyl ether, tetrahydrofuran, ethyl acetate and / or its mixture. Preferably, water-miscible solvent such as ethanol is used for extracting (-)-sclareolide from the solid phase of reaction mixture. The use of ethanol is advantageous because it is easy to handle, nontoxic and environmentally friendly.

[0082] 11. Application of Cell-derived ω-hydroxy fatty acid-derived molecules

[0083] Various applications of ω-hydroxy fatty acids and their derivatives include, but are not limited to, non-enzymatically produced ambrinolide and / or ambrinolide-like molecules, as well as other fragrance molecules for use in fine fragrances or consumer products such as fabric care, toiletries, beauty care, and cleaning products.

[0084] The following non-limiting examples are provided to further illustrate the present method.

[0085] Examples

[0086] Example 1: Generation of cells for the production of C16 ω-hydroxy fatty acids

[0087] Select the Figure 1 The parent strain of Yarrowia lipolytica (ATCC No. 20362) with the biochemical pathway shown in was used for genetic manipulation to engineer a strain that could produce large amounts of ω-hydroxy fatty acids.

[0088] To this end, a parent strain of Yarrowia was engineered to reduce or eliminate lipid storage, β-oxidation, and conversion of ω-hydroxy fatty acids to diacids. As summarized in Table 1, two new modified strains with unique genotypes were generated from the parent wild-type strain ATCC No. 20362. Strain AH007 has a wild-type MFE2 gene. Strain AH085 has a deleted MFE2 gene.

[0089] Table 1. Yarrowia strains used for production of ω-hydroxy fatty acids

[0090]

[0091] H. et al.

[0092] Example 2: Generation of cells for further increasing C16:0 ω-hydroxy fatty acid production

[0093] To construct a Yarrowia strain to further overproduce ω-hydroxy fatty acids, a strain containing two expression cassettes ( Figure 3 ) of artificial plasmids ( Figure 2 ) were transformed into cells of strain AH085 from Example 1 for overexpression of slightly different codon-optimized sequences encoding CYP enzymes derived from Pisum sativum (i.e., VsCYP94A1; GenBank Accession No. AAD10204; SEQ ID NO: 14 encoding SEQ ID NO: 13 and VsCPR; GenBank Accession No. Z26252; SEQ ID NO: 16 encoding SEQ ID NO: 15).

[0094] Restriction endonuclease sites were introduced upstream of the translation start codon and downstream of the stop codon of each codon-optimized sequence to enable excision of the polynucleotide including the VsCYP or VsCPR coding sequence for transformation into AH085 cells.

[0095] The resulting transformed cells were plated onto minimal medium plates and incubated at 30° C. for 2 days. A single colony from each transformation was restreaked onto MM plates. The 24 strains were directly analyzed for ω-hydroxy fatty acid production using a block assay. Specifically, a single colony was restreaked onto an MM plate and then inoculated into a 24-well block containing liquid Y2P1D2-B medium (20 g / L yeast extract, 10 g / L peptone, 20 g / L glucose, 16.37 g / L K2HPO4, 0.82 g / L KH2PO4, and 0.2 ml / L; trace metals (100X): 1 ml / L thiamine-HCl (75 mg / ml), 0.5 ml / L 1 M MgSO4-·7H2O, 1 ml / L, and 50 mg / ml kanamycin; trace metal formula (100X): 10.0 g / L citric acid, 1.5 g / L CaCl2·2H2O, 10.0 g / L FeSO4·7H2O, 0.39 g / L ZnSO4·7H2O, 0.38 g / L CuSO4·5H2O, 0.20 g / L The cultures were then shaken at 30°C and 375 rpm for 20 hours. 0.12 mL of 1 M NaHCO was added to adjust the pH of the cultures to 8.0, after which ethyl palmitate was added directly to the culture medium to a final concentration of 23 mg / mL. The cultures were then shaken at 30°C and 375 rpm for an additional 2 days, after which whole broth samples from each culture were analyzed for ω-hydroxy fatty acids according to known methods.

[0096] GC analysis proved that most transformants from these two groups of transformations produced C16ω-hydroxy fatty acids (data not shown). AH007 and AH085 only produced about 1g / L C16ω-hydroxy fatty acids. A strain named H021 derived from parent strain AH007 produced 8.1g / L C16ω-hydroxy fatty acids. Another strain named H088 derived from AH085 produced 10.1g / L. These data show that mfe2Δ deletion causes C16ω-hydroxy fatty acid levels to increase by about 25%. Similarly, the amount of C16:0 diacid produced by strain H088 also increased by>25% compared to the amount produced by strain H021.

[0097] The fatty acid compositions of whole culture broths obtained from strains H021 and H088 based on a 3-day block assay are summarized in Table 2 .

[0098] Table 2. Fatty acid composition of whole culture broth obtained from strains H021 and H088

[0099]

[0100] Example 3: (Prophetic) Generation of Cells for the Production of C16:1(Δ9)ω-Hydroxy Fatty Acids

[0101] Cells with reduced or eliminated lipid storage, β-oxidation, and conversion of ω-hydroxy fatty acids to diacids can be used to efficiently produce C16:1 (Δ9) ω-fatty acids, such as by overexpressing an ω-hydroxylase complex (Example 2) in combination with a Δ9 desaturase (stearoyl-CoA 9-desaturase) that converts palmitic acid to palmitoleic acid. Figure 4 ), which fatty acids can be produced from vegetable oils, vegetable oil-derived fatty acids, or fatty acid esters.

[0102] Example 4: (Prophetic) Production of Cells for the Production of C16:1(Δ7)ω-Hydroxy Fatty Acids

[0103] Cells with reduced or eliminated lipid storage, β-oxidation, and conversion of ω-hydroxy fatty acids to diacids can be used to efficiently produce C16:1(Δ7)ω-fatty acids, such as by co-expressing an ω-hydroxylase complex (Example 2) with a Δ7 desaturase (i.e., palmitoyl-monogalactosyldiacylglycerol desaturase) that converts palmitic acid to (Z)-7-hexadecenoic acid. Figure 5 ), which fatty acids can be produced from vegetable oils, vegetable oil-derived fatty acids, or fatty acid esters.

Claims

1. A genetically engineered cell having reduced fatty alcohol oxidase activity and reduced fatty acid β-oxidation compared to an otherwise identical parent cell, wherein the genetically engineered cell is engineered to reduce or eliminate the expression or activity of peroxisomal β-oxidation multifunctional enzyme type 2 (MFE2) and to reduce or eliminate the expression or activity of endogenous fatty alcohol oxidase.

2. The genetically engineered cell of claim 1, wherein the fatty alcohol oxidase is fatty alcohol oxidase 1 (FAO1).

3. The genetically engineered cell of claim 1 or 2, wherein the cell is further engineered to reduce or eliminate the expression or activity of one or more diacylglycerol acyltransferases.

4. The genetically engineered cell of claim 3, wherein the diacylglycerol acyltransferase is acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and / or acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) or phospholipid:diacylglycerol acyltransferase (PDAT), which is functionally and / or structurally similar to a protein or homolog or has at least 60% of the amino acid sequence.

5. The genetically engineered cell of any one of claims 1 to 4, wherein the cell is further engineered to reduce or eliminate the expression or activity of a peroxisomal membrane protein.

6. The genetically engineered cell of any one of claims 1 to 5, wherein the cell is further engineered to increase the expression or activity of one or more endogenous cytochrome P450 proteins, or to overexpress one or more exogenous cytochrome P450 proteins.

7. The genetically engineered cell of any one of claims 1-6, wherein the strain is a Yarrowia strain.

8. The genetically engineered cell of any one of claim 7, wherein the strain is a Yarrowia lipolytica strain.

9. A method for producing ω-hydroxy fatty acids from a hydrophobic substrate, the method comprising: (a) providing a genetically engineered cell according to any one of claims 1 to 7; (b) culturing the strain in a suitable culture medium; and (c) contacting the strain with the hydrophobic substrate to form one or more ω-hydroxy fatty acids.

10. The method of claim 9, further comprising (d) isolating the one or more β-hydroxy fatty acids.

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