Recombinant microbial strains comprising reduced beta-oxidation phenotypes and methods thereof

Abstract

Genetically engineered yeast cells having disruption of pex3, pex5 or pex20 genes and combinations thereof having reduced beta-oxidation and methods for increasing polyunsaturated fatty acids are disclosed.

Description

RECOMBINANT MICROBIAL STRAINS COMPRISING REDUCED BETA-OXIDATION PHENOTYPES AND METHODS THEREOFFIELD OF THE INVENTION

[0001] This invention is in the field of biotechnology, it pertains to methods useful for manipulating the beta-oxidation in eukaryotic organisms, based on disruption of peroxisome biogenesis factor (PEX) proteins. More specifically, this invention relates to microbes such as yeast, genetically engineered to have reduced or eliminated betaoxidation.BACKGROUND

[0002] Ambrettolide, iso-Ambrettolide(s), Hexadecanolide and innumerable related molecules are macrocyclic molecules that have exceptional diffusion properties and fine musk characteristics. These fragrance molecules can be produced from C16 omega- hydroxylated (co-hydroxy) fatty acids. Currently, most co-hydroxy fatty acid derivatives are made chemically from petroleum-based starting materials or through the bioconversion of paraffin. The required chemical methods for producing these compounds involve the use of hazardous organic reagents, are energy intensive and are environmentally costly.

[0003] Biotechnological approaches are needed to produce the mentioned molecules that have musk characteristics without the drawbacks of chemical processes as mentioned.

[0004] In order to understand possible biotechnological approaches in an industrially relevant microorganism, an overview of certain metabolic processes in yeast is needed.

[0005] Beta oxidation (p-oxidation) is the catabolic process by which fatty acid molecules are broken down to generate acetyl-CoA, which enters the citric acid cycle, and NADH and FADH2, which are co-enzymes used in the electron transport chain. The overall reaction for one cycle beta-oxidation (FIG. 1 ), for example starting from palmitic acid (C16:0), is as the follows:C16-acyl-CoA + FAD + NAD+ + H2O + CoA 014-acyl-CoA + FADH2 + NADH + H+ + acetyl-CoA

[0006] In yeast Yarrowia lipolytica, beta-oxidation takes place mainly in peroxisomes, that is different from most other eukaryotic organisms in which beta-oxidation happens in mitochondria. Peroxisome is a ubiquitous organelle found in all eukaryotic cells. There are more than 30 known peroxisomal proteins, also known as peroxins (PEXs), involved in peroxisome biogenesis and / or that participate in the process of importing cellular proteinsby means of ATP hydrolysis through the peroxisomal membrane. The acronym of a gene that encodes any of these proteins is “PEX gene”.

[0007] Y. lipolytica is an oleaginous yeast that can produce large amounts of oil inside the cells, wherein Y. lipolytica cells have strong beta-oxidation capability and can use fatty acid or oil as sole carbon source for its growth. However, the strong beta-oxidation capability of Y. lipolytica (FIG. 1) converts most fatty acid substrates to carbon dioxide, resulting in limited amounts of oil produced.

[0008] Hence, there exists a need for safer and more environmentally friendly methods for producing macrocyclic fragrance molecules that ideally use renewable feedstocks such as plant oils or fatty acids and are more cost effective than conventional chemical methods.

[0009] There is a need for modified yeasts, such as modified Yarrowia spp, for example modified Yarrowia lipolytica, which are modified to have a reduced, or even a blocked, fatty acid p-oxidation pathway compared to the parent non-modified yeasts.

[0010] There is a need for modified yeast cells suitable for producing dicarboxylic acids. There is a need for modified yeast cells able to have an increase of dicarboxylic acids production compared to the parent non-modified yeast cells.

[0011] There is a need for modified yeast cells suitable for producing hydroxy fatty acids. There is a need for modified yeast cells able to have an increase of hydroxy fatty acids production compared to the parent non-modified yeast cells.

[0012] The present disclosure has for purpose to meet all or part of these needs.SUMMARY OF THE INVENTION

[0013] In one example, it is disclosed a modified yeast cell having a reduction of betaoxidation activity compared to beta-oxidation activity of an otherwise identical (isogenic) parental yeast cell, wherein the modified cell comprises a genetic modification reducing or eliminating the expression, activity and / or function of one or more native peroxisome biogenesis factor (Pex) proteins encoded by one or more endogenous peroxisome biogenesis factor {PEX) genes.

[0014] In one example, it is disclosed a modified yeast cell having a reduction of betaoxidation activity compared to beta-oxidation activity of a non-modified parental yeast cell, wherein the modified cell comprises a genetic modification reducing or eliminating the expression, activity and / or function of one or more native peroxisome biogenesis factor (Pex) proteins encoded by one or more endogenous peroxisome biogenesis factor (PEX) genes.

[0015] In one example, it is disclosed a modified yeast cell having a reduction of betaoxidation activity, the modified yeast having a reduced ability to grow in an oleic acid- enriched medium compared to a non-modified parental yeast cell, wherein the modified yeast cell comprises a genetic modification reducing or eliminating the expression, activity and / or function of one native peroxisome biogenesis factor (Pex) proteins encoded by one endogenous peroxisome biogenesis factor (PEX gene.

[0016] In one example, it is disclosed a modified yeast cell having a reduction of betaoxidation activity in which less acetyl-CoA is produced as compared to beta-oxidation activity of an otherwise identical (isogenic) parental yeast cell, wherein the modified cell comprises a genetic modification reducing or eliminating the expression, activity and / or function of one or more native peroxisome biogenesis factor (Pex) proteins encoded by one or more endogenous peroxisome biogenesis factor (PEX genes.

[0017] In one example, it is disclosed a method of increasing the weight percent of at least one polyunsaturated fatty acid relative to the weight percent of total fatty acids in a yeast cell, the method comprising genetically modifying said yeast cell for reducing or eliminating the expression, activity and / or function of one or more native peroxisome biogenesis factor (Pex) proteins encoded by one or more endogenous peroxisome biogenesis factor (PEX) genes of said yeast cell.

[0018] The genetic modification results in reduction of beta-oxidation activity.

[0019] In some embodiments, the modified yeast cell may comprise more than one genetic modification reducing or eliminating the expression, activity and / or function of more than one native peroxisome biogenesis factor (Pex) proteins encoded by endogenous peroxisome biogenesis factor (PEX) genes.

[0020] In one example, it is disclosed a method of increasing the weight percent of at least one polyunsaturated fatty acid relative to the weight percent of total fatty acids in a modified yeast cell having a reduction of beta-oxidation activity in which less acetyl-CoA is produced as compared to beta-oxidation activity of an otherwise identical (isogenic) parental yeast cell, the method providing a modified yeast cell comprising a genetic modification by reducing or eliminating the expression, activity and / or function of one or more native peroxisome biogenesis factor (Pex) proteins encoded by one or more endogenous peroxisome biogenesis factor (PEX) genes; and growing the modified yeast cell so as to increase the weight percent of at least one polyunsaturated fatty acid relative to the weight percent of total fatty acids in the total lipid fraction or in the oil fraction in the modified yeast cell when compared to the weight percent of the at least one polyunsaturated fatty acid relative to the weight percent of total fatty acids in the total lipid fraction or in the oil fraction in the otherwise identical (isogenic) parental yeast cell.

[0021] In some embodiments, the genetic modification reducing or eliminating the expression, activity and / or function of one or more native PEX proteins may comprise disrupting, partially deleting, completely deleting, mutating and / or down-regulating the one or more endogenous PEXgenes.

[0022] In some embodiments, the one or more native PEX proteins may be selected from a group consisting of a PEX3 protein, a PEX5 protein and a PEX 20 protein.

[0023] In some embodiments, the one or more genes encoding the native PEX proteins may comprise a PEX3 gene.

[0024] In some embodiments, the one or more genes encoding the native PEX proteins may comprise a PEX5 gene.

[0025] In some embodiments, the one or more genes encoding the native PEX proteins may comprise a PEX20 gene.

[0026] In some embodiments, the genetic modification which eliminates or reduces the activity and / or function of the one more native PEX proteins may comprise disrupting, partially deleting and / or mutating a subsequence of a PEX gene encoding a PEX protein ATP binding site, a PEX protein transmembrane (TM) domain or a PEX protein zinc-finger domain or protein-protein interaction domain, or combinations thereof.

[0027] In some embodiments, the modified yeast cell is Yarrowia lipolytica.BRIEF DESCRIPTION OF FIGURES

[0028] The above objects and other advantages of the invention will become more readily apparent upon reading the following description and drawings, in which:

[0029] Figure 1 shows a cycle of beta-oxidation.

[0030] Figures 2A-2C show examples of the genetically modified Y. lipolytica strains.

[0031] Figure 3 shows illustrations of strain lineages.

[0032] Figure 4 shows a physical and functional map of plasmid pO4L2P3.

[0033] Figure 5 shows a physical and functional map of the Cla\ large fragment of plasmid pO4L2P3.

[0034] Figure 6 depicts the physical and functional map of plasmid pPOX4-LEU2.

[0035] Figure 7 shows physical and functional map of the C / al large fragment of plasmid pPOX4-LEU2.

[0036] Figure 8 is an illustration of pex20 deletion by Pop-in and Pop-out approach.

[0037] Figure 9 is an illustration of pex5 deletion by Pop-in and Pop-out approach.

[0038] Figure 10 depicts the physical and functional map of plasmid pY-P3.

[0039] Figure 11 shows physical and functional map of Sph\ and Asci large fragment of plasmid pY-P3.DETAILED DESCRIPTION OF THE INVENTION

[0040] The examples provided in the detailed description are merely examples and should not be used to limit the scope of the claims in any claim construction or interpretation.1. Definitions and abbreviations

[0041] Prior to describing the variants and methods in detail, the following terms are defined. Unless defined otherwise, 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 dictates otherwise. The present document is organized into a number of 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 different sections of the disclosure should not be construed as limiting.1.1. Definitions

[0042] As used herein, the term “ambrox” refers to (3aR,5aS,9aS,9bR)-dodecahydro- 3a,6,6,9a-tetramethylnaphtho [2,1-b]furan), which is known commercially as AMBROX (Firmenich), Ambroxan (Henkel) AMBROFIX® (Givaudan), AMBERLYN® (Quest), CETALOX® Laevo (Firmenich), AMBERMOR® (International Flavors and Fragrances, and AROMOR® and / or norambrenolide Ether (Pacific). The desirable sensory benefits of ambrox come from the (-) stereoisomer rather than the (+) enantiomer. The odor of the (- ) stereoisomer is described as musk-like, woody, warm or ambery whereas the (+) enantiomer has a relatively weak odor note.

[0043] As used herein, a “functional gene” is a gene capable of being used by cellular components to produce an active gene product, typically a protein. In contrast, a “nonfunctional gene” cannot be used by cellular components to produce an active gene product ( / .e., a functional protein), or has a reduced ability to be used by cellular components to produce an active gene product ( / .e., a functional protein).

[0044] As used herein, a “functional protein” is a protein that possesses a function (or activity), such as an enzymatic function / activity, a binding function / activity (e.g., DNA binding), an ATP-binding and / or hydrolysis function / activity, a surface-active property, signal transduction function / activity or transporter function / activity, and the like, and which has not been mutagenized, truncated, or otherwise modified to abolish or reduce that function / activity. In certain embodiments, a functional protein includes and relates to the ability of a particular protein to function such as normal peroxisomal function (e.g.,peroxisome membrane function). Such function includes ability of proteins to be involved in peroxisome biogenesis and / or that participates in the process of importing cellular proteins into peroxisomes.

[0045] As used herein, the term “activity” means the ability of an enzyme to react with a substrate to provide a target product. The activity can be determined in what is known as an activity test via the increase of the target product, the decrease of the substrate (or starting materials) or via a combination of these parameters as a function of time.

[0046] As used herein, the term “nucleic acid molecule,” refers to polynucleotides of the disclosure which can be DNA, cDNA, genomic DNA, synthetic DNA, or RNA, and can be double-stranded or single-stranded, the sense and / or an antisense strand.

[0047] As used, herein, an “expression vector” includes a recombinant nucleic acid molecule encoding polypeptides, including necessary regulatory regions suitable for expressing the polypeptides.

[0048] 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. The conventional one-letter or three-letter codes for amino acid residues are used herein and all sequences are presented from an N-terminal to C-terminal direction. The polymer can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0049] As used herein, “functionally and / or structurally similar proteins” are considered to be “related proteins,” or “homologs.” Such proteins can be derived from organisms of different genera and / or species, or different classes of organisms (e.g., bacteria and fungi), or artificially designed. Related proteins also encompass homologs determined by primary sequence analysis, determined by secondary or tertiary structure analysis, or determined by immunological cross-reactivity, or determined by their functions.

[0050] As used herein, the term “homologous protein” refers to a protein that has similar activity and / or structure to a reference protein. It is not intended that homologs necessarily be evolutionarily related. Thus, it is intended that the term encompass the same, similar, or corresponding proteins (e.g., enzymes; i.e., in terms of structure and function) obtained from different organisms. In some embodiments, it is desirable to identify a homolog thathas a quaternary, tertiary and / or primary structure similar to the reference protein. In some embodiments, homologous proteins induce similar immunological response(s) as a reference protein. In some embodiments, homologous proteins are engineered to produce enzymes with desired activity(ies).

[0051] 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, Wl); and Devereux et al. (1984) Nucleic Acids Res. 12:387-95).

[0052] For example, PILEUP is a useful program to determine sequence homology levels. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pair-wise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle, (Feng and Doolittle (1987) J. Mol. Evol. 35:351 -60). The method is similar to that described by Higgins and Sharp ((1989) CAB / OS 5:151 -53). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps. 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). One particularly useful BLAST program is the WU-BLAST-2 program (see, e.g., Altschul et al. (1996) Meth. Enzymol. 266:460-80). 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) alignments (B) of 50, expectation (E) of 10, M'5, N'- 4, and a comparison of both strands.

[0053] As used herein, the phrases “substantially similar’’ and “substantially identical,” in the context of at least two nucleic acids or polypeptides, typically means that a polynucleotide or polypeptide comprises a sequence that has at least about 60%% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91 % identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or even at least about 99% identity, or more, compared to the reference (i.e., wild-type) sequence described or referenced in the present patent application.

[0054] Percent sequence identity is calculated using CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the CLUSTAL W algorithm are:Gap opening penalty: 10.0Gap extension penalty: 0.05Protein weight matrix: BLOSUM seriesDNA weight matrix: IUBDelay divergent sequences %: 40Gap separation distance: 8DNA transitions weight: 0.50List hydrophilic residues: GPSNDQEKRUse negative matrix: OFFToggle Residue specific penalties: ON Toggle hydrophilic penalties: ONToggle end gap separation penalty OFF

[0055] 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 a conservative substitution. 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 medium to high stringency).

[0056] As used herein, the term “gene” is synonymous with the term “allele” in referring to a nucleic acid that encodes and directs the expression of a protein or RNA.

[0057] Various vegetative forms of yeast such as yeast of the genus Yarrowia (e.g., Yarrowia lipolytica) are generally haploid, therefore a single copy of a specified gene ( / .e., a single allele) is sufficient to confer a specified phenotype, for example, reduced betaoxidation (reduced Acetyl-CoA production).

[0058] As used herein, the term “expressing a polypeptide” and similar terms refers to the cellular process of producing a polypeptide using the translation machinery (e.g., ribosomes) of the cell.

[0059] As used herein, “over-expressing a polypeptide,” “increasing the expression of a polypeptide,” and similar terms, refer to expressing a polypeptide at higher-than-normal levels compared to those observed with parental or “wild-type cells that do not include a specified genetic modification.

[0060] As used herein, an “expression cassette” refers to a DNA fragment that includes a promoter, and amino acid coding region and a terminator ( / .e., promoter: :amino acid coding region::terminator) and other nucleic acid sequence needed to allow the encoded polypeptide to be produced in a cell. Expression cassettes can be exogenous ( / .e., introduced into a cell) or endogenous ( / .e., extant in a cell).

[0061] As used herein, the terms “wild-type” and “native” may be used interchangeably and refer to genes, proteins or strains found in nature, or that are not intentionally modified for the advantage of the presently described cells / strains.

[0062] “Otherwise identical (isogenic) parental yeast cell” and “non-modified parental yeast cell” are used interchangeably and intend to refer to a yeast cell that is genetically identical to a modified yeast cell except for the specific genetic modification or mutation introduced for the intent of the disclosure.

[0063] As used herein, the terms “modification” and “genetic modification” are used interchangeably and include, but are not limited to: (a) the insertion, substitution, or removal of one or more nucleotides in a gene or an ORF thereof, or the insertion, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of the gene or ORF thereof, (b) a gene disruption, (c) a gene deletion, (d) the down-regulation of a gene, (e) specific mutagenesis and / or (f) random mutagenesis of any one or more the genes disclosed herein. For example, genetic modification eliminates or reduces the activity and / or function of the one more native PEX proteins comprises disrupting, partially deleting and / or mutating a subsequence of a PEX gene encoding a PEX protein ATP binding site, a PEX protein transmembrane (TM) domain, and a PEX protein zinc-finger domain, and protein-protein interaction domain, and the like.

[0064] As used herein, when used to describe the expression of a gene or polynucleotide sequence, the terms "disruption", "down -regulated", "down-regulation", "inhibition", "inactivation", "silencing" and the like are used interchangeably herein to refer to instances when the transcription of the polynucleotide sequence is reduced or eliminated, which results in a reduction or elimination of protein expression derived from the polynucleotide sequence (if the gene comprised an ORF). Alternatively, down-regulation can refer to instances where protein translation from transcripts produced by the polynucleotide sequence is reduced or eliminated. Alternatively, still, down-regulation can refer to instances where a protein expressed by the polynucleotide sequence has reduced activity

[0065] Exemplary methods of disruption include complete gene knockout such that the whole gene is deleted from the genome or partial deletion of any portion of a gene, including a polypeptide-coding sequence, a promoter, an enhancer, or another regulatoryelement, or mutagenesis of the same, where mutagenesis encompasses substitutions, insertions, deletions, inversions, and combinations and variations, thereof, any of which mutations substantially prevent the production of a function gene product. A gene can also be disrupted using CRISPR, RNAi, antisense, or any other method that abolishes gene expression. A gene can be disrupted by deletion or genetic manipulation of non-adjacent control elements. The deletion of a gene refers to the deletion of the coding sequence, and optionally adjacent enhancer elements, including but not limited to, for example, promoter and / or terminator sequences, but does not require the deletion of non-adjacent control elements. Deletion of a gene also refers to the deletion a part of the coding sequence, or a part of promoter immediately or not immediately adjacent to the coding sequence, where there is no functional activity of the interested gene existed in the engineered cell. The reduction in any of the above processes (transcription, translation, protein activity) in a cell can be by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to a corresponding process in a suitable control cell. Downregulation can result from a targeting event (e.g., mdcl, knock-out, knock-in) or from using antisense or RNAi technology, for example.

[0066] As used herein, the terms “genetic manipulation,” “genetic alteration”, “genetic engineering”, and similar terms are used interchangeably and refer to the alteration / change of a nucleic acid sequence. The alteration can include but is not limited to a substitution, deletion, insertion or chemical modification of at least one nucleic acid in the nucleic acid sequence.

[0067] 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 that exhibits an activity characteristic of the wild-type protein. Such modifications include, but are not limited to, deletion or disruption of the gene encoding the protein (as described, herein), modification of the gene such that the encoded polypeptide lacks the aforementioned activity, modification of the gene to affect post-translational processing or stability, altering signal transduction to turn on gene transcription or translation inside the cell, and combinations, thereof.

[0068] “Reducing the expression, activity and / or function of a protein” intends to any method or process, and the results thereof, that decreases the production (expression) of a protein, diminishes its biological activity, or impairs its functional role within a cell. This decrease is perceptible and measurable by comparison with a non-modified parental cell. Methods for measuring a reduction of expression, activity and / or function of a protein, include, but are not limited to, Western Blotting, Quantitative PCR (qPCR), Enzymeactivity assay, Enzyme-Linked Immunosorbent Assay (ELISA), Flow Cytometry, or Reporter Assays.

[0069] “Eliminating the expression, activity and / or function of a protein” intends to any method or process, and the results thereof, that abolishes the production (expression) of a protein, its biological activity, or its functional role within a cell. This elimination is perceptible and measurable by comparison with a non-modified parental cell, and within the limits of the used measuring methods. Methods for measuring an elimination of expression, activity and / or function of a protein, include, but are not limited to, Western Blotting, Quantitative PCR (qPCR), Enzyme activity assay, Enzyme-Linked Immunosorbent Assay (ELISA), Flow Cytometry, or Reporter Assays.

[0070] As used herein, the terms “down-regulation” of gene expression and “upregulation” of gene expression include any method that results in lower (down-regulated) or higher (up-regulated) expression of a gene. For example, the down-regulation of a gene in yeast can be achieved by RNA-induced gene silencing, genetic modifications of control elements such as the promoter, untranslated regions (UTRs), codon changes, and the like.

[0071] For example, in certain embodiments, a modified strain of Yarrowia {e.g., Y. lipolytica) is constructed ( / .e., genetically modified) by use of transcriptional activator like endonucleases (TALENs), zinc-finger endonucleases (ZFNs), homing (mega) endonuclease and the like. More particularly, the portion of the gene to be modified {e.g., a coding region, a non-coding region, a leader sequence, a pro-peptide sequence, a signal sequence, a transcription terminator, a transcriptional activator, or other regulatory elements required for expression of the coding region) is subjected genetic modification by means of ZFN gene editing, TALEN gene editing, homing (mega) endonuclease and the like, which modification methods are well known and available to one skilled in the art.

[0072] In certain other embodiments, a modified strain of Yarrowia e.g., Y. lipolytica) is constructed by means of CRISPR / Cas9 editing. More specifically, compositions and methods for fungal genome modification by CRISPR / Cas9 systems are described and well known in the art (e.g., see, PCT Publication Nos: WO2016 / 100571 , WO2016 / 100568, WO2016 / 100272, WO2016 / 100562 and the like). Thus, a gene encoding a PEX protein can be disrupted, deleted, mutated, or otherwise genetically modified by means of nucleic acid guided endonucleases, that find their target DNA by binding either a guide RNA (e.g., Cas9) or a guide DNA (e.g., NgAgo), which recruits the endonuclease to the target sequence on the DNA, wherein the endonuclease can generate a single or double stranded break in the DNA. This targeted DNA break becomes a substrate for DNA repair and can recombine with a provided editing template to disruptor delete the gene. For example, the gene encoding the nucleic acid guided endonuclease (e.g., a Cas9 from S. pyogenes, or a codon optimized gene encoding the Cas9 nuclease) is operably linked to a promoter active in the Yarrowia (e.g., Y. lipolytics) cell and a terminator active in Yarrowia (e.g., Y. lipolytics) cell, thereby creating a Yarrowia (e.g., Y. lipolytics) Cas9 expression cassette. Likewise, one or more target sites unique to the gene of interest are readily identified by a person skilled in the art.

[0073] For example, to build a DNA construct encoding a gRNA-directed to a target site within the gene of interest, the variable targeting domain (VT) will comprise nucleotides of the target site which are 5' of the (PAM) proto-spacer adjacent motif (TGG), which nucleotides are fused to DNA encoding the Cas9 endonuclease recognition domain for S. pyogenes Cas9 (CER). The combination of the DNA encoding a VT domain and the DNA encoding the CER domain thereby generate a DNA encoding a gRNA. Thus, a filamentous fungal expression cassette for the gRNA is created by operably linking the DNA encoding the gRNA to a promoter active in filamentous fungal cells and a terminator active in filamentous fungal cells.

[0074] In certain embodiments, the DNA break induced by the endonuclease is repaired / replaced with an incoming sequence. For example, to precisely repair the DNA break generated by the Cas9 expression cassette and the gRNA expression cassette described above, a nucleotide editing template is provided, such that the DNA repair machinery of the cell can utilize the editing template. For example, about 500bp 5’ of targeted gene can be fused to about 500bp 3' of the targeted gene to generate an editing template, which template is used by the filamentous fungal host’s machinery to repair the DNA break generated by the RGEN (RNA-guided endonuclease).

[0075] The Cas9 expression cassette, the gRNA expression cassette and the editing template can be co-delivered to filamentous fungal cells using many different methods (e.g., protoplast fusion, electroporation, natural competence, or induced competence). The transformed cells are screened by PCR, by amplifying the target locus with a forward and reverse primer. These primers can amplify the wild-type locus or the modified locus that has been edited by the RGEN. These fragments are then sequenced using a sequencing primer to identify edited colonies.

[0076] As used herein, the term “transformed” refers to the introduction of an exogenous or heterologous DNA into a cell. The transforming DNA may or may not be integrated, i.e., covalently linked into the genome of the cell.

[0077] As used herein, the phrase “engineered cells,” “modified yeast cells” or similar phrases, refer to cells that include genetic modifications and characteristics described herein. Engineered / modified / recombinant yeast do not include naturally occurring yeast.

[0078] The common approach to reduce or eliminate beta-oxidation is to down- regulate enzymes involved in the beta-oxidation process (Fig. 1 ), especially the acyl-CoA oxidase that carries out the first reaction. However, there are six different acyl-CoA oxidases in Y. lipolytica, each has its own substrate specificity, and some also have regulatory functions (http: / / dx.doi.org / 10.1083 / jcb.200305055).

[0079] Reducing or eliminating storage oil amount from external fed fatty acids as substrates increases the yield of the produced precursors of Ambrettolide, iso- Ambrettolide(s) and Hexadecanolide. The diacylglycerol acyltransferases are the key enzymes for oil biosynthesis in Yarrowia, it includes acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1 ) and acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2). Either or both DGAT1 and DGAT2 can be downregulated to reduce oil amounts (Zhang et al., Yeast, 29: 25-38). Down-regulation of DGAT1 or DGAT2 or both can be performed using any of the strategies disclosed herein useful for such as for example (e.g., deletion, insertion, other type of mutation).

[0080] The term “peroxisomes” refers to ubiquitous organelles found in all eukaryotic cells. They have a single lipid bilayer membrane that separates their contents from the cytosol and that contains various membrane proteins essential to the functions described below. Peroxisomes selectively import proteins via an “extended shuttle mechanism”. More specifically, there are at least 32 known peroxisomal proteins, also known as peroxins, which participate in the process of importing proteins by means of ATP hydrolysis through the peroxisomal membrane. Some peroxins comprise a specific protein signal, i.e., a peroxisomal targeting signal or “PTS” at either the N-terminus or C-terminus to signal that importation through the peroxisomal membrane should occur. Once cellular proteins are imported into the peroxisome, they are typically subjected to some means of degradation. For example, peroxisomes contain oxidative enzymes, such as catalase, D- amino acid oxidase and uric acid oxidase, that enable degradation of substances that are toxic to the cell. Alternatively, peroxisomes breakdown fatty acid molecules to produce free molecules of acetyl-CoA which are exported back to the cytosol, in a process called P-oxidation.

[0081] The terms “peroxisome biogenesis factor protein”, “peroxin” and “PEX protein” are interchangeable and refer to proteins involved in peroxisome biogenesis and / or that participate in the process of importing cellular proteins by means of ATP hydrolysis through the peroxisomal membrane. The acronym of a gene that encodes any of these proteins is “PEX gene”. The terms “peroxisome biogenesis factor protein”, “peroxin” and “PEX protein” are interchangeable and refer to proteins involved in peroxisome biogenesis and / or that participate in the process of importing cellular proteins by means of ATPhydrolysis through the peroxisomal membrane. A system for nomenclature of PCX genes is described by Distel et al., J. Cell Biol., 135:1 -3 (1996).

[0082] PEX 3. The term "PEX3" herein refers to a polynucleotide sequence encoding peroxisome biogenesis factor-3 (Pex3 protein ["Pex3p"]; GenBank Acc. No. KAG5359845, YALI0F22539g). PEX3 protein is a peroxisomal integral membrane protein believed to play a role in peroxisomal membrane formation during peroxisome biogenesis (e.g., Baerends et al., J. Biol. Chem. 271 :8887-8894; Bascom et al., Mai. Biol. Cell 14:939- 957).

[0083] PEX 5. The term “PEX5” herein refers to a polynucleotide sequence encoding peroxisome biogenesis factor-5 (Pex5 protein [‘Pex5’], GenBank Acc. No. KAG5370090; YALI0F28457g). PEX5 is a peroxisomal targeting signal receptor; it binds to the C-terminal PTS1 -type tripeptide peroxisomal targeting signal (SKL-type) and plays an essential role in peroxisomal protein import (Szilard and Rachubinski, Biochem. J. 346 (Pt 1 ): 177-184).

[0084] PEX 20. The term “PEX2CT herein refers to a polynucleotide sequence encoding peroxisome biogenesis factor-20 (Pex20 protein [‘Pex20’]; GenBank Acc. No. XP_503644; YALI0_E06831 g). PEX20 is a coreceptor required for the peroxisomal import of proteins, it contains a C-terminal PTS2-type peroxisomal targeting signal, such as 3- oxoacyl-CoA thiolase (Vladimir et al., J. Cell Biol. 142: 403-420; Chang & Rachubinski, Traffic 20: 504-515).

[0085] In one example, the PEX3, PEX5, and PEX20 individually or combining two of them, or all three of them in order to downregulate or delete can be useful strategies to reduce or eliminate beta-oxidation. Down-regulation of PEX3, PEX5 or PEX20, or combining two of them or all three together for downregulation or deletion can be performed using any of the strategies disclosed herein useful for such as for example (e.g., deletion, insertion, other type of mutation).

[0086] The disruption may occur in a PEX gene that encodes a peroxisome biogenesis factor protein that includes the all following PEXs, and two or three of their different combinations: Pexl p, Pex2p, Pex3p, Pex3Bp, Pex4p, Pex5p, Pex5Bp, Pex5Cp, Pex5 / 20p, Pex6p, Pex7p, Pex8p, Pex10p, Pex12p, Pex13p, Pex14p, Pex15p, Pex16p, Pex17p, Pex14 / 17p, Pex18p, Pex19p, Pex20p, Pex21 p, Pex21 Bp, Pex22p, Pex22p-like and PeX26p (US 9,617571 B2). And in any of these methods, the disruption may be a gene knockout or a deletion in a portion of the gene. Described herein is also a pex- disrupted Y. lipolytica, having a disruption in a native gene encoding Pex3p or Pex5p or Pex20p, or Pex3P and PEX5P, or Pex3P and Pex20P, or Pex3p and pex5p and Pex20p together. US Patent 9,617,571 and US Patent 10,626,424, the disclosures of each ofwhich is incorporated by reference, further describe certain genetic modifications of native genes encoding one or more PEX proteins.

[0087] Certain embodiments of the disclosure provide methods for increasing the weight percent of at least one polyunsaturated fatty acid relative to the weight percent of total fatty acids in a modified yeast cell having a reduction of beta-oxidation activity in which less acetyl-CoA is produced as compared to beta-oxidation activity of an otherwise identical (isogenic) parental yeast cell. In certain aspects, such methods include providing a modified (recombinant) yeast cell comprising a genetic modification of one or more native peroxisome biogenesis factor proteins (PEX) encoded by one or more endogenous peroxisome biogenesis factor genes (PEX and growing the modified yeast cell so as to increase the weight percent of at least one polyunsaturated fatty acid relative to the weight percent of total fatty acids in the total lipid fraction or in the oil fraction in the modified yeast cell when compared to the weight percent of the at least one polyunsaturated fatty acid relative to the weight percent of total fatty acids in the total lipid fraction or in the oil fraction in the otherwise identical (isogenic) parental yeast cell.

[0088] As the following will show, the specification discloses an approach to reduce beta-oxidation by disrupting one, or two, or three Peroxisome Biogenesis Factor Proteins (PEX proteins) Disruption of 2 PEX proteins is more effective than disrupting one PEX protein; and disrupting three PEX proteins is the most effective way to reduce betaoxidation. Applicants genetically engineered Y. lipolytica to produce the precursors of Hexadecanolide, Iso-Ambrettolide (Z9), and Ambrettolide (Z7) musk molecules from saturated or monounsaturated C16 fatty acids which needs to be reduced or to have betaoxidation eliminated.

[0089] The following table provides a description of the various Y. lipolytica yeast Strains.Table 1Y. lipolytica Strains

[0090] The following example describes generation of an AH291 (control) strain with genotype as: MATA, dgatl-, dgat2-, and ura3-.

[0091] For example, Yarrowia strains shown in Table 1 with a disruption of a single pex3 gene (AH246), pex5 gene (AH299) or pex20 gene (AH292) can still grow on plates with palmitate as a sole carbon source and produce a good amount of biomass at 72 hours, suggesting that these strains have some beta-oxidation capabilities, and the time courses of their growth are shown in Figures 2A-2C. Figure 2A shows strain growth for 24 hours. Figure 2B shows strain growth for 48 hours. Figure 2C shows strain growth for 72 hours.

[0092] Figure 3 shows an illustration of strain lineages, as the following examples will show.EXAMPLESEXAMPLE 1 : Generation of strain AH291 from strain AH1314 (2738Y-45) for restoration of LEU2, PEX3 and P0X4

[0093] The control strain AH291 (dgatl-, dgat2, ura3-) was generated from strain AH1314 (known as strain 2738Y-45 in US 10,626,424 B2) to restore the following three genes, (1) LEU2 (beta-isopropylmalate dehydrogenase involved in leucine biosynthesis, GenBank Acc. No. M37309; YALI0C00407g, SEQ ID NOs: 2 & 3); (2) PEX3 (SEQ ID NOs: 4 & 5, and (3) POX4 (Acyl-CoA oxidase 4, GenBank Acc. No. AJ001302, YALI0E27654g, SEQ ID Nos: 6 & 7) back to their wild types (Table 1 , Figs. 2 & 3).

[0094] Construct pO4L2P3 (Fig. 4; SEQ ID NO: 1 , Table 2) contains wild type genes of LEU2, PEX3 and POX4, the components of the Cla\ large fragment of plasmid pO4L2P3, used for generation of strain AH291 , are further described in Table 2 and Fig. 5.Table 2- Description of the C / al DNA fragment of Plasmid pO4L2P3 (SEQ ID NO: 1, Fig. 4) integrated into the genome of Strain AH291

[0095] The DNA sequence of the plasmid pO4L2P3 is shown, below, as SEQ IDNO: 1 :

[0096] ATCGATACTTGTAGACATCTACACACTGATGCTCCCAATCGAATATTTTCTACCTACTGTAGAGCACTTGAAACACTTGTCTTGGCGGTACAACAGTAATACAAGTACAAGTACAGTATATACAGTACATACAAGTATATTAGCTACAGTATGTACATACAGAATATCCATATCTGCTTGGGTCTCGAAGGTGACTCCACAGCAAGACCAGAAGCACAGAAGGGCGACATGATCCATCGAGTAAAAGATAACTTTTTCTAAGAGAGTGGAATATCCAAACTGATCCTCCAGCTTCCTCAGACGTCACACTTCGCCACTATATTGTTCAAATACTTTCCCCTTAGTAAACCTCGGTTTACAGTTGAACTATTACTTCAATTTTATTGAGCGGGGCTACACAACGCTGTGTCCACCGTTCTCCTTCATACAGTAGCCTATAACATAACACGACTGATTGCTTCAATTTGATCAAGAACAAACCGTCGATCACCACAGCATTTACCTGAAGTGTGTAAGGTAGCACACATCGTACGCGCAGACTAAAATTATCCAACCAAGTTTCCCCATGTCCACACGGAGATGTGGGAGCTGAAAAATGATGGACTGTTTTTTTTTCTTTTTCTGCATATACATTCATTTAATGTAGTATTATCCGAATGGGTGTGCCATTGTGATGTGATTATTCATCAGTCACAAGTTCGTCGCAACGGCGAGACTTTCATACAGGCACCATAAAACTGAAACGTGCAAATTAGGTCGTAATAACTCTTCATTGCCTCCTCATCTCTTCAGATCAAGCCCTTGGAGAACATCCTTGTATTTACTGTACTGTACCATCCGGACCCAATTTTTAGGCTTCTATGCACTCACCCGTGTTTCGTCTAAAAGTCACAGATCCCATACACTTGCAACATCTGTTGAGAAGCCCATTGTATATTATTAGGATCGTAGCATTATTGTGGCAAAAAATATTCAAGTGCTCATGTGAATTGACACGATCACGTAAATACCTGGTGAAATTGCTAGTATTCGTGATGTTCTAATACAACTCTGTTCAATATTTCCGGCGCTCTCTTGTATACAAGAGCACAAGACATGCACCCCACATTAACCGAGGTCAAGTGTTTATGTATGAAAAGTGACATAAATCGTCCAAAAAAAAGTAGCACATAGTTGTATGGCTGTAAGTTATGTGATTGTCAGTTCTTCGGCCTTCCAACTCCTATGCACCGTCTTCAATCATCTACCCCCGTGCCCCACACCCCGCACTATTAGAGTTTATCACAGTCAGCTAAACTGCTTGCACATCTACACCTCTGACTACACCACCATGGATTTCTTCAGACGGCACCAGAAAAAGGTGCTGGCACTGGTAGGTGTGGCGCTGAGTTCCTACCTGTTTATCGACTATGTGAAGAAAAAGTTCTTCGAGATCCAGGGTCGTTTGAGCTCGGAGCGAACCGCTAAACAGAATCTCCGGCGCCGATTTGAACAGAACCAGCAGGATGCAGATTTTACAATCATGGCTCTGCTATCCAGCTTGACGACACCGGTAATGGAGCGTTACCCCGTCGACCAGATCAAGGCAGAGTTACAGAGCAAGAGACGCCCCACAGACCGGGTTTTGGCTCTCGAGAGCTCCACCTCGTCCTCAGCTACCGCACAAACCGTGCCCACCATGACAAGTGGCGCCACAGAGGAGGGCGAGAAGTCGAAAACACAGTTGTGGCAGGATCTCAAGCGAACGACCATTTCCCGAGCGTTTTCTCTTGTCTATGCAGATGCACTTCTTATTTTCTTCACGCGTTTGCAGCTCAACATTCTAGGACGACGAAACTACGTCAACAGTGTTGTCGCTCTGGCGCAGCAGGGCCGAGAGGGTAATGCCGAGGGTCGAGTGGCGCCCTCGTTTGGTGATCTTGCAGATATGGGCTATTTCGGCGACCTTTCAGGCTCGTCCAGCTTCGGAGAAACTATTGTCGATCCCGATCTGGACGAACAGTACCTTACCTTTTCGTGGTGGCTGCTGAACGAGGGATGGGTGTCGCTGAGCGAGCGAGTGGAGGAAGCGGTTCGTCGAGTGTGGGACCCCGTGTCACCCAAGGCCGAACTTGGATTTGACGAGTTGTCGGAACTGATTGGACGAACACAGATGCTCATTGATCGACCTCTCAATCCCTCGTCGCCACTCAACTTTCTGAGCCAGCTGCTGCCACCACGGGAGCAGGAGGAGTACGTGCTTGCCCAGAACCCCAGCGATACTGCTGCCCCCATTGTAGGACCTACCCTCCGACGGCTTCTGGACGAGACTGCCGACTTCATCGAGTCCCCTAATGCCGCAGAGGTGATTGAGCGACTTGTTCACTCCGGTCTCTCTGTGTTCATGGACAAGCTGGCTGTCACGTTTGGAGCCACACCTGCTGATTCGGGTTCGCCTTATCCTGTGGTGCTGCCTACTGCAAAGGTCAAGCTGCCCTCCATTCTTGCCAACATGGCTCGACAGGCTGGAGGCATGGCCCAGGGATCGCCGGGCGTGGAAAACGAGTACATTGACGTGATGAACCAAGTGCAGGAGCTGACCTCCTTTAGTGCTGTGGTCTATTCATCTTTTGATTGGGCTCTCTAGAGGCTCATTCACGAAAGACACGAAGAACGAAGATGGGGACTGAATACAGCGCTCTCATTTGTACACAAATGATTTATGACAGAGTAACTTGTACATCATGTAGAGCATACATACTGAAGGTGTGATCTCACGGGATATCTTGAAGACCACTCGTAGCTGGAGGCATAGGTAGTGCTAGTACGGATACTTGCACCGTATCCAACATAAGTAGAGGAGCCTCCTAGTGGCTATTGGTACACCGATAAAGATACACATACATGTTATTGCCACTTGTATCTACCAGTACCCGTACTGTATGTACTTGTACAGCACTTACTGTTACTTACATGACCAATGCCTTAGATCGAAATATTTCTGATCAAAGTAGAACCAGGCGTAAGACGAAAATGGACAGGTTTTACCGAAAGCCCACGTGAACAGGTTACTTTCTCAAACCTTGGAGAGAGTGGAGAGCCAGTCACCTGAAACAGAACTCTCATCCTCATTCATCGTACCGTACGTACTTGTACGAGTACATGCCTGGCACAGTCTCATTCTATCCTAACACAATGTACAATGTACAACATTTTTCGGCTCGGCCATGCAGCTTCGGAGACACGCTCGTGAAAAAGGGTCGGACTAATCAACCTCACCGTTGCGTTAGGCTGCCAGGTGAAACAATATTATTTTGACAGCTAGAAAGTCCGGAGATGACACTGGGGTAACGGAACTACTGTACTTCGCACAAGTCGGCGTGAGTTTGTGGTAGTTTATATGAGTAGTTAGCTATAGTCATGCCCCATTATTTGATAATCGGATCACACACGCTGAATAATTACACTGGTCTTATTTGTGCTATCATTCCTACTACCTCCTTCTGTATAATAATACACCTTCGCCGTAAAATGTGGAGAAGAAATCGGCACTAAAAAGTCAGGTAGACTGGAAAATGCGCCATGAAATGAATATCTCTTGCTACAGTAATGCCCAGCATCGAGGGGTATTGTGTCACCAACACTATAGTGGCAGCTGAAGCGCTCGTGATTGTAGTATGAGTCTTTATTGGTGATGGGAAGAGTTCACTCAATATTCTCGTTACTGCCAAAACACCACGGTAATCGGCCAGACACCATGGATGTAGATCACCAAGCCTGTGAATGTTATTCGAGCTAAAATGCACATGGTTGGTGAAAGGAGTAGTTGCTGTCGAATTCCGTCGTCGCCTGAGTCATCATTTATTTACCAGTTGGCCACAAACCCTTGACGATCTCGTATGTCCCCTCCGACATACTCCCGGCCGGCTGGGGTACGTTCGATAGCGCTATCGGCATCGACAAGGTTTGGGTCCCTAGCCGATACCGCACTACCTGAGTCACAATCTTCGGAGGTTTAGTCTTCCACATAGCACGGGCAAAAGTGCGTATATATACAAGAGCGTTTGCCAGCCACAGATTTTCACTCCACACACCACATCACACATACAACCACACACATCCACAATGGAACCCGAAACTAAGAAGACCAAGACTGACTCCAAGAAGATTGTTCTTCTCGGCGGCGACTTCTGTGGCCCCGAGGTGATTGCCGAGGCCGTCAAGGTGCTCAAGTCTGTTGCTGAGGCCTCCGGCACCGAGTTTGTGTTTGAGGACCGACTCATTGGAGGAGCTGCCATTGAGAAGGAGGGCGAGCCCATCACCGACGCTACTCTCGACATCTGCCGAAAGGCTGACTCTATTATGCTCGGTGCTGTCGGAGGCGCTGCCAACACCGTATGGACCACTCCCGACGGACGAACCGACGTGCGACCCGAGCAGGGTCTCCTCAAGCTGCGAAAGGACCTGAACCTGTACGCCAACCTGCGACCCTGCCAGCTGCTGTCGCCCAAGCTCGCCGATCTCTCCCCCATCCGAAACGTTGAGGGCACCGACTTCATCATTGTCCGAGAGCTCGTCGGAGGTATCTACTTTGGAGAGCGAAAGGAGGATGACGGATCTGGCGTCGCTTCCGACACCGAGACCTACTCCGTTCCTGAGGTTGAGCGAATTGCCCGAATGGCCGCCTTCCTGGCCCTTCAGCACAACCCCCCTCTTCCCGTGTGGTCTCTTGACAAGGCCAACGTGCTGGCCTCCTCTCGACTTTGGCGAAAGACTGTCACTCGAGTCCTCAAGGACGAATTCCCCCAGCTCGAGCTCAACCACCAGCTGATCGACTCGGCCGCCATGATCCTCATCAAGCAGCCCTCCAAGATGAATGGTATCATCATCACCACCAACATGTTTGGCGATATCATCTCCGACGAGGCCTCCGTCATCCCCGGTTCTCTGGGTCTGCTGCCCTCCGCCTCTCTGGCTTCTCTGCCCGACACCAACGAGGCGTTCGGTCTGTACGAGCCCTGTCACGGATCTGCCCCCGATCTCGGCAAGCAGAAGGTCAACCCCATTGCCACCATTCTGTCTGCCGCCATGATGCTCAAGTTCTCTCTTAACATGAAGCGCGCCGGTGACGCTGTTGAGGCTGCCGTCAAGGAGTCCGTCGAGGCTGGTATCACTACCGCCGATATCGGAGGCTCTTCCTCGACCTCCGAGGTCGGAGACTTGTTGCCAACAAGGTCAAGGAGCTGCTCAAGAAGGAGTAAGTCGTTTCTACGACGCATTGATGGAAGGAGCAAACTGACGCGCCTGCGGGTTGGTCTACCGGCAGGGTCCGCTAGTGTATAAGACTCTATAAAAAGGGCCCTGCCCTGCTAATGAAATGATGATTTATAATTTACCGGTGTAGCAACCTTGACTAGAAGAAGCAGATTGGGTGTGTTTGTAGTGGAGGACAGTGGTACGTTTTGGAAACAGTCTTCTTGAAAGTGTCTTGTCTACAGTATATTCACTCATAACCTCAATAGCCAAGGGTGTAGTCGGTTTATTAAAGGAAGGGAGTTGTGGCTGATGTGGATAGATATCTTTAAGCTGGCGACTGCACCCAACGAGTGTTCTTTTGCCGATACATATGCACCATTAAATGATTAGAATGCGGGGATTGAGTTTCGGATTTTTCTTACATGCGTTGCGCGTTGGAGGTGATCCGAGCAGGTGGAATAGGTGTATATTGAGCGATTGGGAGAGTTGGTTGTGTACAATTATTTTAATACCTCTTCTGATTGTTTTCTATTGCCTTCCATTTCTATCTTTACCTGCCATCTCACGTCGTGTGTACCATCCCCACATACGGAACCAGTAGGTCTTTTAGGCTCTGAACGTGCAAATGAGTTTGGTGGGGTAGGCAGAGATCGCATAGAGACGGGTAGAATGAGCAGTTAAAAGCTGTGTTGAGTGGTAAAAATTTACAATAAGTGTTCCTCAAGGGATCAAGGAGACGAAATAAGCCATTATGGACACGAACCAACAGTCCCACCACGTTCTAAACACATTCCTCCACTGCCACTCCCAAACACCACGTCCCACATAAACTTCTACCCCACATTTTGACAAGCCTATTCGTTTAATAATCACCCCGAGGAGACAGAAAGCCTAACAGCTGGAGCCACTATATAGTTGCAGTGGCATGCTGTTTGATTGAAACGACCCACAACAATGATCAGCCCAAACCCCGCTAACGACATTGTCCATGACGGCAAGCTCTACGACACCTTCACTGAGCCCCCCAAGCTGATGGCTCAGGAGCGAGCTCAGCTGGACTTCGACCCTAGAGACATCACCTACTTTCTGGATGGCTCTAAGGAGGAGACCGAGCTGCTGGAGTCGCTCATGCTCATGTACGAGCGAGACCCTCTCTTCAACAACCAGAACGAGTACGATGAATCGTTTGAAACACTGCGAGAGCGATCTGTGAAGCGAATTTTCCAGCTGTCCAAGTCCATCGCCATGGACCCCGAGCCCATGTCTTTCCGAAAGATTGGGTTCCTGGGTATTCTTGACATGGGAACGTATGCTCGACTGGGAGTCCACTACGCGCTCTTCTGTAACTCCATCCGGGGCCAGGGAACCCCCGATCAGCTCATGTACTGGCTGGACCAGGGAGCCATGGTCATCAAGGGCTTCTACGGCTGTTTTGCCATGACCGAAATGGGCCATGGATCTAACCTGTCGCGTCTGGAAACCATCGCCACTTTCGACAAAGAGACCGACGAATTTATCATTAACACGCCCCACGTTGGAGCCACAAAGTGGTGGATTGGAGGAGCCGCCCACACTGCTACTCACACACTTGCCTTTGCCCGTCTTCAAGTAGACGGAAAGGACTACGGTGTGAAATCGTTTGTCGTACCTCTCCGAAACCTGGACGACCATTCGCTGCGTCCTGGAATCGCCACAGGTGATATTGGTAAGAAGATGGGTCGAGATGCCGTTGACAACGGCTGGATTCAGTTCACCAACGTCCGAGTGCCCCGAAACTACATGCTCATGAAGCATACCAAGGTTCTTCGAGACGGTACCGTCAAGCAGCCGCCTTTGGCCCAACTGACTTACGGATCTCTCATCACTGGACGAGTCCAGATGACCACTGACTCTCACAATGTGTCCAAAAAGTTCCTCACCATTGCCCTGAGATACGCCACCATCCGACGACAGTTCTCGTCAACTCCAGGAGAGCCCGAAACCCGACTAATTGACTACCTGTACCACCAAAGACGACTCCTGCCTCTTATGGCTTACTCTTACGCCATGAAACTAGCTGGAGATCACGTCCGAGAGCTGTTCTTTGCATCCCAGGAGAAGGCTGAGAGCCTCAAGGAGGACGACAAAGCCGGAGTTGAGTCTTACGTCCAGGATATCAAGGAGCTCTTCTCTGTTTCTGCTGGTCTCAAGGCTGCCACTACATGGGCTTGTGCTGACATCATTGACAAGGCCCGACAGGCGTGTGGAGGCCACGGATACTCTGCCTACAACGGCTTTGGACAGGCCTTCCAGGACTGGGTTGTCCAGTGCACTTGGGAGGGTGACAATACTGTTCTGACTCTATCTGCCGGCCGAGCTCTGATCCAATCTGCTCTCGTCTACCGAAAGGAGGGCAAACTAGGTAACGCCACGAAGTACCTCTCTCGGTCCAAGGAGCTTGCCAACGCCAAGAGAAACGGACGATCCCTGGAAGACCCCAAGCTGCTCGTGGAGGCATGGGAGGCTGTCTCTGCCGGTGCTATCAACGCTGCTACTGACGCTTACGAGGAGCTCTCCAAGCAGGGAGTTTCTGTTGACGAGTGCTTTGAGCAGGTGTCCCAGGAGCGATTCCAGGCTGCCCGAATCCACACTCGACGAGCTCTTATCGAGGCCTTCTACTCACGAATCGCCACTGCTGATGAGAAGGTGAAGCCTCATCTGATCCCTCTGGCCAACCTGTTTGCCCTGTGGTCCATTGAGGAGGACTCTGCTGTGTTCCTGGCTGAGGGCTACTTTGAGCCTGAGGATATCATTGAGGTGACTTCTCTTGTCAACAAGTACTGCGGAATTGTTCGAAAGAACGTTATTGGATACACCGATGCCTTCAACCTGTCCGACTACTTCATCAACGCTGCCATTGGACGATACGACGGAGACGTGTACAAGAACTACTTTGAGAAGGTCAAACAGCAGTACGCTCCTGAGGGTGGCAAGCCTCACTACTACGAGGATGTCATGAAGCCCTTCCTGCATCGAGAGCGAATTCCCGATGTCCCCATGGAGCCCGAGGATATTCAGTAATGATTTTGTAGGTTTTAGTAAGTATTAACGTTTTTTATGATTTGGAGTTGTGTGTAACTTGTACAGGTACACCTACATACTGTACTGTAGGTCCAAAGATAGGTACACTGTGGCAATAATTATGCGAGTACTTGTACCGTCATCGTAGCTGCTGTAAAGAGATCAGACACAGGCACTTTTCCCCACCATGAGATCACCACTCGTCGTCCGAGTACTTCTATGGCACAGCCACAATCACATGTACTTGTGCATGCCAATGTGTGACATCATCATCTAGAGCTATCATCATATTCCCGCTGCAAATGGTCTACGTATTACTATTAAGCAGGGGGGGGGGAGGAATTATGACGACATTGTACGTGTACTCGTACCGGTACTTGTAGCACGCCGAACTGCGGTATTACTGTGCACTGTAATTTCGGACCCCTCTTATAGCCCCAAGTTGGTCTATACATCTGAACCGGTGCAGACTCACTATTAAAAGTGCGGCAGCTAATTTTGCTGACACAGCCTTGTCGATAAAAGTAGCTACTTGTACGATGTACTCTTGTAGCTTATGCGTGGGCTATCATTAATTTTAGACAACAGTGTCCTACCACGCCACGGAACGGGAGAAAGCAGAAGATCCGAACATCTTGTGCGGAGTGTGTTGCGTCTGTGACACCGGTGAAGTTCCGTTGTCCATGACCCGCCGATCTTTTTTTTGGCATATGGTTTGCATCGCCTCGCAGACAAGAAAGGGCGAAGGTAAATGCAGCACATGTTAGAAGTGCCTGGCATGGGACATACACGCGGACGTTTGTGCAGTTGTCAGCAGCGTGTTTATCGATTCTGGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTGTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTT TGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGGCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTT TGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGT ATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCAT GTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGT TGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCA TGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAG AATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACC GCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCG AAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTG CACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAA CAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACA TTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTT AAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCT TATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGCGCTCC CATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGGTGCGGGCCTCT TCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGA CGTAATACGACTCACTATAGGGCGAATTGGCGGAAGGCCGTCAAGGCCGCATT

[0097] The amino acid sequence of the Y. lipolytica LEU2 (GenBank Acc. No. M37309, YALI0C00407g) polypeptide is shown, below, as SEQ ID NO: 2:

[0098] MEPETKKTKTDSKKIVLLGGDFCGPEVIAEAVKVLKSVAEASGTEFVFEDRLIGGAAIEKEGEPITDATLDICRKADSIMLGAVGGAANTVWTTPDGRTDVRPEQGLLKLRK DLNLYANLRPCQLLSPKLADLSPIRNVEGTDFIIVRELVGGIYFGERKEDDGSGVASDTE TYSVPEVERIARMAAFLALQHNPPLPVWSLDKANVLASSRLWRKTVTRVLKDEFPQLEL NHQLIDSAAMILIKQPSKMNGIIITTNMFGDIISDEASVIPGSLGLLPSASLASLPDTNEAFGLYEPCHGSAPDLGKQKVNPIATILSAAMMLKFSLNMKPAGDAVEAAVKESVEAGITTA DIGGSSSTSEVGDLLPTRSRSCSRRSKSFLRRIDGRSKLTRLRVGLPAGSASV

[0099] The DNA sequence of the Y. lipolytica LEU2 (GenBank Acc. No. M37309, YALI0C00407g) is shown, below, as SEQ ID NO: 3:

[0100] ATGGAACCCGAAACTAAGAAGACCAAGACTGACTCCAAGAAGATTGTTCTTCTCGGCGGCGACTTCTGTGGCCCCGAGGTGATTGCCGAGGCCGTCAAGGTGCTCAAGTCTGTTGCTGAGGCCTCCGGCACCGAGTTTGTGTTTGAGGACCGACTCATTGGAGGAGCTGCCATTGAGAAGGAGGGCGAGCCCATCACCGACGCTACTCTCGACATCTGCCGAAAGGCTGACTCTATTATGCTCGGTGCTGTCGGAGGCGCTGCCAACACCGTATGGACCACTCCCGACGGACGAACCGACGTGCGACCCGAGCAGGGTCTCCTCAAGCTGCGAAAGGACCTGAACCTGTACGCCAACCTGCGACCCTGCCAGCTGCTGTCGCCCAAGCTCGCCGATCTCTCCCCCATCCGAAACGTTGAGGGCACCGACTTCATCATTGTCCGAGAGCTCGTCGGAGGTATCTACTTTGGAGAGCGAAAGGAGGATGACGGATCTGGCGTCGCTTCCGACACCGAGACCTACTCCGTTCCTGAGGTTGAGCGAATTGCCCGAATGGCCGCCTTCCTGGCCCTTCAGCACAACCCCCCTCTTCCCGTGTGGTCTCTTGACAAGGCCAACGTGCTGGCCTCCTCTCGACTTTGGCGAAAGACTGTCACTCGAGTCCTCAAGGACGAATTCCCCCAGCTCGAGCTCAACCACCAGCTGATCGACTCGGCCGCCATGATCCTCATCAAGCAGCCCTCCAAGATGAATGGTATCATCATGACCAGCAACATGTTTGGCGATATCATCTCCGACGAGGCCTCCGTCATCCCCGGTTCTCTGGGTCTGCTGCCCTCCGCCTCTCTGGCTTCTCTGCCCGACACCAACGAGGCGTTCGGTCTGTACGAGCCCTGTCACGGATCTGCCCCCGATCTCGGCAAGCAGAAGGTCA ACCCCATTGCCACCATTCTGTCTGCCGCCATGATGCTCAAGTTCTCTCTTAACATGAAGCCCGCCGGTGACGCTGTTGAGGCTGCCGTCAAGGAGTCCGTCGAGGCTGGTATCACTACCGCCGATATCGGAGGCTCTTCCTCCACCTCCGAGGTCGGAGACTTGTTG CCAACAAGGTCAAGGAGCTGCTCAAGAAGGAGTAAGTCGTTTCTACGACGCATTGA TGGAAGGAGCAAACTGACGCGCCTGCGGGTTGGTCTACCGGCAGGGTCCGCTAGTGTATAA

[0101] The amino acid sequence of the Y. lipolytica PEX3 (GenBank Acc. No. KAG5359845, YALI0F22539g) polypeptide is shown, below, as SEQ ID NO: 4:

[0102] MDFFRRHQKKVLALVGVALSSYLFIDYVKKKFFEIQGRLSSERTAKQNLRRRFEQNQQDADFTIMALLSSLTTPVMERYPVDQIKAELQSKRRPTDRVLALESSTSSSATAQTVPTMTSGATEEGEKSKTQLWQDLKRTTISRAFSLVYADALLIFFTRLQLNILGRRNYVNSVVALAQQGREGNAEGRVAPSFGDLADMGYFGDLSGSSSFGETIVDPDLDEQYLTF SWWLLNEGWVSLSERVEEAVRRVWDPVSPKAELGFDELSELIGRTQMLIDRPLNPSSPLNFLSQLLPPREQEEYVLAQNPSDTAAPIVGPTLRRLLDETADFIESPNAAEVIERLVHSGLSVFMDKLAVTFGATPADSGSPYPVVLPTAKVKLPSILANMARQAGGMAQGSPGVE NEYIDVMNQVQELTSFSAVVYSSFDWAL

[0103] The DNA sequence of the Y. lipolytica PEX3 (GenBank Acc. No. KAG5359845, YALI0F22539g) is shown, below, as SEQ ID NO: 5:

[0104] ATGGATTTCTTCAGACGGCACCAGAAAAAGGTGCTGGCACTGGTAGGT GTGGCGCTGAGTTCCTACCTGTTTATCGACTATGTGAAGAAAAAGTTCTTCGAGATC CAGGGTCGTTTGAGCTCGGAGCGAACCGCTAAACAGAATCTCCGGCGCCGATTTG AACAGAACCAGCAGGATGCAGATTTTACAATCATGGCTCTGCTATCCAGCTTGACG ACACCGGTAATGGAGCGTTACCCCGTCGACCAGATCAAGGCAGAGTTACAGAGCA AGAGACGCCCCACAGACCGGGTTTTGGCTCTCGAGAGCTCCACCTCGTCCTCAGC TACCGCACAAACCGTGCCCACCATGACAAGTGGCGCCACAGAGGAGGGCGAGAA GTCGAAAACACAGTTGTGGCAGGATCTCAAGCGAACGACCATTTCCCGAGCGTTTT CTCTTGTCTATGCAGATGCACTTCTTATTTTCTTCACGCGTTTGCAGCTCAACATTCT AGGACGACGAAACTACGTCAACAGTGTTGTCGCTCTGGCGCAGCAGGGCCGAGAG GGTAATGCCGAGGGTCGAGTGGCGCCCTCGTTTGGTGATCTTGCAGATATGGGCT ATTTCGGCGACCTTTCAGGCTCGTCCAGCTTCGGAGAAACTATTGTCGATCCCGAT CTGGACGAACAGTACCTTACCTTTTCGTGGTGGCTGCTGAACGAGGGATGGGTGT CGGTGAGCGAGCGAGTGGAGGAAGCGGTTCGTCGAGTGTGGGACCCCGTGTCAC CCAAGGCCGAACTTGGATTTGACGAGTTGTCGGAACTCATTGGACGAACACAGATG CTCATTGATCGACCTCTCAATCCCTCGTCGCCACTCAACTTTCTGAGCCAGCTGCT GCCACCACGGGAGCAGGAGGAGTACGTGCTTGCCCAGAACCCCAGCGATACTGC TGCCCCCATTGTAGGACCTACCCTCCGACGGCTTCTGGACGAGACTGCCGACTTC ATCGAGTCCCCTAATGCCGCAGAGGTGATTGAGCGACTTGTTCACTCCGGTCTCTC TGTGTTCATGGACAAGCTGGCTGTCACGTTTGGAGCCACACCTGCTGATTCGGGTT CGCCTTATCCTGTGGTGCTGCCTACTGCAAAGGTCAAGCTGCCCTCCATTCTTGCC AACATGGCTCGACAGGCTGGAGGCATGGCCCAGGGATCGCCGGGCGTGGAAAAC GAGTACATTGACGTGATGAACCAAGTGCAGGAGCTGACCTCCTTTAGTGCTGTGGT CTATTCATCTTTTGATTGGGCTCTCTAG

[0105] The amino acid sequence of the Y. lipolytica POX4 (Acyl-CoA oxidase 4; GenBank Acc. No.: AJ001302, YALI0E27654g) polypeptide is shown, below, as SEQ ID NO: 6:MITPNPANDIVHDGKLYDTFTEPPKLMAQERAQLDFDPRDITYFLDGSKEETELLESLML MYERDPLFNNQNEYDESFETLRERSVKRIFQLSKSIAMDPEPMSFRKIGFLGILDMGTY ARLGVHYALFCNSIRGQGTPDQLMYWLDQGAMVIKGFYGCFAMTEMGHGSNLSRLET IATFDKETDEFIINTPHVGATKWWIGGAAHTATHTLAFARLQVDGKDYGVKSFVVPLRN LDDHSLRPGIATGDIGKKMGRDAVDNGWIQFTNVRVPRNYMLMKHTKVLRDGTVKQPPLAQLTYGSLITGRVQMTTDSHNVSKKFLTIALRYATIRRQFSSTPGEPETRLIDYLYHQ RRLLPLMAYSYAMKLAGDHVRELFFASQEKAESLKEDDKAGVESYVQDIKELFSVSAG LKAATTWACADIIDKARQACGGHGYSAYNGFGQAFQDWVVQCTWEGDNTVLTLSAG RALIQSALVYRKEGKLGNATKYLSRSKELANAKRNGRSLEDPKLLVEAWEAVSAGAINAATDAYEELSKQGVSVDECFEQVSQERFQAARIHTRRALIEAFYSRIATADEKVKPHLIPL ANLFALWSIEEDSALFLAEGYFEPEDIIEVTSLVNKYCGIVRKNVIGYTDAFNLSDYFINAA IGRYDGDVYKNYFEKVKQQYPPEGGKPHYYEDVMKPFLHRERIPDVPMEPEDIQ

[0106] The DNA sequence of the Y. lipolytica P0X4 (Acyl-CoA oxidase 4; GenBank Acc. No. AJ001302, YALI0E27654g) is shown, below, as SEQ ID NO: 7:

[0107] ATGATCACCCCAAACCCCGCTAACGACATTGTCCATGACGGCAAGCTCT ACGACACCTTCACTGAGCCCCCCAAGCTGATGGCTCAGGAGCGAGCTCAGCTGGA CTTCGACCCTAGAGACATCACCTACTTTCTGGATGGCTCTAAGGAGGAGACCGAGCTGCTGGAGTCGCTCATGCTCATGTACGAGCGAGACCCTCTCTTCAACAACCAGAAC GAGTACGATGAATCGTTTGAAACACTGCGAGAGCGATCTGTGAAGCGAATTTTCCA GCTGTCCAAGTCCATCGCCATGGACCCCGAGCCCATGTCTTTCCGAAAGATTGGGTTCCTGGGTATTCTTGACATGGGAACGTATGCTCGACTGGGAGTCCACTACGCGCTC TTCTGTAACTCCATCCGGGGCCAGGGAACCCCCGATCAGCTCATGTACTGGCTGG AGCAGGGAGGCATGGTCATCAAGGGCTTCTAGGGCTGTTTTGCCATGACCGAAATG GGCCATGGATCTAACCTGTCGCGTCTGGAAACCATCGCCACTTTCGACAAAGAGAC CGACGAATTTATCATTAACACGCCCCACGTTGGAGCCACAAAGTGGTGGATTGGAG GAGCCGCCCACACTGCTACTCACACACTTGCCTTTGCCCGTCTTCAAGTAGACGGA AAGGACTACGGTGTGAAATCGTTTGTCGTACCTCTCCGAAACCTGGACGACCATTC GCTGCGTCCTGGAATCGCCACAGGTGATATTGGTAAGAAGATGGGTCGAGATGCC GTTGACAACGGCTGGATTCAGTTCACCAACGTCCGAGTGCCCCGAAACTACATGCT CATGAAGCATACCAAGGTTCTTCGAGACGGTACCGTCAAGCAGCCGCCTTTGGCC CAACTGACTTACGGATCTCTCATCACTGGACGAGTCCAGATGACCACTGACTCTCA CAATGTGTCCAAAAAGTTCCTCACCATTGCCCTGAGATACGCCACCATCCGACGAC AGTTCTCGTCAACTCCAGGAGAGCCCGAAACCCGACTAATTGACTACCTGTACCAC CAAAGACGACTCCTGCCTCTTATGGCTTACTCTTACGCCATGAAACTAGCTGGAGA TCACGTCCGAGAGCTGTTCTTTGCATCCCAGGAGAAGGCTGAGAGCCTCAAGGAG GACGACAAAGCCGGAGTTGAGTCTTACGTCCAGGATATCAAGGAGCTCTTCTCTGT TTCTGCTGGTCTCAAGGCTGCCACTACATGGGCTTGTGCTGACATCATTGACAAGG CCCGACAGGCGTGTGGAGGCCACGGATACTCTGCCTACAACGGCTTTGGACAGGC CTTCCAGGACTGGGTTGTCCAGTGCACTTGGGAGGGTGACAATACTGTTCTGACTC TATCTGCCGGCCGAGCTCTGATCCAATCTGCTCTCGTCTACCGAAAGGAGGGCAAA CTAGGTAACGCCACGAAGTACCTCTCTCGGTCCAAGGAGCTTGCCAACGCCAAGAGAAACGGAGGATCCGTGGAAGACCCCAAGCTGCTCGTGGAGGCATGGGAGGCTG TCTCTGCCGGTGCTATCAACGCTGCTACTGACGCTTACGAGGAGCTCTCCAAGCAG GGAGTTTCTGTTGACGAGTGCTTTGAGCAGGTGTCCCAGGAGCGATTCCAGGCTG CCCGAATCCACACTCGACGAGCTCTTATCGAGGCCTTCTACTCACGAATCGCCACT GCTGATGAGAAGGTGAAGCCTCATCTGATCCCTCTGGCCAACCTGTTTGCCCTGTG GTCCATTGAGGAGGACTCTGCTCTGTTCCTGGCTGAGGGCTACTTTGAGCCTGAG GATATCATTGAGGTGACTTCTCTTGTCAACAAGTACTGCGGAATTGTTCGAAAGAAC GTTATTGGATACACCGATGCCTTCAACCTGTCCGACTACTTCATCAACGCTGCCATT GGACGATACGACGGAGACGTGTACAAGAACTACTTTGAGAAGGTCAAACAGCAGTA CCCTCCTGAGGGTGGCAAGCCTCACTACTACGAGGATGTCATGAAGCCCTTCCTG CATCGAGAGCGAATTCCCGATGTCCCCATGGAGCCCGAGGATATTCAGTAA

[0108] The C / al DNA fragment of plasmid pO4L2P3 containing LEU2, PEX3 and POX4 wild type genes (Fig. 5, SEQ ID NO: 8) was transformed into strain AH1314.

[0109] The transformed cells were growing on Complete Media (CM) minus leucin plates (0.13% Amino acid dropout powder minus leucine, 0.17% yeast nitrogen base, 0.5% ammonium sulfate, 2.0% glucose, 2.1% agar). The colonies from transformation were analyzed by PGR.

[0110] A PGR fragment of 426 bp (SEQ ID NO. 9) was produced with primers of AH088 (GAACGACCATTTCCCGAGCGTTTTC), referred to as SEQ ID. NO. 10 and P216 (GTCGATCAATGAGCATCTGTGTTCGTC), referred to as SEQ ID. No. 11 indicating the DNA fragment with PEX3 was integrated into cells.

[0111] A PCR fragment of 517 bp (SEQ ID NO. 12) was produced with primers of P189 (CGTTATTGGATACACCGATGCCTTCAAC), referred to as SEQ ID. NO. 13 and P190 (AGCGGGAATATGATGATAGCTCTA-GATGATG), referred to as SEQ ID. NO. 14 indicating the DNA fragment of POX4 was integrated into cells.

[0112] Thus, a colony containing the PEX3 and POX4 genes was designated as strain AH291 (dgatl-, dgat2-, ura3-, table 1 ).

[0113] Figure 5 shows a physical and functional map of the C / al large fragment of plasmid pO4L2P3.

[0114] The following shows the DNA sequence of the C / al large DNA fragment (Fig.5, table 2) of plasmid pO4L2P3 is shown, below, as SEQ ID NO: 8:

[0115] CGATACTTGTAGACATCTACACACTGATGCTCCCAATCGAATATTTTCTA CCTACTGTAGAGCACTTGAAACACTTGTCTTGGCGGTACAACAGTAATACAAGTACA AGTACAGTATATACAGTACATACAAGTATATTAGCTACAGTATGTACATACAGAATAT CCATATCTGCTTGGGTCTCGAAGGTGACTCCACAGCAAGACCAGAAGCACAGAAGGGCGACATGATCCATCGAGTAAAAGATAACTTTTTCTAAGAGAGTGGAATATCCAAACTGATCCTCCAGCTTCCTCAGACGTCACACTTCGCCACTATATTGTTCAAATACTTTCCCCTTAGTAAACCTCGGTTTACAGTTGAACTATTACTTCAATTTTATTGAGCGGGGCTACACAACGCTGTGTCCACCGTTCTCCTTCATACAGTAGCCTATAACATAACACGAGTGATTGCTTCAATTTGATGAAGAACAAACGGTCGATCACCACAGCATTTACCTGAAGTGTGTAAGGTAGCACACATCGTACGCGCAGACTAAAATTATCCAACCAAGTTTCCCCATGTCCACACGGAGATGTGGGAGCTGAAAAATGATGGACTGTTTTTTTTTCTTTTTCTGCATATACATTCATTTAATGTAGTATTATCCGAATGGGTGTGCCATTGTGATGTGATTATTCATCAGTCACAAGTTCGTCGCAACGGCGAGACTTTCATACAGGCACCATAAAACTGAAACGTGCAAATTAGGTCGTAATAACTCTTCATTGCCTCCTCATCTCTTCAGATCAAGCCCTTGGAGAACATCCTTGTATTTACTGTACTGTACCATCCGGACCCAATTTTTAGGCTTCTATGCACTCACCCGTGTTTCGTCTAAAAGTCACAGATCCCATACACTTGCAACATCTGTTGAGAAGCCCATTGTATATTATTAGGATCGTAGCATTATTGTGGCAAAAAATATTCAAGTGCTCATGTGAATTGACACGATCACGTAAATACCTGGTGAAATTGCTAGTATTCGTGATGTTCTAATACAACTCTGTTCAATATTTCCGGCGCTCTCTTGTATACAAGAGCACAAGACATGCACCCCACATTAACCGAGGTCAAGTGTTTATGTATGAAAAGTGACATAAATCGTCCAAAAAAAAGTAGCACATAGTTGTATGGCTGTAAGTTATGTGATTGTCAGTTCTTCGGCCTTCCAACTCCTATGCACCGTCTTCAATCATCTACCCCCGTGCCCCACACCCCGCACTATTAGAGTTTATCACAGTCAGCTAAACTGCTTGCACATCTACACCTCTGACTACACCACCATGGATTTCTTCAGACGGCACCAGAAAAAGGTGCTGGCACTGGTAGGTGTGGCGCTGAGTTCCTACCTGTTTATCGACTATGTGAAGAAAAAGTTCTTCGAGATCCAGGGTCGTTTGAGCTCGGAGCGAACCGCTAAACAGAATCTCCGGCGCCGATTTGAACAGAACCAGCAGGATGCAGATTTTACAATCATGGCTCTGCTATCCAGCTTGACGACACCGGTAATGGAGCGTTACCCCGTCGACCAGATCAAGGCAGAGTTACAGAGCAAGAGACGCCCCACAGACCGGGTTTTGGCTCTCGAGAGCTCCACCTCGTCCTCAGCTACCGCACAAACCGTGCCCACCATGACAAGTGGCGCCACAGAGGAGGGCGAGAAGTCGAAAACACAGTTGTGGCAGGATCTCAAGCGAACGACCATTTCCCGAGCGTTTTCTCTTGTCTATGCAGATGCACTTCTTATTTTCTTCACGCGTTTGCAGCTCAACATTCTAGGACGACGAAACTACGTCAACAGTGTTGTCGCTCTGGCGCAGCAGGGCCGAGAGGGTAATGCCGAGGGTCGAGTGGCGCCCTCGTTTGGTGATCTTGCAGATATGGGCTATTTCGGCGACCTTTCAGGCTCGTCCAGCTTCGGAGAAACTATTGTCGATCCCGATCTGGACGAACAGTACCTTACCTTTTCGTGGTGGCTGCTGAACGAGGGATGGGTGTCGCTGAGCGAGCGAGTGGAGGAAGCGGTTCGTCGAGTGTGGGACCCCGTGTCACCCAAGGCCGAACTTGGATTTGACGAGTTGTCGGAACTCATTGGACGAACACAGATGCTCATTGATCGACCTCTCAATCCCTCGTCGCCACTCAACTTTCTGAGCCAGCTGCTGCCACCACGGGAGCAGGAGGAGTACGTGCTTGCCCAGAACCCCAGCGATACTGCTGCCCCCATTGTAGGACCTACCCTCCGACGGCTTCTGGACGAGACTGCCGACTTCATCGAGTCCCCTAATGCCGCAGAGGTGATTGAGCGACTTGTTCACTCCGGTCTCTCTGTGTTCATGGACAAGCTGGCTGTCACGTTTGGAGCCACACCTGCTGATTCGGGTTCGCCTTATCCTGTGGTGCTGCCTACTGCAAAGGTCAAGCTGCCCTCCATTCTTGCCAACATGGCTCGACAGGGTGGAGGCATGGCCCAGGGATCGCCGGGCGTGGAAAACGAGTACATTGACGTGATGAACCAAGTGCAGGAGCTGACCTCCTTTAGTGCTGTGGTCTATTCATCTTTTGATTGGGCTCTCTAGAGGCTCATTCACGAAAGACACGAAGAACGAAGATGGGGACTGAATACAGCGCTCTCATTTGTACACAAATGATTTATGACAGAGTAACTTGTACATCATGTAGAGCATACATACTGAAGGTGTGATCTCACGGGATATCTTGAAGACCACTCGTAGCTGGAGGCATAGGTAGTGCTAGTACGGATACTTGCACCGTATCCAACATAAGTAGAGGAGCCTCCTAGTGGCTATTGGTACACCGATAAAGATACACATACATGTTATTGCCACTTGTATCTACCAGTACCCGTACTGTATGTACTTGTACAGCACTTACTGTTACTTACATGACCAATGCCTTAGATCGAAATATTTCTGATCAAAGTAGAACCAGGCGTAAGACGAAAATGGACAGGTTTTACCGAAAGCCCACGTGAACAGGTTACTTTCTCAAACCTTGGAGAGAGTGGAGAGCCAGTCACCTGAAACAGAACTCTCATCCTCATTCATCGTACCGTACGTACTTGTACGAGTACATGCCTGGCACAGTCTCATTCTATCCTAACACAATGTACAATGTACAACATTTTTCGGCTCGGCCATGCAGCTTCGGAGACACGCTCGTGAAAAAGGGTCGGACTAATCAACCTCACCGTTGCGTTAGGCTGCCAGGTGAAACAATATTATTTTGACAGCTAGAAAGTCCGGAGATGACACTGGGGTAACGGAACTACTGTACTTCGCACAAGTCGGCGTGAGTTTGTGGTAGTTTATATGAGTAGTTAGCTATAGTCATGCCCCATTATTTGATAATCGGATCACACACGCTGAATAATTACACTGGTCTTATTTGTGCTATCATTCCTACTACCTCCTTCTGTATAATAATACACCTTCGCCGTAAAATGTGGAGAAGAAATCGGCACTAAAAAGTCAGGTAGACTGGAAAATGCGCCATGAAATGAATATCTCTTGCTACAGTAATGCCCAGCATCGAGGGGTATTGTGTCACCAACACTATAGTGGCAGCTGAAGCGCTCGTGATTGTAGTATGAGTCTTTATTGGTGATGGGAAGAGTTCACTCAATATTCTCGTTACTGCCAAAACACCACGGTAATCGGCCAGACACCATGGATGTAGATGACCAAGCCTGTGAATGTTATTCGAGCTAAAATGCACATGGTTGGTGAAAGGAGTAGTTGCTGTCGAATTCCGTCGTCGCCTGAGTCATCATTTATTTACCAGTTGGCCACAAACCCTTGACGATCTCGTATGTCCCCTCCGACATACTCCCGGCCGGCTGGGGTACGTTCGATAGCGCTATCGGCATCGACAAGGTTTGGGTCCCTAGCCGATACCGCACTACCTGAGTCACAATCTTCGGAGGTTTAGTCTTCCACATAGCACGGGCAAAAGTGCGTATATATACAAGAGCGTTTGCCAGCCACAGATTTTCACTCCACACACCACATCACACATACAACCACACACATCCACAATGGAACCCGAAACTAAGAAGACCAAGACTGACTCCAAGAAGATTGTTCTTCTCGGCGGCGACTTCTGTGGCCCCGAGGTGATTGCCGAGGCCGTCAAGGTGCTCAAGTCTGTTGCTGAGGCCTCCGGCACCGAGTTTGTGTTTGAGGACCGACTCATTGGAGGAGCTGCCATTGAGAAGGAGGGCGAGCCCATCACCGACGCTACTCTCGACATCTGCCGAAAGGCTGACTCTATTATGCTCGGTGCTGTCGGAGGCGCTGCCAACACCGTATGGACCACTCCCGACGGACGAACCGACGTGCGACCCGAGCAGGGTCTCCTCAAGCTGCGAAAGGACGTGAACCTGTACGCCAACCTGCGACCCTGCCAGCTGCTGTCGCCCAAGCTCGCCGATCTCTCCCCCATCCGAAACGTTGAGGGCACCGACTTCATCATTGTCCGAGAGCTCGTCGGAGGTATCTACTTTGGAGAGCGAAAGGAGGATGACGGATCTGGCGTCGCTTCCGACACCGAGACCTACTCCGTTCCTGAGGTTGAGCGAATTGCCCGAATGGCCGCCTTCCTGGCCCTTCAGCACAACCCCCCTCTTCCCGTGTGGTCTCTTGACAAGGCCAACGTGCTGGCCTCCTCTCGACTTTGGCGAAAGACTGTCACTCGAGTCCTCAAGGACGAATTCCCCCAGCTCGAGCTCAACCACCAGCTGATCGACTCGGCCGCCATGATCCTCATCAAGCAGCCCTCCAAGATGAATGGTATCATCATCACCACCAACATGTTTGGCGATATCATCTCCGACGAGGCCTCCGTCATCCCCGGTTCTCTGGGTCTGCTGCCCTCCGCCTCTCTGGCTTCTCTGCCCGACACCAACGAGGCGTTCGGTCTGTACGAGCCCTGTCACGGATCTGCCCCCGATCTCGGCAAGCAGAAGGTCAACCCCATTGCCACCATTCTGTCTGCCGCCATGATGCTCAAGTTCTCTCTTAACATGAAGCCCGCCGGTGACGCTGTTGAGGCTGCCGTCAAGGAGTCCGTCGAGGCTGGTATCACTACCGCCGATATCGGAGGCTCTTCCTCCACCTCCGAGGTCGGAGACTTGTTGCCAACAAGGTCAAGGAGCTGCTCAAGAAGGAGTAAGTCGTTTCTACGACGCATTGATGGAAGGAGCAAACTGACGCGCCTGCGGGTTGGTCTACCGGCAGGGTCCGCTAGTGTATAAGACTCTATAAAAAGGGCCCTGCCCTGCTAATGAAATGATGATTTATAATTTACCGGTGTAGCAACCTTGACTAGAAGAAGCAGATTGGGTGTGTTTGTAGTGGAGGACAGTGGTACGTTTTGGAAACAGTCTTCTTGAAAGTGTCTTGTCTACAGTATATTCACTCATAACCTCAATAGCCAAGGGTGTAGTCGGTTTATTAAAGGAAGGGAGTTGTGGCTGATGTGGATAGATATCTTTAAGCTGGCGACTGCACCCAACGAGTGTTCTTTTGCCGATACATATGCACCATTAAATGATTAGAATGCGGGGATTGAGTTTCGGATTTTTCTTACATGCGTTGCGCGTTGGAGGTGATCCGAGCAGGTGGAATAGGTGTATATTGAGCGATTGGGAGAGTTGGTTGTGTACAATTATTTTAATACCTCTTCTGATTGTTTTCTATTGCCTTCCATTTCTATCTTTACCTGCCATCTCACGTCGTGTGTACCATCCCCACATACGGAACCAGTAGGTCTTTTAGGCTCTGAACGTGCAAATGAGTTTGGTGGGGTAGGCAGAGATCGCATAGAGACGGGTAGAATGAGCAGTTAAAAGCTGTGTTGAGTGGTAAAAATTTACAATAAGTGTTCCTCAAGGCATCAAGGAGACGAAATAAGCCATTATGGACACGAACCAACAGTCCCACCACGTTCTAAACACATTCCTCCACTGCCACTCCCAAACACCACGTCCCACATAAACTTCTACCCCACATTTTGACAAGCCTATTCGTTTAATAATCACCCCGAGGAGACAGAAAGCCTAACAGCTGGAGCCACTATATAGTTGCAGTGGCATGCTCTTTCATTGAAACGACCCACAACAATGATCACCCCAAACCCCGCTAACGACATTGTCCATGACGGCAAGCTCTACGACACCTTCACTGAGCCCCCCAAGCTGATGGCTCAGGAGCGAGCTCAGCTGGAGTTCGACCCTAGAGACATCACCTACTTTCTGGATGGCTCTAAGGAGGAGACCGAGCTGCTGGAGTCGCTCATGCTCATGTACGAGCGAGACCCTCTCTTCAACAACCAGAACGAGTACGATGAATCGTTTGAAACACTGCGAGAGCGATCTGTGAAGCGAATTTTCCAGCTGTCCAAGTCCATCGCCATGGACCCCGAGCCCATGTCTTTCCGAAAGATTGGGTTCCTGGGTATTCTTGACATGGGAACGTATGCTCGACTGGGAGTCCACTACGCGCTCTTCTGTAACTCCATCCGGGGCCAGGGAACCCCCGATCAGCTCATGTACTGGCTGGACCAGGGAGCCATGGTCATCAAGGGCTTCTACGGCTGTTTTGCCATGACCGAAATGGGCCATGGATCTAACCTGTCGCGTCTGGAAACCATCGCCACTTTCGACAAAGAGACCGACGAATTTATCATTAACACGCCCCACGTTGGAGCCACAAAGTGGTGGATTGGAGGAGCCGCCCACACTGCTACTCACACACTTGCCTTTGCCCGTCTTCAAGTAGACGGAAAGGACTACGGTGTGAAATCGTTTGTCGTACCTCTCCGAAACCTGGACGACCATTCGCTGCGTCCTGGAATCGCCACAGGTGATATTGGTAAGAAGATGGGTCGAGATGCCGTTGACAACGGCTGGATTCAGTTCACCAACGTCCGAGTGCCCCGAAACTACATGCTCATGAAGCATACCAAGGTTCTTCGAGACGGTACCGTCAAGCAGCCGCCTTTGGCCCAACTGACTTACGGATCTCTCATCACTGGACGAGTCCAGATGACCACTGACTCTCACAATGTGTCCAAAAAGTTCCTCACCATTGCCCTGAGATACGCCACCATCCGACGACAGTTCTCGTCAACTCCAGGAGAGCCCGAAACCCGACTAATTGACTACCTGTACCACCAAAGACGACTCCTGCCTCTTATGGCTTACTCTTACGCCATGAAACTAGCTGGAGATCACGTCCGAGAGCTGTTCTTTGCATCCCAGGAGAAGGCTGAGAGCCTCAAGGAGGACGACAAAGCCGGAGTTGAGTCTTACGTCCAGGATATCAAGGAGCTCTTCTCTGTTTCTGCTGGTCTCAAGGCTGCCACTACATGGGCTTGTGCTGACATCATTGACAAGGCCCGACAGGCGTGTGGAGGCCACGGATACTCTGCCTACAACGGCTTTGGACAGGCCTTCCAGGACTGGGTTGTCCAGTGCACTTGGGAGGGTGACAATACTGTTCTGACTCTATCTGCCGGCCGAGCTCTGATCCAATCTGCTCTCGTCTACCGAAAGGAGGGCAAACTAGGTAACGCCACGAAGTACCTCTCTCGGTCCAAGGAGCTTGCCAACGCCAAGAGAAACGGACGATCCCTGGAAGACCCCAAGCTGCTCGTGGAGGCATGGGAGGCTGTCTCTGCCGGTGCTATCAACGCTGCTACTGACGCTTACGAGGAGCTCTCCAAGCAGGGAGTTTCTGTTGACGAGTGCTTTGAGCAGGTGTCCCAGGAGCGATTCCAGGCTGCCCGAATCCACACTCGACGAGCTCTTATCGAGGCCTTCTACTCACGAATCGCCACTGCTGATGAGAAGGTGAAGCCTCATCTGATCCCTCTGGCCAACCTGTTTGCCCTGTGGTCCATTGAGGAGGACTCTGCTCTGTTCCTGGCTGAGGGCTACTTTGAGCCTGAGGATATCATTGAGGTGACTTCTCTTGTCAACAAGTACTGCGGAATTGTTCGAAAGAACGTTATTGGATACACCGATGCCTTCAACCTGTCCGACTACTTCATCAACGCTGCCATTGGACGATACGACGGAGACGTGTACAAGAACTACTTTGAGAAGGTCAAACAGCAGTACCCTCCTGAGGGTGGCAAGCCTCACTACTACGAGGATGTCATGAAGCCCTTCCTGCATCGAGAGCGAATTCCCGATGTCCCCATGGAGCCCGAGGATATTCAGTAATGATTTTGTAGGTTTTAGTAAGTATTAACGTTTTTTATGATTTGGAGTTGTGTGTAACTTGTACAGGTACACCTACATACTGTACTGTAGGTCCAAAGATAGGTACACTGTGGCAATAATTATGCGAGTACTTGTACCGTCATCGTAGCTGCTGTAAAGAGATCAGACACAGGCACTTTTCCCCACCATGAGATCACCACTCGTCGTCCGAGTACTTCTATGGCACAGGCACAATCACATGTACTTGTGGATGCCAATGTGTGACATCATCATCTAGAGCTATCATCATATTCCCGCTGCAAATGGTCTACGTATTACTATTAAGCAGGGGGGGGGGAGGAATTATGACGACATTGTACGTGTACTCGTACCGGTACTTGTAGCACGCCGAACTGCGGTATTACTGTGCACTGTAATTTCGGACCCCTCTTATAGCCCCAAGTTGGTCTATACATCTGAACCGGTGCAGACTCACTATTAAAAGTGCGGCAGCTAATTTTGCTGACACAGCCTTGTCGATAAAAGTAGCTACTTGTACGATGTACTCTTGTAGCTTATGCGTGGGCTATCATTAATTTTAGACAACAGTGTCCTACCACGCCACGGAACGGGAGAAAGCAGAAGATCCGAACATCTTGTGCGGAGTGTGTTGCGTCTGTGACACCGGTGAAGTTCCGTTGTCCATGACCCGCCGATCTTTTTTTTGGCATATGGTTTGCATCGCCTCGCAGACAAGAAAGGGCGAAGGTAAATGCAGCACATGTTAGAAGTGCCTGGCATGGGACATACACGCGGACGTTTGTGCAGTTGTCAGCAGCGTGT TTATCG

[0116] The 426 bp DNA sequence of the PGR fragment is shown, below, as SEQ IDNO: 9:GAACGACCATTTCCCGAGCGTTTTCTCTTGTCTATGCAGATGCACTTCTTATTTTCTTCACGCGTTTGCAGCTCAACATTCTAGGACGACGAAACTACGTCAACAGTGTTGTCGCTCTGGCGCAGCAGGGCCGAGAGGGTAATGCCGAGGGTCGAGTGGCGCCCTCGTTTGGTGATCTTGCAGATATGGGCTATTTCGGCGACCTTTCAGGCTCGTCCAGCTTCGGAGAAACTATTGTCGATCCCGATCTGGACGAACAGTACCTTACCTTTTCGTGGTGGCTGCTGAACGAGGGATGGGTGTCGCTGAGCGAGCGAGTGGAGGAAGCGGTTCGTCGAGTGTGGGACCCCGTGTCACCCAAGGCCGAACTTGGATTTGACGAGTTGTCG GAACTCATTGGACGAACACAGATGCTCATTGATCGAC

[0117] The 517 bp DNA sequence of the PGR fragment is shown, below, as SEQ I D NO: 12:GTTATTGGATACACCGATGCCTTCAACCTGTCCGACTACTTCATCAACGCTGCCATTGGACGATACGACGGAGACGTGTACAAGAACTACTTTGAGAAGGTCAAACAGCAGTACCCTCCTGAGGGTGGCAAGCCTCACTACTACGAGGATGTCATGAAGCCCTTCCTGCATCGAGAGCGAATTCCCGATGTCCCCATGGAGCCCGAGGATATTCAGTAATGATTTTGTAGGTTTTAGTAAGTATTAACGTTTTTTATGATTTGGAGTTGTGTGTAACTTGTACAGGTACACCTACATACTGTACTGTAGGTCCAAAGATAGGTACACTGTGGCAATAATTATGCGAGTACTTGTACCGTCATCGTAGCTGCTGTAAAGAGATCAGACACAGGCACTTTTCCCCACCATGAGATCACCACTCGTCGTCCGAGTACTTCTATGGCACAGCCACAATCACATGTACTTGTGCATGCCAATGTGTGACATCATCATCTAGAGCTATCATCA TATTCCCGCTEXAMPLE 2: Generation of strain AH246 from strain AH1314 (2738Y-45) for restoration of LEU2 and P0X4 genes

[0118] Strain AH246 (dgatl-, dgat2-, pex3-, ura3-) was generated from strain AH1314 in order to restore the LEU2 and P0X4 genes back to their wild types (Table 1 , Fig. 2 & 3).

[0119] Construct pPOX4-Leu2 (Fig. 6; SEQ ID No: 15, Table 3) contains wild type genes of LEU2, and P0X4, the components of the C / al large fragment of plasmid pPOX4- Leu2, used for generation of strain AH246, are further described in Table 3, Fig. 7 and SEQ ID NO: 16

[0120] TABLE 3: Description of the C / al DNA large fragment (Fig. 7 and SEQ ID NO: 16) of Plasmid pPOX4-LEU2 (SEQ ID NO: 15, Fig. 6) integrated into the genome of Strain AH246

[0121] Figure 6 depicts the physical and functional map of plasmid pPOX4-LEU2.

[0122] The following shows the DNA sequence of the plasmid pPOX4-LEU2 as shown, below, as SEQ ID NO: 15:

[0123] ATCGATACTTGTAGACATCTACACACTGATGCTCCCAATCGAATATTTTCTACCTACTGTAGAGCACTTGAAACACTTGTCTTGGCGGTACAACAGTAATACAAGTACAAGTACAGTATATACAGTACATACAAGTATATTAGCTACAGTATGTACATACAGAATATCCATATCTGCTTGGGTCTCGAAGGTGACTCCACAGCAAGACCAGAAGCACAGAAGGGCGACATGATCCATCGAGTAAAAGATAACTTTTTCTAAGAGAGTGGAATATCCAA ACTGATCCTCCAGCTTCCTCAGACGTCACACTTCGCCACTATATTGTTCAAATACTTTCCCCTTAGTAAACCTCGGTTTACAGTTGAACTATTACTTCAATTTTATTGAGCGGG GCTACACAACGCTGTGTCCACCGTTCTCCTTCATACAGTAGCCTATAACATAACACGAGTGATTGCTTCAATTTGATGAAGAACAAACGGTCGATCACCACAGCATTTACCTGAAGTGTGTAAGGTAGCACACATCGTACGTACTTGTACGAGTACATGCCTGGCACAGTCTCATTCTATCCTAACACAATGTACAATGTACAACATTTTTCGGCTCGGCCATGCAGCTTCGGAGACACGCTCGTGAAAAAGGGTCGGACTAATCAACCTCACCGTTGCGTTAGGCTGCCAGGTGAAACAATATTATTTTGACAGCTAGAAAGTCCGGAGATGACACTGGGGTAACGGAACTACTGTACTTCGCACAAGTCGGCGTGAGTTTGTGGTAGTTTATATGAGTAGTTAGCTATAGTCATGCCCCATTATTTGATAATCGGATCACACACGCTGAATAATTACACTGGTCTTATTTGTGCTATCATTCCTACTACCTCCTTCTGTATAATAATACACCTTCGCCGTAAAATGTGGAGAAGAAATCGGCACTAAAAAGTCAGGTAGACTGGAAAATGCGCCATGAAATGAATATCTCTTGCTACAGTAATGCCCAGCATCGAGGGGTATTGTGTCACCAACACTATAGTGGCAGCTGAAGCGCTCGTGATTGTAGTATGAGTCTTTATTGGTGATGGGAAGAGTTCACTCAATATTCTCGTTACTGCCAAAACACCACGGTAATCGGCCAGACACCATGGATGTAGATCACCAAGCCTGTGAATGTTATTCGAGCTAAAATGCACATGGTTGGTGAAAGGAGTAGTTGCTGTCGAATTCCGTCGTCGCCTGAGTCATCATTTATTTACCAGTTGGCCACAAACCCTTGACGATCTCGTATGTCCCCTCCGACATACTCCCGGCCGGCTGGGGTACGTTCGATAGCGCTATCGGCATCGACAAGGTTTGGGTCCCTAGCCGATACCGCACTACCTGAGTCACAATCTTCGGAGGTTTAGTCTTCCACATAGCACGGGCAAAAGTGCGTATATATACAAGAGCGTTTGCCAGCCACAGATTTTCACTCCACACACCACATCACACATACAACCACACACATCCACAATGGAACCCGAAACTAAGAAGACCAAGACTGACTCCAAGAAGATTGTTCTTCTCGGCGGCGACTTCTGTGGCCCCGAGGTGATTGCCGAGGCCGTCAAGGTGCTCAAGTCTGTTGCTGAGGCCTCCGGCACCGAGTTTGTGTTTGAGGACCGACTCATTGGAGGAGCTGCCATTGAGAAGGAGGGCGAGCCCATCACCGACGCTACTCTCGACATCTGCCGAAAGGCTGACTCTATTATGCTCGGTGCTGTCGGAGGCGCTGCCAACACCGTATGGACCACTCCCGACGGACGAACCGACGTGCGACCCGAGCAGGGTCTCCTCAAGCTGCGAAAGGACCTGAACCTGTACGCCAACCTGCGACCCTGCCAGCTGCTGTCGCCCAAGCTCGCCGATCTCTCCCCCATCCGAAACGTTGAGGGCACCGACTTCATCATTGTCCGAGAGCTCGTCGGAGGTATCTACTTTGGAGAGCGAAAGGAGGATGACGGATCTGGCGTCGCTTCCGACACCGAGACCTACTCCGTTCCTGAGGTTGAGCGAATTGCCCGAATGGCCGCCTTCCTGGCCCTTCAGCACAACCCCCCTCTTCCCGTGTGGTCTCTTGACAAGGCCAACGTGCTGGCCTCCTCTCGACTTTGGCGAAAGACTGTCACTCGAGTCCTCAAGGACGAATTCCCCCAGCTCGAGCTCAACCACCAGCTGATCGACTCGGCCGCCATGATCCTCATCAAGCAGCCCTCCAAGATGAATGGTATCATCATCACCACCAACATGTTTGGCGATATCATCTCCGACGAGGCCTCCGTCATCCCCGGTTCTCTGGGTCTGCTGCCCTCCGCCTCTCTGGCTTCTCTGCCCGACACCAACGAGGCGTTCGGTCTGTACGAGCCCTGTCACGGATCTGCCCCCGATCTCGGCAAGCAGAAGGTCAACCCCATTGCCACCATTCTGTCTGCCGCCATGATGCTCAAGTTCTCTCTTAACATGAAGCCCGCCGGTGACGCTGTTGAGGCTGCCGTCAAGGAGTCCGTCGAGGCTGGTATCACTACCGCCGATATCGGAGGCTCTTCCTCCACCTCCGAGGTCGGAGACTTGTTGCCAACAAGGTCAAGGAGCTGCTCAAGAAGGAGTAAGTCGTTTCTACGACGCATTGATGGAAGGAGCAAACTGACGCGGCTGCGGGTTGGTCTACCGGCAGGGTGCGCTAGTGTATAAGACTCTATAAAAAGGGCCCTGCCCTGCTAATGAAATGATGATTTATAATTTACCGGTGTAGCAACCTTGACTAGAAGAAGCAGATTGGGTGTGTTTGTAGTGGAGGACAGTGGTACGTTTTGGAAACAGTCTTCTTGAAAGTGTCTTGTCTACAGTATATTCACTCATAACCTCAATAGCCAAGGGTGTAGTCGGTTTATTAAAGGAAGGGAGTTGTGGCTGATGTGGATAGATATCTTTAAGCTGGCGACTGCACCCAACGAGTGTTCTTTTGCCGATACATATGCACCATTAAATGATTAGAATGCGGGGATTGAGTTTCGGATTTTTCTTACATGCGTTGCGCGTTGGAGGTGATCCGAGCAGGTGGAATAGGTGTATATTGAGCGATTGGGAGAGTTGGTTGTGTACAATTATTTTAATACCTCTTCTGATTGTTTTCTATTGCCTTCCATTTCTATCTTTACCTGCCATCTCACGTCGTGTGTACCATCCCCACATACGGAACCAGTAGGTCTTTTAGGCTCTGAACGTGCAAATGAGTTTGGTGGGGTAGGCAGAGATCGCATAGAGACGGGTAGAATGAGCAGTTAAAAGCTGTGTTGAGTGGTAAAAATTTACAATAAGTGTTCCTCAAGGCATCAAGGAGACGAAATAAGCCATTATGGACACGAACCAACAGTCCCACCACGTTCTAAACACATTCCTCCACTGCCACTCCCAAACACCACGTCCCACATAAACTTCTACCCCACATTTTGACAAGCCTATTCGTTTAATAATCACCCCGAGGAGACAGAAAGCCTAACAGCTGGAGCCACTATATAGTTGCAGTGGCATGCTCTTTCATTGAAACGACCCACAACAATGATCACCCCAAACCCCGCTAACGACATTGTCCATGACGGCAAGCTCTACGACACCTTCACTGAGCCCCCCAAGCTGATGGCTCAGGAGCGAGCTCAGCTGGACTTCGACCCTAGAGACATCACCTACTTTCTGGATGGCTCTAAGGAGGAGACCGAGCTGCTGGAGTCGCTCATGCTCATGTACGAGCGAGACCCTCTCTTCAACAACCAGAACGAGTACGATGAATCGTTTGAAACACTGCGAGAGCGATCTGTGAAGCGAATTTTCCAGCTGTCCAAGTCCATCGCCATGGACCCCGAGCCCATGTCTTTCCGAAAGATTGGGTTCCTGGGTATTCTTGACATGGGAACGTATGCTCGACTGGGAGTCCACTACGCGCTCTTCTGTAACTCCATCCGGGGCCAGGGAACCCCCGATCAGCTCATGTACTGGCTGGACCAGGGAGCCATGGTCATCAAGGGCTTCTACGGCTGTTTTGCCATGACCGAAATGGGCCATGGATCTAACCTGTCGCGTCTGGAAACCATCGCCACTTTCGACAAAGAGACCGACGAATTTATCATTAACACGCCCCACGTTGGAGCCACAAAGTGGTGGATTGGAGGAGCCGCCCACACTGCTACTCACACACTTGCCTTTGCCCGTCTTCAAGTAGACGGAAAGGACTACGGTGTGAAATCGTTTGTCGTACCTCTCCGAAACCTGGACGACCATTCGCTGCGTCCTGGAATCGCCACAGGTGATATTGGTAAGAAGATGGGTCGAGATGCCGTTGACAACGGCTGGATTCAGTTCACCAACGTCCGAGTGCCCCGAAACTACATGCTCATGAAGCATACCAAGGTTCTTCGAGACGGTACCGTCAAGCAGCCGCCTTTGGCCCAACTGACTTACGGATCTCTCATCACTGGACGAGTCCAGATGACCACTGACTCTCACAATGTGTCCAAAAAGTTCCTCACCATTGCCCTGAGATACGCCACCATCCGACGACAGTTCTCGTCAACTCCAGGAGAGCCCGAAACCCGACTAATTGACTACCTGTACCACCAAAGACGACTCCTGCCTCTTATGGCTTACTCTTACGCCATGAAACTAGCTGGAGATCACGTCCGAGAGCTGTTCTTTGCATCCCAGGAGAAGGCTGAGAGCCTCAAGGAGGACGACAAAGCCGGAGTTGAGTCTTACGTCCAGGATATCAAGGAGCTCTTCTCTGTTTCTGCTGGTCTCAAGGCTGCCACTACATGGGCTTGTGCTGACATCATTGACAAGGCCCGACAGGCGTGTGGAGGCCACGGATACTCTGCCTACAACGGCTTTGGACAGGCCTTCCAGGACTGGGTTGTCCAGTGCACTTGGGAGGGTGACAATACTGTTCTGACTCTATCTGCCGGCCGAGCTCTGATCCAATCTGCTCTCGTCTACCGAAAGGAGGGCAAACTAGGTAACGCCACGAAGTACCTCTCTCGGTCCAAGGAGCTTGCCAACGCCAAGAGAAACGGACGATCCCTGGAAGACCCCAAGCTGCTCGTGGAGGCATGGGAGGCTGTCTCTGCCGGTGCTATCAACGCTGCTACTGACGCTTACGAGGAGCTCTCCAAGCAGGGAGTTTCTGTTGACGAGTGCTTTGAGCAGGTGTCCCAGGAGCGATTCCAGGCTGCCCGAATCCACACTCGACGAGCTCTTATCGAGGCCTTCTACTCACGAATCGCCACTGCTGATGAGAAGGTGAAGCCTCATCTGATCCCTCTGGCCAACCTGTTTGCCCTGTGGTCCATTGAGGAGGACTCTGCTCTGTTCCTGGCTGAGGGCTACTTTGAGCCTGAGGATATCATTGAGGTGACTTCTCTTGTCAACAAGTACTGCGGAATTGTTCGAAAGAACGTTATTGGATACACCGATGCCTTCAACCTGTCCGACTACTTCATCAACGCTGCCATTGGACGATACGACGGAGACGTGTACAAGAACTACTTTGAGAAGGTCAAACAGCAGTACCCTCCTGAGGGTGGCAAGCCTCACTACTACGAGGATGTCATGAAGCCCTTCCTGCATCGAGAGCGAATTCCCGATGTCCCCATGGAGCCCGAGGATATTCAGTAATGATTTTGTAGGTTTTAGTAAGTATTAACGTTTTTTATGATTTGGAGTTGTGTGTAACTTGTACAGGTACACCTACATACTGTACTGTAGGTCCAAAGATAGGTACACTGTGGCAATAATTATGCGAGTACTTGTACCGTCATCGTAGCTGCTGTAAAGAGATCAGACACAGGCACTTTTCCCCACCATGAGATCACCACTCGTCGTCCGAGTACTTCTATGGCACAGCCACAATCACATGTACTTGTGCATGCCAATGTGTGACATCATCATCTAGAGGTATCATCATATTGCCGCTGCAAATGGTCTACGTATTACTATTAAGCAGGGGGGGGGGAGGAATTATGACGACATTGTACGTGTACTCGTACCGGTACTTGTAGCACGCCGAACTGCGGTATTACTGTGCACTGTAATTTCGGACCCCTCTTATAGCCCCAAGTTGGTCTATACATCTGAACCGGTGCAGACTCACTATTAAAAGTGCGGCAGCTAATTTTGCTGACACAGCCTTGTCGATAAAAGTAGCTACTTGTACGATGTACTCTTGTAGCTTATGCGTGGGCTATCATTAATTTTAGACAACAGTGTCCTACCACGCCACGGAACGGGAGAAAGCAGAAGATCCGAACATCTTGTGCGGAGTGTGTTGCGTCTGTGACACCGGTGAAGTTCCGTTGTCCATGACCCGCCGATCTTTTTTTTGGCATATGGTTTGCATCGCCTCGCAGACAAGAAAGGGCGAAGGTAAATGCAGCACATGTTAGAAGTGCCTGGCATGGGACATACAGGCGGACGTTTGTGCAGTTGTCAGCAGCGTGTTTATCGATTCTGGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGGCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAG CTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGCGCTCCCATTCGCCATTCAGGCT GCGCAACTGTTGGGAAGGGCGTTTCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAATTGGCGGAAGGCCGTCAAGGCCGCATT

[0124] The DNA sequence of the Cla\ large fragment of plasmid pPOX4-LEU2 is shown, below, as SEQ ID NO: 16:

[0125] CGATACTTGTAGACATCTACACACTGATGCTCCCAATCGAATATTTTCTACCTACTGTAGAGCACTTGAAACACTTGTCTTGGCGGTACAACAGTAATACAAGTACAAGTACAGTATATACAGTACATACAAGTATATTAGCTACAGTATGTACATACAGAATAT CCATATCTGCTTGGGTCTCGAAGGTGACTCCACAGCAAGACCAGAAGCACAGAAGGGCGACATGATCCATCGAGTAAAAGATAACTTTTTCTAAGAGAGTGGAATATCCAAACTGATCCTCCAGCTTCCTCAGACGTCACACTTCGCCACTATATTGTTCAAATACTTTCCCCTTAGTAAACCTCGGTTTACAGTTGAACTATTACTTCAATTTTATTGAGCGGGG CTACACAACGCTGTGTCCACCGTTCTCCTTCATACAGTAGCCTATAACATAACACGACTGATTGCTTCAATTTGATCAAGAACAAACCGTCGATCACCACAGCATTTACCTGAAGTGTGTAAGGTAGCACACATCGTACGTACTTGTACGAGTAGATGCCTGGCACAGTCTCATTCTATCCTAACACAATGTACAATGTACAACATTTTTCGGCTCGGCCATGCAGC TTCGGAGACACGCTCGTGAAAAAGGGTCGGACTAATCAACCTCACCGTTGCGTTAGGCTGCCAGGTGAAACAATATTATTTTGACAGCTAGAAAGTCCGGAGATGACACTGGGGTAACGGAACTACTGTACTTCGCACAAGTCGGCGTGAGTTTGTGGTAGTTTATAT GAGTAGTTAGCTATAGTCATGCCCCATTATTTGATAATCGGATCACACACGCTGAATAATTACACTGGTCTTATTTGTGCTATCATTCCTACTACCTCCTTCTGTATAATAATAC ACCTTCGCCGTAAAATGTGGAGAAGAAATCGGCACTAAAAAGTCAGGTAGACTGGAAAATGCGCCATGAAATGAATATCTCTTGCTACAGTAATGCCCAGCATCGAGGGGTATTGTGTCACCAACACTATAGTGGCAGCTGAAGCGCTCGTGATTGTAGTATGAGTCT TTATTGGTGATGGGAAGAGTTCACTCAATATTCTCGTTACTGCCAAAACACCACGGTAATCGGCCAGACACCATGGATGTAGATCACCAAGCCTGTGAATGTTATTCGAGCTA AAATGCACATGGTTGGTGAAAGGAGTAGTTGCTGTCGAATTCCGTCGTCGCCTGAGTCATCATTTATTTACCAGTTGGCCACAAACCCTTGACGATCTCGTATGTCCCCTCCG ACATACTCCCGGCCGGCTGGGGTACGTTCGATAGCGCTATCGGCATCGACAAGGT TTGGGTCCCTAGCCGATACCGCACTACCTGAGTCACAATCTTCGGAGGTTTAGTCT TCCACATAGCACGGGCAAAAGTGCGTATATATACAAGAGCGTTTGCCAGCCACAGATTTTCACTCCACACACCACATCACACATACAACCACACACATCCACAATGGAACCCGAAACTAAGAAGACCAAGACTGACTCCAAGAAGATTGTTCTTCTCGGCGGCGACTTCTGTGGCCCCGAGGTGATTGCCGAGGCCGTCAAGGTGCTCAAGTCTGTTGCTGAGGCCTCCGGCACCGAGTTTGTGTTTGAGGACCGACTCATTGGAGGAGCTGCCATTGAGAAGGAGGGCGAGCCCATCACCGACGCTACTCTCGACATCTGCCGAAAGGCTGACTCTATTATGCTCGGTGCTGTCGGAGGCGCTGCCAACACCGTATGGACGACTCCCGACGGACGAACCGACGTGCGACCCGAGCAGGGTCTCCTCAAGCTGCGAAAGGACCTGAACCTGTACGCCAACCTGCGACCCTGCCAGCTGCTGTCGCCCAAGCTCGCCGATCTCTCCCCCATCCGAAACGTTGAGGGCACCGACTTCATCATTGTCCGAGAGCTCGTCGGAGGTATCTACTTTGGAGAGCGAAAGGAGGATGACGGATCTGGCGTCGCTTCCGACACCGAGACCTACTCCGTTCCTGAGGTTGAGCGAATTGCCCGAATGGCCGCCTTCCTGGCCCTTCAGCACAACCCCCCTCTTCCCGTGTGGTCTCTTGACAAGGCCAACGTGCTGGCCTCCTCTCGACTTTGGCGAAAGACTGTCACTCGAGTCCTCAAGGACGAATTCCCCCAGCTCGAGCTCAACCACCAGCTGATCGACTCGGCCGCCATGATCCTCATCAAGCAGCCCTCCAAGATGAATGGTATCATCATCACCACCAACATGTTTGGCGATATCATCTCCGACGAGGCCTCCGTCATCCCCGGTTCTCTGGGTCTGCTGCCCTCCGCCTCTCTGGCTTCTCTGCCCGACACCAACGAGGCGTTCGGTCTGTACGAGCCCTGTCACGGATCTGCCCCCGATCTCGGCAAGCAGAAGGTCAACCCCATTGCCACCATTCTGTCTGCCGCCATGATGCTCAAGTTCTCTCTTAACATGAAGCCCGCCGGTGACGCTGTTGAGGCTGCCGTCAAGGAGTCCGTCGAGGCTGGTATCACTACCGCCGATATCGGAGGCTCTTCCTCCACCTCCGAGGTCGGAGACTTGTTGCCAACAAGGTCAAGGAGCTGCTCAAGAAGGAGTAAGTCGTTTCTACGACGCATTGATGGAAGGAGCAAACTGACGCGCCTGCGGGTTGGTCTACCGGCAGGGTCCGCTAGTGTATAAGACTCTATAAAAAGGGCCCTGCCCTGCTAATGAAATGATGATTTATAATTTACCGGTGTAGCAACCTTGACTAGAAGAAGCAGATTGGGTGTGTTTGTAGTGGAGGACAGTGGTACGTTTTGGAAACAGTCTTCTTGAAAGTGTCTTGTCTACAGTATATTCACTCATAACCTCAATAGCCAAGGGTGTAGTCGGTTTATTAAAGGAAGGGAGTTGTGGCTGATGTGGATAGATATCTTTAAGCTGGCGACTGCACCCAACGAGTGTTCTTTTGCCGATACATATGCACCATTAAATGATTAGAATGCGGGGATTGAGTTTCGGATTTTTCTTACATGCGTTGCGCGTTGGAGGTGATCCGAGCAGGTGGAATAGGTGTATATTGAGCGATTGGGAGAGTTGGTTGTGTACAATTATTTTAATACCTCTTCTGATTGTTTTCTATTGCCTTCCATTTCTATCTTTACCTGCCATCTCACGTCGTGTGTACCATCCCCACATACGGAACCAGTAGGTCTTTTAGGCTCTGAACGTGCAAATGAGTTTGGTGGGGTAGGCAGAGATCGCATAGAGACGGGTAGAATGAGCAGTTAAAAGCTGTGTTGAGTGGTAAAAATTTACAATAAGTGTTCCTCAAGGCATCAAGGAGACGAAATAAGCCATTATGGACACGAACCAACAGTCCCACCACGTTCTAAACACATTCCTCCACTGCCACTCCCAAACACCACGTCCCACATAAACTTCTACCCCACATTTTGACAAGCCTATTCGTTTAATAATCACCCCGAGGAGACAGAAAGCCTAACAGCTGGAGCCACTATATAGTTGCAGTGGCATGCTCTTTCATTGAAACGACCCACAACAATGATCACCCCAAACCCCGCTAACGACATTGTCCATGACGGCAAGCTCTACGACACCTTCACTGAGCCCCCCAAGCTGATGGCTCAGGAGCGAGCTCAGCTGGACTTCGACCCTAGAGACATCACCTACTTTCTGGATGGCTCTAAGGAGGAGACCGAGCTGCTGGAGTCGCTCATGCTCATGTAGGAGCGAGACCCTCTCTTCAACAACCAGAACGAGTACGATGAATCGTTTGAAACACTGCGAGAGCGATCTGTGAAGCGAATTTTCCAGCTGTCCAAGTCCATCGCCATGGACCCCGAGCCCATGTCTTTCCGAAAGATTGGGTTCCTGGGTATTCTTGACATGGGAACGTATGCTCGACTGGGAGTCCACTACGCGCTCTTCTGTAACTCCATCCGGGGCCAGGGAACCCCCGATCAGCTCATGTACTGGCTGGACCAGGGAGCCATGGTCATCAAGGGCTTCTACGGCTGTTTTGCCATGACCGAAATGGGCCATGGATCTAACCTGTCGCGTCTGGAAACCATCGCCACTTTCGACAAAGAGACCGACGAATTTATCATTAACACGCCCCACGTTGGAGCCACAAAGTGGTGGATTGGAGGAGCCGCCCACACTGCTACTCACACACTTGCCTTTGCCCGTCTTCAAGTAGACGGAAAGGACTACGGTGTGAAATCGTTTGTCGTACCTCTCCGAAACCTGGACGACCATTCGCTGCGTCCTGGAATCGCCACAGGTGATATTGGTAAGAAGATGGGTCGAGATGCCGTTGACAACGGCTGGATTCAGTTCACCAACGTCCGAGTGCCCCGAAACTACATGCTCATGAAGCATACCAAGGTTCTTCGAGACGGTACCGTCAAGCAGCCGCCTTTGGCCCAACTGACTTACGGATCTCTCATCACTGGACGAGTCCAGATGACCACTGACTCTCACAATGTGTCCAAAAAGTTCCTCACCATTGCCCTGAGATACGCCACCATCCGACGACAGTTCTCGTCAACTCCAGGAGAGCCCGAAACCCGACTAATTGACTACCTGTACCACCAAAGACGACTCCTGCCTCTTATGGCTTACTCTTACGCCATGAAACTAGCTGGAGATCACGTCCGAGAGCTGTTCTTTGCATCCCAGGAGAAGGCTGAGAGGCTCAAGGAGGACGACAAAGCCGGAGTTGAGTCTTACGTCCAGGATATCAAGGAGCTCTTCTCTGTTTCTGCTGGTCTCAAGGCTGCCACTACATGGGCTTGTGCTGACATCATTGACAAGGCCCGACAGGCGTGTGGAGGCCACGGATACTCTGCCTACAACGGCTTTGGACAGGCCTTCCAGGACTGGGTTGTCCAGTGCACTTGGGAGGGTGACAATACTGTTCTGACTCTATCTGCCGGCCGAGCTCTGATCCAATCTGCTCTCGTCTACCGAAAGGAGGGCAAACTAGGTAACGCCACGAAGTACCTCTCTCGGTCCAAGGAGCTTGCCAACGCCAAGAGAAACGGACGATCCCTGGAAGACCCCAAGCTGCTCGTGGAGGCATGGGAGGCTGTCTCTGCCGGTGCTATCAACGCTGCTACTGACGCTTACGAGGAGCTCTCCAAGCAGGGAGTTTCTGTTGACGAGTGCTTTGAGCAGGTGTCCCAGGAGCGATTCCAGGCTGCCCGAATCCACACTCGACGAGCTCTTATCGAGGCCTTCTACTCACGAATCGCCACTGCTGATGAGAAGGTGAAGCCTCATCTGATCCCTCTGGCCAACCTGTTTGCCCTGTGGTCCATTGAGGAGGACTCTGCTCTGTTCCTGGCTGAGGGCTACTTTGAGCCTGAGGATATCATTGAGGTGACTTCTCTTGTCAACAAGTACTGCGGAATTGTTCGAAAGAACGTTATTGGATACACCGATGCCTTCAACCTGTCCGACTACTTCATCAACGCTGCCATTGGACGATACGACGGAGACGTGTAGAAGAACTACTTTGAGAAGGTCAA ACAGCAGTACCCTCCTGAGGGTGGCAAGCCTCACTACTACGAGGATGTCATGAAG CCCTTCCTGCATCGAGAGCGAATTCCCGATGTCCCCATGGAGCCCGAGGATATTCA GTAATGATTTTGTAGGTTTTAGTAAGTATTAACGTTTTTTATGATTTGGAGTTGTGTG TAACTTGTAGAGGTAGACCTACATAGTGTAGTGTAGGTCCAAAGATAGGTACACTGT GGCAATAATTATGCGAGTACTTGTACCGTCATCGTAGCTGCTGTAAAGAGATCAGA CACAGGCACTTTTCCCCACCATGAGATCACCACTCGTCGTCCGAGTACTTCTATGG CACAGCCACAATCACATGTACTTGTGCATGCCAATGTGTGACATCATCATCTAGAG CTATCATCATATTCCCGCTGCAAATGGTCTACGTATTACTATTAAGCAGGGGGGGG GGAGGAATTATGACGACATTGTACGTGTACTCGTACCGGTACTTGTAGCACGCCGA ACTGCGGTATTACTGTGCACTGTAATTTCGGACCCCTCTTATAGCCCCAAGTTGGT CTATACATCTGAACCGGTGCAGACTCACTATTAAAAGTGCGGCAGCTAATTTTGCTG ACACAGCCTTGTCGATAAAAGTAGCTACTTGTACGATGTACTCTTGTAGCTTATGCG TGGGCTATCATTAATTTTAGACAACAGTGTCCTACCACGCCACGGAACGGGAGAAA GCAGAAGATCCGAACATCTTGTGCGGAGTGTGTTGCGTCTGTGACACCGGTGAAG TTCCGTTGTCCATGACCCGCCGATCTTTTTTTTGGCATATGGTTTGCATCGCCTCGC AGACAAGAAAGGGCGAAGGTAAATGCAGCACATGTTAGAAGTGCCTGGCATGGGA CATACACGCGGACGTTTGTGCAGTTGTCAGCAGCGTGTTTATCG

[0126] The C / al DNA fragment of plasmid pPOX4-LEU2 containing LEU2, P0X4 genes (Fig. 7, SEQ ID NO: 16, table 3) was transformed into strain AH1314 (Table 1 ). The transformed cells were growing on CM glucose minus leucin plates (0.13% Amino acid dropout powder minus leucine, 0.17% yeast nitrogen base, 0.5% ammonium sulfate, 2.0% glucose, 2.1% agar). The colonies from transformation were analyzed by PGR.

[0127] A PGR fragment of 2,743 bp (SEQ ID NO: 17) was produced with primers of P240 (AATGATAGCCCACGCATAAGCTACAAGAG), referred to as SEQ ID No. 18 and P241 (CCTAACA-GCTGGAGCCACTATATAGTTG), referred to as SEQ ID. No. 19, indicating the DNA fragment (Fig. 7) with wild type POX4 integrated into cells. Thus, a colony with PCR positive was designated as strain AH246 (dgatl-, dgat2-, pex3-, ura3-).

[0128] Figure 7 provides a physical and functional map of the C / al large fragment of plasmid pPOX4-LEU2

[0129] The DNA sequence of the 2,743 bp PCR fragment is shown, below, as SEQ ID NO: 17:GCCTAACAGCTGGAGCCACTATATAGTTGCAGTGGCATGCTCTTTCATTGAAACGA CCCACAACAATGATCACCCCAAACCCCGCTAACGACATTGTCCATGACGGCAAGCT CTACGACACCTTCACTGAGCCCCCCAAGCTGATGGCTCAGGAGCGAGCTCAGCTG GACTTCGACCCTAGAGACATCACCTACTTTCTGGATGGCTCTAAGGAGGAGACCGAGCTGCTGGAGTCGCTCATGCTCATGTACGAGGGAGACCGTCTCTTCAACAACCAGAACGAGTACGATGAATCGTTTGAAACACTGCGAGAGCGATCTGTGAAGCGAATTTTCCAGCTGTCCAAGTCCATCGCCATGGACCCCGAGCCCATGTCTTTCCGAAAGATTGGGTTCCTGGGTATTCTTGACATGGGAACGTATGCTCGACTGGGAGTCCACTACGCGCTCTTCTGTAACTCCATCCGGGGCCAGGGAACCCCCGATCAGCTCATGTACTGGCTGGACCAGGGAGCCATGGTCATCAAGGGCTTCTACGGCTGTTTTGCCATGACCGAAATGGGCCATGGATCTAACCTGTCGCGTCTGGAAACCATCGCCACTTTCGACAAAGAGACCGACGAATTTATCATTAACACGCCCCACGTTGGAGCCACAAAGTGGTGGATTGGAGGAGCCGCCCACACTGCTACTCACACACTTGCCTTTGCCCGTCTTCAAGTAGACGGAAAGGACTACGGTGTGAAATCGTTTGTCGTACCTCTCCGAAACCTGGACGACCATTCGCTGCGTCCTGGAATCGCCACAGGTGATATTGGTAAGAAGATGGGTCGAGATGCCGTTGACAACGGCTGGATTCAGTTCACCAACGTCCGAGTGCCCCGAAACTACATGCTCATGAAGCATACCAAGGTTCTTCGAGACGGTACCGTCAAGCAGCCGCCTTTGGCCCAACTGACTTACGGATCTCTCATCACTGGACGAGTCCAGATGACCACTGACTCTCACAATGTGTCCAAAAAGTTCCTCACCATTGCCCTGAGATACGCCACCATCCGACGACAGTTCTCGTCAACTCCAGGAGAGCCCGAAACCCGACTAATTGACTACCTGTACCACCAAAGACGACTCCTGCCTCTTATGGCTTACTCTTACGCCATGAAACTAGCTGGAGATCACGTCCGAGAGCTGTTCTTTGCATCCCAGGAGAAGGCTGAGAGCCTCAAGGAGGACGACAAAGCCGGAGTTGAGTCTTACGTCCAGGATATCAAGGAGCTCTTCTCTGTTTCTGCTGGTCTCAAGGCTGCCACTACATGGGCTTGTGCTGACATCATTGACAAGGCCCGACAGGCGTGTGGAGGCCACGGATACTCTGCCTACAACGGCTTTGGACAGGCCTTCCAGGACTGGGTTGTCCAGTGCACTTGGGAGGGTGACAATACTGTTCTGACTCTATCTGCCGGCCGAGCTCTGATCCAATCTGCTCTCGTCTACCGAAAGGAGGGCAAACTAGGTAACGCCACGAAGTACCTCTCTCGGTCCAAGGAGCTTGCCAACGCCAAGAGAAACGGACGATCCCTGGAAGACCCCAAGCTGCTCGTGGAGGCATGGGAGGCTGTCTCTGCCGGTGCTATCAACGCTGCTACTGACGCTTACGAGGAGCTCTCCAAGCAGGGAGTTTCTGTTGACGAGTGCTTTGAGCAGGTGTCCCAGGAGCGATTCCAGGCTGCCCGAATCCACACTCGACGAGCTCTTATCGAGGCCTTCTACTCACGAATCGCCACTGCTGATGAGAAGGTGAAGCCTCATCTGATCCCTCTGGCCAACCTGTTTGCCCTGTGGTCCATTGAGGAGGACTCTGCTCTGTTCCTGGCTGAGGGCTACTTTGAGCCTGAGGATATCATTGAGGTGACTTCTCTTGTCAACAAGTACTGCGGAATTGTTCGAAAGAACGTTATTGGATACACCGATGCCTTCAACCTGTCCGACTACTTCATCAACGCTGCCATTGGACGATACGACGGAGACGTGTACAAGAACTACTTTGAGAAGGTCAAACAGCAGTACCCTCCTGAGGGTGGCAAGCCTCACTACTACGAGGATGTCATGAAGCCCTTCCTGCATCGAGAGCGAATTCCCGATGTCCCCATGGAGCCCGAGGATATTCAGTAATGATTTTGTAGGTTTTAGTAAGTATTAACGTTTTTTATGATTTGGAGTTGTGTGTAACTTGTACAGGTACAGCTACATACTGTACTGTAGGTGCAAAGATAGGTACACTGTGGCA ATAATTATGCGAGTACTTGTACCGTCATCGTAGCTGCTGTAAAGAGATCAGACACA GGCACTTTTCCCCACCATGAGATCACCACTCGTCGTCCGAGTACTTCTATGGCACA GCCACAATCACATGTACTTGTGCATGCCAATGTGTGACATCATCATCTAGAGCTATC ATCATATTCCCGCTGCAAATGGTCTACGTATTACTATTAAGCAGGGGGGGGGGAGG AATTATGACGACATTGTACGTGTACTCGTACCGGTACTTGTAGCACGCCGAACTGC GGTATTACTGTGCACTGTAATTTCGGACCCCTCTTATAGCCCCAAGTTGGTCTATAC ATCTGAACCGGTGCAGACTCACTATTAAAAGTGCGGCAGCTAATTTTGCTGACACA GCCTTGTCGATAAAAGTAGCTACTTGTACGATGTACTCTTGTAGCTTATGCGTGGG CTATCATEXAMPLE 3: Generation of strain AH292 from strain AH291 for deletion of PEX 20

[0130] Strain AH292 (table 1 , Figs. 2 & 3) with pex20 deletion was generated from strain AH291 (dgatl-, dgat2-, ura3-, table 1 ) by deletion of PEX20 gene encoding proteins (SEQ ID NOs: 20 & 21).

[0131] The amino acid sequence of the Y. lipolytica PEX20 (GenBank Acc. No. XP_503644, YALI0_E06831g) polypeptide is shown, below, as SEQ ID NO: 20:

[0132] MASCGPSNALQNLSKHASADRSLQHDRMAPGGAPGAQRQQFRSQTQGG QLNNEFQQFAQAGPAHNSFEQSQMGPHFGQQHFGQPHQPQMGQHAPMAHGQQSD WAQSFSQLNLGPQTGPQHTQQSNWGQDFMRQSPQSHQVQPQMANGVMGSMSGM SSFGPMYSNSQLMNSTYGLQTEHQQTHKTETKSSQDAAFEAAFGAVEESITKTSDKGK EVEKDPMEQTYRYDQADALNRQAEHISDNISREEVDIKTDENGEFASIARQIASSLEEA DKSKFEKSTFMNLMRRIGNHEVTLDGDKLVNKEGEDIREEVRDELLREGASQENGFQS EAQQTAPLPVHHEAPPPEQIHPHTETGDKQLEDPMVYIEQEAARRAAESGRTVEEEKL NFYSPFEYAQKLGPQGVAKQSNWEEDYDF

[0133] The DNA sequence of the Y. / / joolytica PEX20 (GenBank Acc. No. XP_503644, YALI0_E06831g) is shown, below, as SEQ ID NO: 21 : ATGGCATCTTGCGGACCTTCTAACGCCCTACAAAACCTGTCGAAACATGCGTCAGC AGATCGATCGCTTCAGCATGACCGAATGGCCCCCGGTGGCGCTCCTGGCGCTCAG CGACAGCAGTTTCGATCTCAGACCCAAGGAGGACAGCTCAACAATGAGTTCCAGCA GTTTGCCCAAGCAGGACCGGCCCATAACTCATTTGAACAGTCTCAGATGGGCCCAC ATTTTGGCCAGCAACATTTTGGCCAGCCCCATCAACCCCAGATGGGTCAGCACGCC CCCATGGCACACGGTCAACAAAGTGACTGGGCCCAGTCTTTCAGTCAACTGAACCT GGGTCCTCAAACCGGCCCTCAGCATACTCAGCAGTCCAACTGGGGTCAAGATTTTA TGCGACAAAGTCCCCAGTCACACCAGGTCCAGCCCCAGATGGCTAACGGTGTGAT GGGCAGCATGTCTGGTATGTCCAGCTTTGGACCCATGTACTCCAACTCACAGCTCATGAACTGCACGTACGGCCTTCAAACCGAACATCAGCAGACACACAAGACGGAGAC GAAGAGCTCCCAGGATGCAGCGTTCGAGGCCGCCTTTGGAGCAGTGGAGGAGTC CATCACCAAGACGTCAGACAAGGGCAAGGAGGTGGAAAAGGACCCCATGGAGCAA ACATACCGCTACGACCAGGCTGACGCTCTCAACCGACAGGCCGAGCACATTTCGG ACAATATTTCGCGAGAAGAGGTGGATATCAAGAGGGACGAGAACGGCGAGTTTGC ATCGATTGCGCGCCAGATTGCGTCTTCGCTGGAAGAGGCCGATAAGTCGAAGTTT GAAAAGTCAACCTTCATGAACCTGATGCGGCGAATCGGCAACCACGAAGTCACTTT GGATGGAGACAAATTGGTCAACAAGGAAGGAGAGGACATCCGAGAGGAAGTCAGA GACGAGCTACTTCGAGAGGGTGCTTCTCAGGAGAATGGATTCCAGTCCGAGGCTC AACAGACTGCTCCCCTTCCTGTTCATCATGAAGCCCCTCCACCTGAACAAATTCATC CTCATACCGAGACTGGAGACAAACAGCTGGAGGATCCCATGGTGTACATTGAGCA GGAGGCAGCTCGTCGGGCTGCCGAGTCTGGACGAACTGTCGAGGAGGAAAAGCT CAACTTCTACTCGCCCTTTGAGTACGCCCAGAAGCTGGGCCCTCAGGGCGTTGCT AAGCAGAGCAACTGGGAGGAGGACTACGACTTTTGA

[0134] Pop-in and pop-out approach. The PEX20 was deleted by the pop-in and pop- out approach disclosed in Leon, et al., Yeast, 19:1003-1014), the disclosure of which is incorporated by reference herein and illustrated in Fig. 8.

[0135] Specifically, plasmid pYRH-YPEX20-5-3N (SEQ ID NO: 22) was linearized with restriction enzyme BglW and transformed into strain AH291 Table 1 , Fig. 2 & 3).

[0136] The DNA sequence of the plasmid pYRH-YPEX20-5-3N is shown, below, as SEQ ID NO: 22:

[0137] CCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTC CTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCT CACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAA CATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTG GCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAG TCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGA AGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCG CCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTC AGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTC AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGA CACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGT ATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGA AGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGT TGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACGTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGCCCGACGTCGCGATCGCGAGTATCTGTCTGACTCGTCATTGCCGCCTTTGGAGTACGACTCCAACTATGAGTGTGCTTGGATCACTTTGACGATACATTCTTCGTTGGAGGCTGTGGGTCTGACAGCTGCGTTTTCGGCGCGGTTGGCCGACAACAATATCAGCTGCAACGTCATTGCTGGCTTTCATCATGATCACATTTTTGTCGGCAAAGGCGACGCCCAGAGAGCCATTGACGTTCTTTCTAATTTGGACCGATAGCCGTATAGTCCAGTCTATCTATAAGTTCAACTAACTCGTAACTATTACCATAACATATACTTCACTGCCCCAGATAAGGTTCCGATAAAAAGTTCTGCAGACTAAATTTATTTCAGTCTCCTCTTCACCACCAAAATGCCCTCCTACGAAGCTCGAGCTAACGTCCACAAGTCCGCCTTTGCCGCTCGAGTGCTCAAGCTCGTGGCAGCCAAGAAAACCAACCTGTGTGCTTCTCTGGATGTTACCACCACCAAGGAGCTCATTGAGCTTGCCGATAAGGTCGGACCTTATGTGTGCATGATCAAAACCCATATCGACATCATTGACGACTTCACCTACGCCGGCACTGTGCTCCCCCTCAAGGAACTTGCTCTTAAGCACGGTTTCTTCCTGTTCGAGGACAGAAAGTTCGCAGATATTGGCAACACTGTCAAGCACCAGTACCGGTGTCACCGAATCGCCGAGTGGTCCGATATCACCAACGCCCACGGTGTACCCGGAACCGGAATCATTGCTGGCCTGCGAGCTGGTGCCGAGGAAACTGTCTCTGAACAGAAGAAGGAGGACGTCTCTGACTACGAGAACTCCCAGTACAAGGAGTTCCTAGTCCCCTCTCCCAACGAGAAGCTGGCCAGAGGTCTGCTCATGCTGGCCGAGCTGTCTTGCAAGGGCTCTCTGGCCACTGGCGAGTACTCCAAGCAGACCATTGAGCTTGCCCGATCCGACCCCGAGTTTGTGGTTGGCTTCATTGCCCAGAACCGACCTAAGGGCGACTCTGAGGACTGGCTTATTCTGACCCCCGGGGTGGGTCTTGACGACAAGGGAGACGCTCTCGGACAGCAGTACCGAACTGTTGAGGATGTCATGTCTACCGGAACGGATATCATAATTGTCGGCCGAGGTCTGTACGGCCAGAACCGAGATCCTATTGAGGAGGCCAAGCGATACCAGAAGGCTGGCTGGGAGGCTTACCAGAAGATTAACTGTTAGAGGTTAGACTATGGATATGTAATTTAACTGTGTATATAGAGAGCGTGCAAGTATGGAGCGCTTGTTCAGCTTGTATGATGGTCAGACGACCTGTCTGATCGAGTATGTATGATACTGCAGAACCTGTGTATCCGCATGATCTGTCCAATGGGGCATGTTGTTGTGTTTCTCGATACGGAGATGCTGGGTACAGTGCTAATACGTTGAACTACTTATACTTATATGAGGCTCGAAGAAAGCTGACTTGTGTATGACGCATGCGTCACTTGGAGGTTGCCCAATTGTATTTGCTGTGACGGAACTCTATTCTGACTAAGCTAACTCCCAACCTTGGAAAAAGGTGACCAATGAGAGGGGACAGTTGGAGGGGGTCTGTCGGGAATGCCACAGAAACGACTCAGAAAAAGAAAAGGATGATCTGGTGTGATCATGAAATTTGAGTGTGGGTCCTCTATTTGACTTTGTCAGTGGCACGTCTCCTTCACCAATTACAAGGACATGTTTGGACAACATAGAGTTGAGGGAGTTACTAAGCCGCCTTCATTGTAAGGAAGTACAGTACTGTAGAGCTGTGTGTTTCTGAGAGTCGAGAGTGCGATAAGAAGACCAAAATCAATATTGGCCGTTGTTGCTACAAGTAGTTGATTCCTTTTCTGAAAACCTCCGGCTTTCATTCTTTCTAGCTTTGAAAACTGAAATTGGTCGTTATGAATCGTCCATACTTCATCTACTGCTACACACATGCTGCAAGTAATATATACACATCGATATAATCTCCAGGTGAGCTGCTACAAAGTGGAATTGCTTTTGCTTCTATAATGCTACTACAGTATGTACATCTCCTCCCGCAACACGGCATCCCCGGCAAAAACGCCAAAGGGGTCAAGTAAGTTACGTTTGTTGAGGGTGTCACTACTACAAGTACAATACGAGTAGTAAAGCTCACCCCAGCAGATCTCTAAAGTACACAGCTGCAACAATTTCTACAAACTTCACTTCACCGCTGGTTTCTCTATCACGTGACAACCCCTTCGCCTGTACCTCGGTATCTCATCTCAAATCACGATGGTTAGTAAACTTTTATGAAATAGGCTCGGCGAGCCGTTAATCAGGCCAGACAAGTTCCGACAGGTAACCGAGGTATAATTTGGGGGCTTGGTGGCGACTGTGTAAGAATAATAAGGGTCTGTGTGTATTTTTCATGGGGTTCCAAATGTATTGATTATCTAAAATCTGAACATTTTCCCCTCGCGTACCCCACATTTGACCCTACCTCTCACTTCTCCACATTCATTCACGGACACAAGGTTCACCGAACCCCACAGAAATATAACCACAACCTAAAGCTAGCGACAGCTGAAGTACAGTAACTATTTAGACATCAGCTTGACAGACAACTTGACCCGCCATACCCCAGTCACTGAGCGCAGTTAGACGGAGAGAAGAGCTACGCAACCACAAGGAGCTCACGACACAACACAGACTCGAACTGTCCTGATACACCACACACATCGCGCAAGCTGATTCACACACACAACCATGGCATCTTGCGGACCTTCTACTACGACTTTTGAGGAAGTGTGGATGGGGAAGTGAGTGCCCGGTTCTGTGTGCACAATTGGCAATCCAAGATGGATGGATTCAACACAGGGATATAGCGAGCTACGTGGTGGTGCGAGGATATAGCAACGGATATTTATGTTTGACACTTGAGAATGTACGATACAAGCACTGTCCAAGTACAATACTAAACATACTGTACATACTCATACTCGTACCCGGGCAACGGTTTCACTTGAGTGCAGTGGCTAGTGCTCTTACTCGTACAGTGTGCAATACTGCGTATCATAGTCTTTGATGTATATCGTATTCATTCATGTTAGTTGCGTCAATCGTCAAATATAGCTATGCGTGAGTTTGTATCTCTATCGCACCTGTGCACAACTACTGTAACATGCAATCTGCTTATGTTGGCATGGTCTGCTCATGCATCTGGTTAGAATCTTGTTCTTTCCTGTTCAGCAGGAACCTCATAGGTATTTATCTTTGGATACTTCTCCATCTCGTAGTCTGTATCGTCCACTGACCTGTTCACATAATAGTCAGGAGCAAAGTTGTTATTGGACTGGTTTCCGTAAACCGGCTGTGGGTGATAATTGTATGCCTGGTAAGGATTTGAAGCTGGGTACATGGCTGGGTTATTTGCTGCTGCGTAGTTACCTTGGTTGGCCTCTCTGTTGATCTTATCGTTACAGCAGCAGGCACAGCAAGAGCACAGCGCACAACAGGCGCTGAAAGCAGATACTCCAAAGAAGCAGCATCTGAAAAAGGCTGTGAGTACCCACAGAACCAGCATTCCTCCCAGAACAATTCCCACAATCGCCACAATCTTGCAAAACGTGTTATCCATGGATGTATCCCAGCTCTTGAAAGCTTCTCCAGTTGATTTGGCAGTTTGTGCTAATCCTCTTTTTGAGAAGCTGATGAGTGGTTGGGCCATAGCCTGTGGACAGAGTTGGATCATTGGAGCCATGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGTGTGTGGAACTGTGTGTGTAAGTTGATTCGGTATTGCGTGCCTGAAACTGCAGATGGTGTATCAGTTATAGGTCTTCCAGAAGTGATATAAACAGTATGATTACAAGCAATGAAAATGACAGGTCAACACTCTATATAATATCTACGGTGCCACATTTCTATGTTCCCAAAATAGACCATGCCAAGTTAGCTTGGGACCAACACGGCTGTGCACTTAGTGTTATGGAGGCTATGCTGGGCGGTGTAGATGGGGGTTGTACATGCCATGAGGTGAACAAAAAAGCAGAGGAATCACGTGACTGTATCTTTAAAGGACATTTCCTGGGCTAAATCCGTCTATAACTCATTACTCCCACACCTTCCTATCAACCTCTCCCCCCACAAGTCGTATTATGAAACC CAACTCCTATTACAGTCGAGATGCGTTATGAAACACGAGTTTATTTGTGCGTTTGCC TTTCATGGCAACCTGGAGAAGCGGTTGAGTACAGTGCGTAGTTGGAGGCGCTAGT GTGTGGATTGGTGGGTGCTTTTAGAGACAGCTTCTGTACGTTTGTCACGTGAATAA TGTAGAGTGTCGCTCATCTTCATATGTACATATTGATACTACTGTAGGTGAATACTT GAACAGACGTTTGTCAACTGAACTCAATCAGTGAAGTGATGACATCACATGCATGTA ATTTCACATAGTCATTGGTATCTGTAAAGATGATGTAACATCAATGGCAGGTAAAAA TCTCAGTCTTGTACCACTGTTAGTGATTCCAATTTTAGATCGGTTCGGCGCGCCAG CTGCATTAATGAATCGG

[0138] Figure 8 is an illustration of PEX20 deletion by a Pop-in and Pop-out approach.

[0139] The transformed cells were growing on CM glucose minus uracil plates (0.13% amino acid dropout mixture minus uracil, with 0.17% yeast nitrogen base, 2.0% glucose, 0.5% ammonium sulfate, and 2.1% agar). The colonies from transformation were analyzed by PCR with primers of YPEX20-5-confirm (GATTCTAGTATATCCAAATACC), referred to as SEQ ID. No. 23 and YPEX20-3-confirmA (AAATATCCGTTGCTATATCCTC), referred to as SEQ ID No. 24.

[0140] Transformant with a PCR fragment of 1 ,436 bp (SEQ ID No. 25) indicates the BglW digested fragment integrated into right chromosome position (Fig. 8).

[0141] The DNA sequence of the 1 ,436 bp PCR fragment is shown, below, as SEQ ID NO: 25:GATTCTAGTATATCCAAATACCAAATATTCCTGCGTCACTTGGAGGTTGCCCAATTG TATTTGCTGTGACGGAACTCTATTCTGACTAAGCTAACTCCCAACCTTGGAAAAAGG TGACCAATGAGAGGGGACAGTTGGAGGGGGTCTGTCGGGAATGCCACAGAAACGA CTCAGAAAAAGAAAAGGATGATCTGGTGTGATCATGAAATTTGAGTGTGGGTCCTC TATTTGACTTTGTCAGTGGCACGTCTCCTTCACCAATTACAAGGACATGTTTGGACA ACATAGAGTTGAGGGAGTTACTAAGCCGCCTTCATTGTAAGGAAGTACAGTACTGT AGAGCTGTGTGTTTCTGAGAGTCGAGAGTGCGATAAGAAGACCAAAATCAATATTG GCCGTTGTTGCTACAAGTAGTTGATTCCTTTTCTGAAAACCTCCGGCTTTCATTCTT TCTAGCTTTGAAAACTGAAATTGGTCGTTATGAATCGTCCATACTTCATCTACTGCTA CACACATGCTGCAAGTAATATATACACATCGATATAATCTCCAGGTGAGCTGCTACA AAGTGGAATTGCTTTTGCTTCTATAATGCTACTACAGTATGTACATCTCCTCCCGCA ACACGGCATCCCCGGCAAAAACGCCAAAGGGGTCAAGTAAGTTACGTTTGTTGAG GGTGTCACTACTACAAGTACAATACGAGTAGTAAAGCTCACCCCAGCAGATCTCTA AAGTACACAGCTGCAACAATTTCTACAAACTTCACTTCACCGCTGGTTTCTCTATCA CGTGACAACCCCTTCGCCTGTACCTCGGTATCTCATCTCAAATCACGATGGTTAGT AAACTTTTATGAAATAGGCTCGGCGAGCCGTTAATCAGGCCAGACAAGTTCCGACAGGTAACCGAGGTATAATTTGGGGGCTTGGTGGCGACTGTGTAAGAATAATAAGGGT CTGTGTGTATTTTTCATGGGGTTCCAAATGTATTGATTATCTAAAATCTGAACATTTT CCCCTCGCGTACCCCACATTTGACCCTACCTCTCACTTCTCCACATTCATTCACGGA CACAAGGTTCACCGAACCCCACAGAAATATAACGACAACCTAAAGCTAGCGACAGC TGAAGTACAGTAACTATTTAGACATCAGCTTGACAGACAACTTGACCGGCCATACCC CAGTCACTGAGCGCAGTTAGACGGAGAGAAGAGCTACGCAACCACAAGGAGCTCA CGACACAACACAGACTCGAACTGTCCTGATACACCACACACATCGCGCAAGCTGAT TCACACACACAACCGGAAGTGTGGATGGGGAAGTGAGTGCCCGGTTCTGTGTGCA CAATTGGCAATCCAAGATGGATGGATTCAACACAGGGATATAGCGAGCTACGTGGT GGTGCGAGGATATAGCAACGGATATTT

[0142] The following describes some definitions for some of the reagents used.

[0143] YPD: (Yeast extract, Peptone and Dextrose (glucose)

[0144] FOA (5-Fluoroorotic Acid)

[0145] To pop-out the fragment containing the PEX20, URA3, and Amp genes, a PGR positive transformant was growing in 2ml of YPD liquid media at 32° C overnight, then 20 ul cells were spread on CM with uracil and FOA (450 ug / L) plate, and the plate was incubated at 32° C for 3 days. The ura3 phenotype would indicate that the two PEX20-3’ arms had popped out.

[0146] Only popped-out cells could grow on CM with uracil and FOA (450 ug / L) plate. Colonies were re-patched on a new CM with uracil and FOA (450 ug / L) plate.

[0147] Re-patched colonies were analyzed by PCR with primers of YPEX20-5- confirmA (SEQ ID NO: 26) and YPEX20-3-confirmA (SEQ ID NO: 27).

[0148] Strains with a PCR fragment of 249 bp (SEQ ID NO: 28) indicate the pex20 was deleted. Thus, a colony with PCR positive was designated as strain AH292 (dgatl-, dgat2-, pex20-, ura3-).

[0149] The DNA sequence of the 249 bp PCR fragment is shown, below, as SEQ ID NO: 28:CAGTCACTGAGCGCAGTTAGACGGAGAGAAGAGCTACGCAACCACAAGGAGCTCA CGACACAACACAGACTCGAACTGTCCTGATACACCACACACATCGCGCAAGCTGAT TCACACACACAACCGGAAGTGTGGATGGGGAAGTGAGTGCCCGGTTCTGTGTGCA CAATTGGCAATCCAAGATGGATGGATTCAACACAGGGATATAGCGAGCTACGTGGT GGTGCGAGGATATAGCAACGGATATTTEXAMPLE 4: Generation of strain AH256 having double deletion of pex3 and pex5 from strain AH246

[0150] Strain AH256 (Table 1 , Fig. 2 & 3) with double deletion of pex3 (SEQ ID NO: 4 and 5) and pex5 (SEQ ID NO: 29 and 30) was generated from strain AH246 (dgatl-, dgat2-, pex3-, ura3-) by deletion of pex5 gene.

[0151] The pex5 was deleted by Pop-in and pop-out approach as illustrated in Fig. 9. Specifically, the Sa / I DNA fragment containing PEX5-5’ and PEX5-3’ arms of plasmid pYRH-PEX5-5-3N (SEQ ID NO.31 , Fig. 9) was transformed into strain AH246.

[0152] The amino acid sequence of the Yarrowia PEX5 gene (GenBank Acc. NO. KAG5370090, YALI0F28457g) is shown, below, as SEQ ID NO: 29:

[0153] MSFMRGGSECSTGRNPLSQFTKHTAEDRSLQHDRVAGPSGGRVGGMRS NTGEMSQQDREMMARFGAAGPEQSSFNYEQMRHELHNMGAQGGQIPQVPSQQGAA NGGQWARDFGGQQTAPGAAPQDAKNWNAEFQRGGSPAEAMQQQGPGPMQGGMGMGGMPMYGMARPMYSGMSANMAPQFQPQQANARVVELDEQNWEEQFKQMDSAVG KGKEVEEQTAETATATETVTETETTTEDKPMDIKNMDFENIWKNLQVNVLDNMDEWLE ETNSPAWERDFHEYTHNRPEFADYQFEENNQFMEHPDPFKIGVELMETGGRLSEAAL AFEAAVQKNTEHAEAWGRLGACQAQNEKEDPAIRALERCIKLEPGNLSALMNLSVSYT NEGYENAAYATLERWLATKYPEVVDQARNQEPRLGNEDKFQLHSRVTELFIRAAQLSP DGANIDADVQVGLGVLFYGNEEYDKAIDCFNAAIAVRPDDALLWNRLGATLANSHRSE EAIDAYYKALELRPSFVRARYNLGVSCINIGCYKEAAQYLLGALSMHKVEGVQDDVLAN QSTNLYDTLKRVFLGMDRRDLVAKVGNGMDVNQFRNEFEF

[0154] The DNA sequence of the Yarrowia PEX5 gene (GenBank Acc. NO. KAG5370090, YALI0F28457g) is shown, below, as SEQ ID NO: 30:

[0155] ATGTCGTTTATGAGAGGAGGAAGCGAATGCTCTACGGGCAGAAACCCC CTGAGCCAGTTCACCAAACACACCGCTGAGGACCGATCCCTCCAGCATGATCGGG TGGCGGGTCCCTCTGGAGGCCGAGTTGGAGGTATGCGATCCAACACTGGCGAGATGTCACAGCAGGACCGAGAGATGATGGCGCGATTCGGTGCTGCCGGACCCGAGCA GTCGTCTTTCAACTACGAGCAGATGCGACATGAGCTCCACAACATGGGTGCCCAAG GAGGCCAGATTCCCCAGGTTCCCAGCCAGCAGGGCGCTGCTAACGGAGGACAGT GGGCCCGAGACTTTGGAGGACAACAGACCGCTCCCGGCGCTGCTCCCCAGGACG CCAAGAAGTGGAACGCCGAGTTCCAGGGAGGAGGATCTCGTGCAGAGGCCATGCA ACAGCAGGGTCCCGGCCCCATGCAAGGCGGCATGGGTATGGGGGGAATGCCCAT GTACGGCATGGCTCGTCCCATGTACTCTGGAATGAGTGCCAACATGGCTCCTCAGT TCCAGCCCCAGCAGGCTAACGCACGAGTTGTCGAGCTGGACGAGCAGAACTGGGA GGAGCAGTTCAAGCAGATGGACTCTGCCGTTGGCAAGGGTAAGGAGGTCGAGGA GCAGACTGCCGAGACTGCTACTGCCACCGAGACTGTCACCGAGACTGAAACCACTAGTGAGGACAAGCCTATGGATATCAAGAACATGGACTTTGAAAACATCTGGAAGAACCTCCAGGTCAACGTTCTCGACAACATGGACGAGTGGCTGGAGGAGACCAACTCGCCCGCGTGGGAGCGAGACTTCCATGAGTATACCCACAACCGGCCTGAGTTTGCCGACTACCAGTTCGAGGAGAACAACCAGTTCATGGAGCACCCTGATCCCTTCAAGATTGGAGTCGAGCTCATGGAGACTGGCGGTCGACTTTCGGAGGCTGCTCTGGCCTTCGAGGCAGCTGTTCAGAAGAACACTGAGCACGCCGAGGCTTGGGGACGACTTGGAGCCTGCCAGGCCCAGAATGAAAAGGAGGACCCTGCTATCCGAGCTCTGGAACGATGCATCAAGCTGGAGCCTGGTAACCTTTCTGCTCTGATGAACTTGTCTGTTTCTTACACCAACGAAGGATACGAGAATGCCGCATATGCTACTCTGGAGCGATGGCTTGCCACCAAGTACCCCGAGGTTGTGGACCAGGCCCGAAACCAGGAGCCTCGACTCGGCAACGAGGATAAGTTCCAGCTGCACTCTCGGGTCACTGAGCTGTTTATCCGAGCTGCCCAACTGTCCCCTGACGGAGCTAACATTGACGCTGATGTCCAAGTTGGTCTCGGTGTTCTGTTCTACGGAAACGAGGAATACGATAAGGCCATTGACTGTTTCAACGCCGCCATTGCTGTTCGACCCGATGATGCTCTTCTGTGGAATAGACTCGGAGCCACCCTTGCCAACTCCCACCGATCTGAGGAGGCCATTGATGCTTACTACAAAGCTCTCGAGCTGCGTCCCTCTTTTGTGCGTGCTCGATACAACCTTGGTGTGTCGTGCATTAACATTGGCTGCTACAAGGAGGCTGCCCAGTATCTTCTGGGGGCTCTGTCCATGCACAAGGTTGAGGGAGTCCAGGATGATGTTTTGGCCAACCAGTCCACTAACCTGTACGATACCCTGAAGCGAGTTTTCCTGGGTATGGACCGACGAGATCTGGTGGCCAAGGTTGGAAACGGAATGGACGTCAACCAGTTCCGAAATGAGTTTGAATTTTAGTATATAGTAATTGATTATTTAAGGATGAGCGAGATGATACATATACAGTATTTACTCGTAACTTTCTCACTTCATGCAAGAACCATGCCTTAAAATTGCAGTACTGTACAGTACAATTGTAGTGTATGTACTTGA

[0156] The DNA sequence of the Sa / I linearized plasmid pYRH-PEX5-5-3N is shown, below, as SEQ ID NO: 31 :

[0157] TCGACTCATGGCAGCTCCTGTTTCCAATCTCAAGATCAAGTTTTCGGACAAGGGTAATGCCAAGGTAGTTATTCCTTCTCCTCTACAGTCGCATCAGTCGATGTTCGATGTGACCATGTTCAAGGGTACCTCTCAGGAGACTGTGACTAGCCATACTGGGTTCCATGAGCTGGTCGAGTTGGAAGAGTGGGTGCGGTGGGCTGCCGATAGATCTGAGCCCAAAAAGAGTAACTAAGTGGCCGACTCCACTCAAGTTTAGTTGACACGAGTTGGCTCACTTTTAACTATTACTTATGCTGTCGTTTTCAGCTGATCCATTAATTATATATGCTGTACTAATGGGAGTCAGCCGACAGTTAGGAATTGTAACAATACACACTTTGTATAAAGCCACTGTATTATTCCCACGCACACTTGTATTCGTACTTATCCTGTCATACAGTAGTTATTTCATGATCCTCTTGTCAACTCGCGCTCGGATCTACAGTAGATAAGCTCTACCCCATCTCTGGATTTACACGTGATGGTGAACTCTGTCAGGAGGGAATAAGATAATAATCCGGCAAACGGTCGGCACAGAACCAAAACTTGAGGGGCAGGTAACGGACTGTCTGACAGACAATGAATAATAGGAAAAGACTGAGTGTCGCCCGTCTGGTTAGTAATCGTCGTCCCTTCCATTGGGTCTGCGAGTACTCCATTAAATTAAATACCACTCCCTGACAATCTTCCAGCATGTTCTGTATGGCGCTCGGCCTTAACAAGGCTCCCCCACATTTTAAGTATCCTCGGCCGCCAGCGCGGTAGTTGCAACCTTATCCCCCAACTGCTCTGGCTTCGTTAGTAAGAAACTTTTGGAACAGGCCAACGTGAAAAATTGAGTGTATTCGAAGTCGTCAGATTGTGGGGTAATAGGGGCGTGAAAGACAAAACTTTAAGGATCTCAATATGATGAGATTGGCGCAGAGTACCACTCTGTGCAGTCGCCACTGAATCCACGTTGCGCACGTGGCCTTATGCCTCGGCTCAAATTCCCCAGATACCCCATTCAACAAGGGTAAGGGCACTCAGTCGTGGGGGAACCTTAATGTTACTGCAGTGACTGCCAGCAGAGAACTATAGACTTGCATATTACACATGATTTCCTTTGGACTGAAATAACTAAACTGTGACTCTTGCCAACCACTTTTCTTGCACACAGCACACACAGAGACCACCAAAGCAAGCAGGAAACATCATTGTAATTGATTATTTAAGGATGAGCGAGATGATACATATACAGTATTTACTCGTAACTTTCTCACTGCATGCAAGAACCATGCCTTAAAATTGCAGTACTGTACAGTACAATTGTACTGTATGTACTTGAGCTGATACAGGCCTGCTTCAACACTGTAATACGTTTCATATATAAATACCTAATTACCCCTTATGTGTATATAAATAATGGCTTGGGATGGTAGCAGCCATGGATTATTGGCCAATAGTCCCGGTATGGTTTGGAGGCCCAAGGTTATTAGTGAGTCCCAGATCGGTTAATGAGCTCTTTATCAGTTAGTTGGTATGTTTTGTCTCAGTGCTGTCTCCACTTTGACCACTCACGATGCTGTTGGATCTAGGGGCCGTTCTCTCACTGTTCCCTGAAATGGTACTTGTGCCAAAAATCAGTTCGCGCGCAAACCCATCGGATAGTTGTTCGGCATCCATAATGGCACTAGGTGCTTGGTCCTCGAATCCAAGTAGGTCGTTCCTAGACCGTAGAGAGCCCGAGCTTTCGCTTCTATCGGCGTAGTTCATGGGCCTCTGGTCCTCTGGTTCAGGCAGTCTATGACCCCTGCCGCCGAATAACCCCCAGGACCAGCCTGACCATCCGGACCATGCCGTGCTAGCCTGAACAGCCAGACGCGAAAGCCATTGCCTCACGTTTGTGTCCCCTATGGAGTGTTCAATGCCGAATTCTCCCGGGTTGGCGGCCGCAACATACTCTCCAAACTCATTGGCCGGTTCAGCCCCCTCCTGAGACAGCAGGGCCTGGGCCTCCTCCTCCTCCTCGTACATGTCGAACTGGATGGTATCGTTCTGTCGCTGACAACACCCCAGGCAGGAGCTATATATTTCCTTTAGTGTCATAGCCGTGGGTGTTCTTTCGAGTGACCTCTTGTAAAGTCGCGTTGTATATTGTTGTTTCTGCCGTATTGCAGCGCAATAGTCGGAAAGTGCAATTCTGGCCGGTAGTTCGGTTCTGGGTTCTCAATATGTCGTATTCTCGCCCTTATGAGTGTCAGAGCTCGTCGGCGGTCTGGTCCTTGTCTGGAACGGCGCGTAGCTCGTTATCTTAGTCGCCTCTGAGATGTGTGTGGGGTCACTCACGATTACTCAACTTTAGTCTAAAACGGCAACTTAATGAATTCTACTGGCAACGGAGAATACTCAAGCCTCTCATAATTCGGGCTGACGAGTAAATTAAGAGGTTCTCTAAAATGAACGCCAGCCACTGGTGTTGGGTCTATATGAAGGCATACTCTAGATTCCTTCATTGATATTCACATAGTGCCATGGACTGACTGTACATCAGCAGACAAACAGCGTACTAATGAATCCCAAGCTTTTGACTGGTCAGGAGGATATAGGAGATGACGTGATTCCACATTACACAATTTCCCCAGGGATGTGAGAGATAACCCTGACAATTGAAAGAATTATAGTCACTTTTATGGCTGTCACTAAACTCGACTCGATCCAATTACTTGTACCCATACTATCCCCATCGAGCGATTTCTGTCTCATTCATACCTCACCCCATTTTATTTACAGTTCCGAACCACAAATTCTGTCGTGGTGAGAGATTGCCACAGGTACATACTGTAGTGTAGTGGAGCTGTACTGGCTGGGACCATACTCGTATGTACTGTAACCATGGTTACGGATGTACAGGAATACATGGATCACAGTCTACCTGTACGAATAATTTCAATTGAACAATTATAAAGCACCATTATACTTGTGCGAGTATTACACTATACAGTATTATGAAGTTTGCTATTTTATTTACTGTAATTCTCTAGACTACTGTAACTACTGTATTTCTACTGTACTTGTACATACTTGTGTACAGTAGTTCCTCCCCATCACTTCTCATGTAAAAGATTTACACCAGAGCTCGCCTTAATATACAGTACAGATGAAACCCATTGTTCTACACCTAAAGAAACAAACACAGATATATATTCCCTCGGCGCGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGCCCGACGTCGCGATCGCGAGTATCTGTCTGACTCGTCATTGCCGCCTTTGGAGTACGACTCCAACTATGAGTGTGCTTGGATCACTTTGACGATACATTCTTCGTTGGAGGCTGTGGGTCTGACAGCTGCGTTTTCGGCGCGGTTGGCCGACAACAATATCAGCTGCAACGTCATTGCTGGCTTTCATCATGATCACATTTTTGTCGGCAAAGGCGACGCCCAGAGAGCCATTGACGTTCTTTCTAATTTGGACCGATAGCCGTATAGTCCAGTCTATCTATAAGTTCAACTAACTCGTAACTATTACCATAACATATACTTCACTGCCCCAGATAAGGTTCCGATAAAAAGTTCTGCAGACTAAATTTATTTCAGTCTCCTCTTCACCACCAAAATGCCCTCCTACGAAGCTCGAGCTAACGTCCACAAGTCCGCCTTTGCCGCTCGAGTGCTCAAGCTCGTGGCAGCCAAGAAAACCAACCTGTGTGCTTCTCTGGATGTTACCACCACCAAGGAGCTCATTGAGCTTGCCGATAAGGTCGGACCTTATGTGTGCATGATCAAAACCCATATCGACATCATTGACGACTTCACCTACGCCGGCACTGTGCTCCCCCTCAAGGAACTTGCTCTTAAGCACGGTTTCTTCCTGTTCGAGGACAGAAAGTTCGCAGATATTGGCAACACTGTCAAGCACCAGTACCGGTGTCACCGAATCGCCGAGTGGTCCGATATCACCAACGCCCACGGTGTACCCGGAACCGGAATCATTGCTGGCCTGCGAGCTGGTGCCGAGGAAACTG TCTCTGAACAGAAGAAGGAGGACGTCTCTGACTACGAGAACTCCCAGTACAAGGA GTTCCTAGTCCCCTCTCCCAACGAGAAGCTGGCCAGAGGTCTGCTCATGCTGGCC GAGCTGTCTTGCAAGGGCTCTCTGGCCACTGGCGAGTACTCCAAGCAGACCATTG AGCTTGCCCGATCCGACCGCGAGTTTGTGGTTGGCTTCATTGCCCAGAACCGACCT AAGGGCGACTCTGAGGACTGGCTTATTCTGACCCCCGGGGTGGGTCTTGACGACA AGGGAGACGCTCTCGGACAGCAGTACCGAACTGTTGAGGATGTCATGTCTACCGG AACGGATATCATAATTGTCGGCCGAGGTCTGTACGGCCAGAACCGAGATCCTATTG AGGAGGCCAAGCGATACCAGAAGGCTGGCTGGGAGGCTTACCAGAAGATTAACTG TTAGAGGTTAGACTATGGATATGTAATTTAACTGTGTATATAGAGAGCGTGCAAGTA TGGAGCGCTTGTTCAGCTTGTATGATGGTCAGACGACCTGTCTGATCGAGTATGTA TGATACTGCACAACCTGTGTATCCGCATGATCTGTCCAATGGGGCATGTTGTTGTG TTTCTCGATACGGAGATGCTGGGTACAGTGCTAATACGTTGAACTACTTATACTTAT ATGAGGCTCGAAGAAAGCTGACTTGTGTATGACGCATGCCCTCTATTGCTGCGACT GGCGAGGTTCTTTTCAAGGTGCGAAATTCTGATACCCGAGGGCTAGGCATCAAGTA TGGTTTTGGAGACGCTGGAAGTAAATATCGTGATCCAGGTATCGGTGTGTTGAAGC GTTCGGCTACAGGAACTGTGGACTTCGAGCCCAAGGAGGAGGTGCAAAAGAAGTT TGTCAGAGTGACTATTAAGTCTGAAGAGGGAGAAGTCACCAAAAGTGTCAGCAATG TATATACCAGGGACTCTAAGCCGCCAACTGACCCGACGCTAGCTGCTATTCACGAG ACTCGCCATGCACTGTTTGAAGAAGAGTTGTTCTTTGAGATTGCCAGAGAGGCAAG ACTGCTGACCTCGAGAAAGGTGACCGTTGCTGATGGAGCTGTAACGGTGGATCTG GGCGAAGGGGACATGGTTGTGATTGAGTGGGTGGAGGTCCCAGAAGAGCCTACAA CTACATTTGCACCTTCTCTAGGAAACTTATTTGTGCTGGCGCTGAGACTGCTGCTG GCTAATGCTCATCGACAGCAATTGGAGAAGATGAGAACTCCCCCTGCTCCGTTGCA ATCTAAGGGTGGTCCCAATCCCAACCCTCCTCTGCCTATACTAAGACCTCTTCTCG CACACATTCTGCACAAGCGGCTGGTGAGTCGCGCCAGAAGATCCCTCTATCTTCTG TCTACAACCCATTCCGGTCTCAGCTTCGAGATAACCACAAACAACTCTACAGATAAGGAATCTTCTTCTTTGGGTCGA

[0158] The transformed cells were growing on CM glucose minus uracil plates (0.13% amino acid dropout mixture minus uracil, with 0.17% yeast nitrogen base, 2.0% glucose, 0.5% ammonium sulfate, and 2.1% agar).

[0159] The colonies from transformation were analyzed by PCR with primers of AH117 (GGTGCGAAATTCTGATACCCGAG), referred to as SEQ ID No. 32, and AH118 (CGTATTACAGTGTTGAAGCAGGC), referred to as SEQ ID No. 33.

[0160] Transformants with PCR fragment of 2,139 bp (SEQ ID NO: 34) indicates that the Sall digested fragment integrated into right chromosome position (Fig. 9).

[0161] The DNA sequence of the 2,139 bp PCR fragment is shown, below, as SEQ ID NO: 34:

[0162] GGTGCGAAATTCTGATACCCGAGGGCTAGGCATCAAGTATGGTTTTGGA GACGCTGGAAGTAAATATCGTGATCCAGGTATCGGTGTGTTGAAGCGTTCGGCTAC AGGAACTGTGGACTTCGAGCCCAAGGAGGAGGTGCAAAAGAAGTTTGTCAGAGTGACTATTAAGTCTGAAGAGGGAGAAGTCACCAAAAGTGTCAGCAATGTATATACCAG GGACTCTAAGCCGCCAACTGACCCGACGCTAGCTGCTATTCACGAGACTCGCCAT GCACTGTTTGAAGAAGAGTTGTTCTTTGAGATTGCCAGAGAGGCAAGACTGCTGAC CTCGAGAAAGGTGACCGTTGCTGATGGAGCTGTAACGGTGGATCTGGGCGAAGGG GACATGGTTGTGATTGAGTGGGTGGAGGTCCCAGAAGAGCCTACAACTACATTTGC ACCTTCTCTAGCAAACTTATTTGTGCTGGCGCTGAGACTGCTGCTGGCTAATGCTC ATCGACAGCAATTGGAGAAGATGAGAACTCCCCCTGCTCCGTTGCAATCTAAGGGT GGTCCCAATCCCAACCCTCCTCTGCCTATACTAAGACCTCTTCTCGCACACATTCTG CACAAGCGGCTGGTGAGTCGCGCCAGAAGATCCCTCTATCTTCTGTCTACAACCCA TTCCGGTCTCAGCTTCGAGATAACCACAAACAACTCTACAGATAAGGAATCTTCTTC TTTGGGTCGACTCATGGCAGCTCCTGTTTCCAATCTCAAGATCAAGTTTTCGGACAA GGGTAATGCCAAGGTAGTTATTCCTTCTCCTCTACAGTCGCATCAGTCGATGTTCG ATGTGACCATGTTCAAGGGTACCTCTCAGGAGACTGTGACTAGCCATACTGGGTTC CATGAGCTGGTCGAGTTGGAAGAGTGGGTGCGGTGGGCTGCCGATAGATCTGAGC CCAAAAAGAGTAACTAAGTGGCCGACTCCACTCAAGTTTAGTTGACACGAGTTGGC TCACTTTTAACTATTACTTATGCTGTCGTTTTCAGCTGATCCATTAATTATATATGCT GTACTAATGGGAGTCAGCCGACAGTTAGGAATTGTAACAATACACACTTTGTATAAA GCCACTGTATTATTCCCACGCACACTTGTATTCGTACTTATCCTGTCATACAGTAGT TATTTCATGATCCTCTTGTCAACTCGCGCTCGGATCTACAGTAGATAAGCTCTACCC CATCTCTGGATTTACACGTGATGGTGAACTCTGTCAGGAGGGAATAAGATAATAATC CGGCAAACGGTCGGCACAGAACCAAAACTTGAGGGGCAGGTAACGGACTGTCTGA CAGACAATGAATAATAGGAAAAGACTGAGTGTCGCCCGTCTGGTTAGTAATCGTCG TCCCTTCCATTGGGTCTGCGAGTACTCCATTAAATTAAATACCACTCCCTGACAATC TTCCAGCATGTTCTGTATGGCGCTCGGCCTTAACAAGGCTCCCCCACATTTTAAGT ATCCTCGGCCGCCAGCGCGGTAGTTGCAACCTTATCCCCCAACTGCTCTGGCTTC GTTAGTAAGAAACTTTTGGAACAGGCCAACGTGAAAAATTGAGTGTATTCGAAGTC GTCAGATTGTGGGGTAATAGGGGCGTGAAAGACAAAACTTTAAGGATCTCAATATG ATGAGATTGGCGCAGAGTACCACTCTGTGCAGTCGCCACTGAATCCACGTTGCGC ACGTGGCCTTATGCCTCGGCTCAAATTCCCCAGATACCCCATTCAACAAGGGTAAGGGCACTCAGTCGTGGGGGAACCTTAATGTTACTGCAGTGACTGCCAGCAGAGAAC TATAGACTTGCATATTACACATGATTTCCTTTGGACTGAAATAACTAAACTGTGACTC TTGCCAACCACTTTTCTTGCACACAGCACACACAGAGACCACCAAAGCAAGCAGGA AACATCATTTTAATTAATATATAGTAATTGATTATTTAAGGATGAGCGAGATGATACA TATAGAGTATTTACTCGTAACTTTCTCACTGCATGCAAGAACCATGCCTTAAAATTGC AGTACTGTACAGTACAATTGTACTGTATGTACTTGAGCTGATACAGGCCTGCTTCAA CACTGTAATACG

[0163] To pop out the DNA fragment containing the PEX20, URA3, and Amp genes, a PCR positive transformant was growing in 2 ml of YPD at 32° C overnight, then 20 ul of cultures were spread on CM with uracil and FOA (450 ug / L) plate, and the plate was incubated at 32° C for 3 days.

[0164] The ura3 phenotype indicates that the two PEX5-3’ arms popped out in this step.

[0165] Only popped out cells could grow on CM with uracil and FOA (450 ug / L) plate. Colonies were re-patched on a new CM with uracil and FOA (450 ug / L) plate. Re-patched colonies were analyzed by PCR with primers of AH117 (SEQ ID NO. 32), and AH118 (SEQ ID NO. 33).

[0166] Thus, a strain with a PCR fragment of 2,139 bp (SEQ ID NO. 34, Fig. 9) indicates that the pex5 was deleted, was designated as strain AH256 (dgatl-, dgat2-, pex3-, pex5- ura3-).EXAMPLE 5: Generation of strain AH293 having double deletion of pex3 and pex20 from strain AH246

[0167] Strain AH293 (Table 1 , Fig. 2 & 3) with double deletion of pex3 (SEQ ID NO:4 and 5) and pex20 (SEQ ID NO: 20 and 21) was generated from strain AH246 (dgatl-, dgat2-, pex3-, ura3-, table 1 , section 2) by deletion of pex20 gene.

[0168] The pex20 was deleted by Pop-in and pop-out approach as illustrated in Fig. 8.

[0169] Specifically, The Bgl\\ DNA fragment containing PEX20-5' and PEX20-3’ arms of plasmid pYRH-PEX20-5-3N (SEQ ID NO: 22) was transformed into AH246.

[0170] The transformed cells were growing on CM glucose minus uracil plates (0.13% amino acid dropout mixture minus uracil, with 0.17% yeast nitrogen base, 2.0% glucose, 0.5% ammonium sulfate, and 2.1% agar). The colonies from transformation were analyzed by PCR with primers of YPEX20-5-confirm (SEQ ID NO: 23) and YPEX20-3- confirmA (SEQ ID NO: 24).

[0171] Transformants with a PGR fragment of 1 ,436 bp (SEQ ID NO: 25) indicate the BglW digested fragment integrated into right chromosome position (Fig. 8).

[0172] To lose the PEX20, UR A3, and Amp genes, a PCR positive transformant was growing in 2ml of YPD at 32° C overnight, then 20 ill cells were spread on CM with uracil and FOA (450 ug / L) plate, and the plate was incubated at 32° C for 3 days. The ura3 phenotype would indicate that the two PEX20-3’ arms had popped out.

[0173] Only popped out cells could grow on CM with uracil and FOA (450 ug / L) plate. Colonies were re-patched on a new CM with uracil and FOA (450 ug / L) plate.

[0174] Re-patched colonies were analyzed by PCR with primers of YPEX20-5- confirmA (SEQ ID NO: 23) and YPEX20-3-confirmA (SEQ ID NO: 24). Strains with a PCR fragment of 249 bp (SEQ ID NO 28) indicate the pex20 was deleted.

[0175] Thus, a colony with PCR positive was designated as strain AH293 (dgatl-, dgat2-, pex3, pex20-, ura3-).EXAMPLE 6: Generation of strain AH294 having triple deletion of pex3, pex5 and pex 20 from strain AH256

[0176] Strain AH294 (Table 1 , Fig. 2 & 3) with triple deletion of pex3 (SEQ ID NO: 4 and 5), pex5 (SEQ ID NO: 29 & 30) and pex20 (SEQ ID NO: 20 and 21) was generated from strain AH256 (dgatl-, dgat2-, pex3-, pex5-, ura3-) by deletion of pex20ger\e.

[0177] The pex20 was deleted by Pop-in and pop-out approach as illustrated in Fig.8

[0178] Specifically, The BglW DNA fragment containing PEX20-5’ and PEX20-3’ arms of plasmid pYRH-PEX20-5-3N (Fig. 8; SEQ ID NO 22) was transformed into AH256.

[0179] The transformed cells were growing on CM glucose minus uracil plates (0.13% amino acid dropout mixture minus uracil, with 0.17% yeast nitrogen base, 2.0% glucose, 0.5% ammonium sulfate, and 2.1% agar).

[0180] The colonies from transformation were analyzed by PCR with primers of YPEX20-5-confirm (SEQ ID No. 23) and YPEX20-3-confirmA (SEQ ID No. 24).

[0181] Transformants with a PCR fragment of 1 ,436 bp (SEQ ID NO: 25) indicate the BglW digested fragment integrated into right chromosome position (Fig. 8).

[0182] To lose the PEX20, URA3, and Amp genes, a PCR positive transformant was growing in 2 ml of YPD 32° C overnight, then 20 ul cells were spread on CM with uracil and FOA (450 ug / L) plate, and the plate was incubated at 32° C for 3 days. The ura3 phenotype would indicate that the two PEX20-3’ arms had popped out.

[0183] Only popped out cells could grow on CM with uracil and FOA (450 ug / L) plate. Colonies were re-patched on a new CM with uracil and FOA (450 ug / L) plate.

[0184] Re-patched colonies were analyzed by PCR with primers of YPEX20-5- confirmA (SEQ ID NO: 23) and YPEX20-3-confirmA (SEQ ID NO: 24).

[0185] Strains with a PCR fragment of 249 bp (SEQ ID No. 28) indicate the pex20 was deleted. Thus, a colony with PCR positive was designated as strain AH294 (dgatl-, dgat2-, pex3, pex5, pex20-, ura3-).EXAMPLE 7: Generation of strain AH299 having a single deletion of pex5 from strain AH256

[0186] Strain AH299 (Table 1 , Fig. 2 & 3) with single pex5 deletion was generated from strain AH256 (dgatl-, dgat2-, pex3-, pex5, ura3-) and to restore the PEX3 gene to wild type (SEQ ID NO: 4 and 5).

[0187] Construct pY-P3 (SEQ ID NO: 35, Fig. 10) contains wild type genes of URA3 (SEQ ID NO: 36 & 37) and PEX3 (SEQ ID NO: 4 & 5), and the components of the Sph\ and Asci large fragment (SEQ ID NO: 38, Fig. 11 ) of plasmid pY-P3, used for generation of strain AH299, are further described in Table 4.Table 4: Description of the Sph\ and Asci large fragment of Plasmid pY-P3 (SEQ ID NO: 34) integrated into the genome of Strain AH299

[0188] The Sph\ and Asci fragment of plasmid pY-P3 containing URA3 and PEX3 genes was transformed into strain AH256 (Table 1 , Fig. 2 & 3).

[0189] The transformed cells were growing on CM glucose minus uracil plates (0.13% amino acid dropout mixture minus uracil, with 0.17% yeast nitrogen base, 2.0% glucose, 0.5% ammonium sulfate, and 2.1% agar).

[0190] The colonies from transformation were analyzed by PCR. Strains have a PCR fragment of 1 ,435 bp (SEQ ID NO. 39) with primers of P211 (GCTCTCTTGTATACAAGAGCACAAGAC), referred to as SEQ ID No. 40 and P215 (CTGTCGAGCC-ATGTTGGCAAGAATG), referred to as SEQ ID No. 41 indicate the PEX3 integrated into its chromosome.

[0191] Thus, a colony with PCR positive was designated as strain AH299 (dgatl-, dgat2-, pex5-).

[0192] The DNA sequence of the plasmid pY-P3 is shown, below, as SEQ ID NO: 35:

[0193] AATTGTAATACGACTCACTATAGGGCGAATTGGGCCCGACGTCGCATGC ATTCCGACAGCAGCGACTGGGCACCATGATCAAGCGAAACACCTTCCCCCAGCTG CCCTGGCAAACCATCAAGAACCCTACTTTCATCAAGTGCAAGAACGGTTCTACTCTT CTCACCTCCGGTGTCTACGGCTGGTGCCGAAAGCCTAACTACACCGCTGATTTCAT CATGTGCCTCACCTGGGCTCTCATGTGCGGTGTTGCTTCTCCCCTGCCTTACTTCT ACCCGGTCTTCTTCTTCCTGGTGCTCATCCACCGAGCTTACCGAGACTTTGAGCGA CTGGAGCGAAAGTACGGTGAGGACTACCAGGAGTTCAAGCGACAGGTCCCTTGGA TCTTCATCCCTTATGTTTTCTAAACGATAAGCTTAGTGAGCGAATGGTGAGGTTACT TAATTGAGTGGCCAGCCTATGGGATTGTATAACAGACAGTCAATATATTACTGAAAA GACTGAACAGCCAGACGGAGTGAGGTTGTGAGTGAATCGTAGAGGGCGGCTATTA CAGCAAGTCTACTCTACAGTGTACTAACACAGCAGAGAACAAATACAGGTGTGCAT TCGGCTATCTGAGAATTAGTTGGAGAGCTCGAGACCCTCGGCGATAAACTGCTCCT CGGTTTTGTGTCCATACTTGTACGGACCATTGTAATGGGGCAAGTCGTTGAGTTCT CGTCGTCCGACGTTCAGAGCACAGAAACCAATGTAATCAATGTAGCAGAGATGGTT CTGCAAAAGATTGATTTGTGCGAGCAGGTTAATTAAGTACGATGAATGAGGATGAG AGTTCTGTTTCAGGTGACTGGCTCTCCACTCTCTCCAAGGTTTGAGAAAGTAACCT GTTCACGTGGGCTTTCGGTAAAACCTGTCCATTTTCGTCTTACGCCTGGTTCTACTT TGATCAGAAATATTTCGATCTAAGGCATTGGTCATGTAAGTAACAGTAAGTGCTGTA CAAGTACATACAGTACGGGTACTGGTAGATACAAGTGGCAATAACATGTATGTGTAT CTTTATCGGTGTACCAATAGCCACTAGGAGGCTCCTCTACTTATGTTGGATACGGT GCAAGTATCCGTACTAGCACTACCTATGCCTCCAGCTACGAGTGGTCTTCAAGATA TCCCGTGAGATCACACCTTCAGTATGTATGCTCTACATGATGTACAAGTTACTCTGT CATAAATCATTTGTGTACAAATGAGAGCGCTGTATTCAGTCCCCATCTTCGTTCTTC GTGTCTTTCGTGAATGAGCCTCTAGAGAGCCCAATCAAAAGATGAATAGACCACAG CACTAAAGGAGGTCAGCTCCTGCACTTGGTTCATCACGTCAATGTACTCGTTTTCCACGGCCGGCGATCCCTGGGCCATGCCTCCAGCCTGTCGAGCCATGTTGGCAAGAATGGAGGGCAGCTTGACCTTTGCAGTAGGCAGCACCACAGGATAAGGCGAACCCGAATCAGCAGGTGTGGCTCCAAACGTGACAGCCAGCTTGTCCATGAACACAGAGAGACCGGAGTGAACAAGTCGCTCAATCACCTCTGCGGCATTAGGGGACTCGATGAAGTCGGCAGTCTCGTCCAGAAGCCGTCGGAGGGTAGGTCCTAGAATGGGGGCAGCAGTATCGCTGGGGTTCTGGGCAAGCACGTACTCCTCCTGCTCCCGTGGTGGCAGCAGCTGGCTCAGAAAGTTGAGTGGCGACGAGGGATTGAGAGGTCGATCAATGAGCATCTGTGTTCGTCCAATGAGTTCCGACAACTCGTCAAATCCAAGTTCGGCCTTGGGTGACACGGGGTCCCACACTCGACGAACCGCTTCCTCCACTCGCTCGCTCAGCGACACCCATCCCTCGTTCAGCAGCCACCACGAAAAGGTAAGGTACTGTTCGTCCAGATCGGGATCGACAATAGTTTCTCCGAAGCTGGACGAGCCTGAAAGGTCGCCGAAATAGCCCATATCTGCAAGATCACCAAACGAGGGCGCCACTCGACCCTCGGCATTACCCTCTCGGCCCTGCTGCGCCAGAGCGACAACACTGTTGACGTAGTTTCGTCGTCCTAGAATGTTGAGCTGCAAACGCGTGAAGAAAATAAGAAGTGCATCTGCATAGACAAGAGAAAACGCTCGGGAAATGGTCGTTCGCTTGAGATCCTGCCACAACTGTGTTTTCGACTTCTCGCCCTCCTCTGTGGCGCCACTTGTCATGGTGGGCACGGTTTGTGCGGTAGCTGAGGACGAGGTGGAGCTCTCGAGAGCCAAAACCCGGTCTGTGGGGCGTCTCTTGCTCTGTAACTCTGCCTTGATCTGGTCGACGGGGTAACGCTCCATTACCGGTGTCGTCAAGCTGGATAGCAGAGCCATGATTGTAAAATCTGCATCCTGCTGGTTCTGTTCAAATCGGCGCCGGAGATTCTGTTTAGCGGTTCGCTCCGAGCTCAAACGACCCTGGATCTCGAAGAACTTTTTCTTCACATAGTCGATAAACAGGTAGGAACTCAGCGCCACACCTACCAGTGCCAGCACCTTTTTCTGGTGCCGTCTGAAGAAATCCATGGTGGTGTAGTCAGAGGTGTAGATGTGCAAGCAGTTTAGCTGACTGTGATAAACTCTAATAGTGCGGGGTGTGGGGCACGGGGGTAGATGATTGAAGACGGTGCATAGGAGTTGGAAGGCCGAAGAACTGACAATCACATAACTTACAGCCATACAACTATGTGCTACTTTTTTTTGGACGATTTATGTCACTTTTCATACATAAACACTTGACCTCGGTTAATGTGGGGTGCATGTCTTGTGCTCTTGTATACAAGAGAGCGCCGGAAATATTGAACAGAGTTGTATTAGAACATCACGAATACTAGCAATTTCACCAGGTATTTACGTGATCGTGTCAATTCACATGAGCACTTGAATATTTTTTGCCACAATAATGCTACGATCCTAATAATATACAATGGGCTTCTCAACAGATGTTGCAAGTGTATGGGATCTGTGACTTTTAGACGAAACACGGGTGAGTGCATAGAAGCCTAAAAATTGGGTCCGGATGGTACAGTACAGTAAATACAAGGATGTTCTCCAAGGGCTTGATCTGAAGAGATGAGGAGGCAATGAAGAGTTATTACGACCTAATTTGCACGTTTCAGTTTTATGGTGCCTGTATGAAAGTCTCGCCGTTGCGACGAACTTGTGACTGATGAATAATCACATCACAATGGCACACCCATTCGGATAATACTACATTAAATGAATGTATATGCAGAAAAAGAAAAAAAAACAGTCCATCATTTTTCAGCTCCCACATCTCCGTGTGGACATGGGGAAACTTGGTTGGATAATTTTAGTCTGCGGAATTCTCTCTCTTGAGCTTTTCCATAACAAGTTCTTCTGCCTCCAGGAAGTCCATGGGTGGTTTGATCATGGTTTTGGTGTAGTGGTAGTGCAGTGGTGGTATTGTGACTGGGGATGTAGTTGAGAATAAGTCATACACAAGTCAGCTTTCTTCGAGCCTCATATAAGTATAAGTAGTTCAACGTATTAGCACTGTACCCAGCATCTCCGTATCGAGAAACACAACAACATGCCCGATTGGACAGATCATGCGGATACACAGGTTGTGCAGTATCATACATACTCGATCAGACAGGTCGTCTGACCATCATACAAGCTGAACAAGCGCTCCATACTTGCACGCTCTCTATATACACAGTTAAATTACATATCCATAGTCTAACCTCTAACAGTTAATCTTCTGGTAAGCCTCCCAGCCAGCCTTCTGGTATCGCTTGGCCTCCTCAATAGGATCTCGGTTCTGGCCGTACAGACCTCGGCCGACAATTATGATATCCGTTCCGGTAGACATGACATCCTCAACAGTTCGGTACTGCTGTCCGAGAGCGTCTCCCTTGTCGTCAAGACCCACCCCGGGGGTCAGAATAAGCCAGTCCTCAGAGTCGCCCTTAGGTCGGTTCTGGGCAATGAAGCCAACCACAAACTCGGGGTCGGATCGGGCAAGCTCAATGGTCTGCTTGGAGTACTCGCCAGTGGCCAGAGAGCCCTTGCAAGACAGCTCGGCCAGCATGAGCAGACCTCTGGCCAGCTTCTCGTTGGGAGAGGGGACTAGGAACTCCTTGTACTGGGAGTTCTCGTAGTCAGAGACGTCCTCCTTCTTCTGTTCAGAGACAGTTTCCTCGGCACCAGCTCGCAGGCCAGCAATGATTCCGGTTCCGGGTACACCGTGGGCGTTGGTGATATCGGACCACTCGGCGATTCGGTGACACCGGTACTGGTGCTTGACAGTGTTGCCAATATCTGCGAACTTTCTGTCCTCGAACAGGAAGAAACCGTGCTTAAGAGCAAGTTCCTTGAGGGGGAGCACAGTGCCGGCGTAGGTGAAGTCGTCAATGATGTCGATATGGGTTTTGATCATGCACACATAAGGTCCGACCTTATCGGCAAGCTCAATGAGCTCCTTGGTGGTGGTAACATCCAGAGAAGCACACAGGTTGGTTTTCTTGGCTGCCACGAGCTTGAGCACTCGAGCGGCAAAGGCGGACTTGTGGACGTTAGCTCGAGCTTGGTAGGAGGGCATTTTGGTGGTGAAGAGGAGACTGAAATAAATTTAGTCTGCAGAACTTTTTATCGGAACCTTATCTGGGGCAGTGAAGTATATGTTATGGTAATAGTTACGAGTTAGTTGAACTTATAGATAGACTGGACTATACGGCTATCGGTCCAAATTAGAAAGAACGTCAATGGCTCTCTGGGCGTCGCCTTTGCCGACAAAAATGTGATCATGATGAAAGCCAGCAATGACGTTGCAGCTGATATTGTTGTCGGCCAACCGCGCCGAAAACGCAGCTGTCAGACCCACAGCCTCCAACGAAGAATGTATCGTCAAAGTGATCCAAGCACACTCATAGTTGGAGTCGTACTCCAAAGGCGGCAATGACGAGTCAGACAGATACTCGTCGACCTTTTCCTTGGGAACCACCACCGTCAGCCCTTCTGACTCACGTATTGTAGCCACCGACACAGGCAACAGTCCGTGGATAGCAGAATATGTCTTGTCGGTCCATTTCTCACCAACTTTAGGCGTCAAGTGAATGTTGCAGAAGAAGTATGTGCCTTCATTGAGAATCGGTGTTGCTGATTTCAATAAAGTCTTGAGATCAGTTTGGCCAGTCATGTTGTGGGGGGTAATTGGATTGAGTTATCGCCTACAGTCTGTACAGGTATACTCGCTGCCCACTTTATACTTTTTGATTCCGCTGCACTTGAAGCAATGTCGTTTACCAAAAGTGAGAATGCTCCACAGAACACACCCCAGGGTATGGTTGAGCAAAAAATAAACACTCCGATACGGGGAATCGAACCCCGGTCTCCACGGTTCTCAAGAAGTATTCTTGATGAGAGCGTATCGATGAGCCTAAAATGAACCCGAGTATATCTCATAAAATTCTCGGTGAGAGGTCTGTGACTGTCAGTACAAGGTGCCTTCATTATGCCCTCAACCTTACCATACCTCACTGAATGTAGTGTACCTCTAAAAATGAAATACAGTGCCAAAAGCCAAGGCACTGAGCTCGTCTAACGGACTTGATATACAACGAATTAAAAGAAATGAAAAGAAATACAGTTCTTTGTATCATTTGTAACAATTACCCTGTACAAACTAAGGTATTGAAATCCCACAATATTCCCAAAGTCCACCCCTTTCCAAATTGTCATGCCTACAACTCATATACCAAGCACTAACCTACCGTTTAAATGTAACGAAACTGAAATTTGACCAGATATTGTGTCCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAAGCTTCCACACAACGTACGTTGATTGAGGTGGAGCCAGATGGGCTATTGTTTCATATATAGACTGGCAGCCACCTCTTTGGCCCAGCATGTTTGTATACCTGGAAGGGAAAACTAAAGAAGCTGGCTAGTTTAGTTTGATTATTATAGTAGATGTCCTAATCACTAGAGATTAGAATGTCTTGGCGATGATTAGTCGTCGTCCCCTGTATCATGTCTAGACCAACTGTGTCATGAAGTTGGTGCTGGTGTTTTACCTGTGTACTACAAGTAGGTGTCCTAGATCTAGTGTACAGAGCCGTTTAGACCCATGTGGACTTCACCATTAACGATGGAAAATGTTCATTATATGACAGTATATTACAATGGACTTGCTCCATTTCTTCCTTGCATCACATGTTCTCCACCTCCATAGTTGATCAACACATCATAGTAGCTAAGGCTGCTGCTCTCCCACTACAGTCCACCACAAGTTAAGTAGCACCGTCAGTACAGCTAAAAGTACACGTCTAGTACGTTTCATAACTAGTCAAGTAGCCCCTATTACAGATATCAGCACTATCACGCACGAGTTTTTCTCTGTGCTATCTAATCAACTTGCCAAGTATTCGGAGAAGATACACTTTCTTGGCATCAGGTATACGAGGGAGCCTATCAGATGAAAAAGGGTATATTGGATCCATTCATATCCACCTACACGTTGTCATAATCTCCTCATTCACGTGATTCATTTCGTGACACTAGTTTCTCACTTTCCCCCCCGCACCTATAGTCAACTTGGCGGACACGCTACTTGTAGCTGACGTTGATTTATAGACCCAATCAAAGCGGGTTATCGGTCAGGTAGCACTTATCATTCATCGTTCATACTACGATGAGCAATCTCGGGCATGTCCGGAAAAGTGTCGGGCGCGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCAGTGGCAGGAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGT G

[0194] The amino acid sequence of Y. lipolytica URA3 (GenBank #: AJ306421) polypeptide is shown, below, as SEQ ID NO: 36:

[0195] MPSYEARANVHKSAFAARVLKLVAAKKTNLCASLDVTTTKELIELADKVGPY VCMIKTHIDIIDDFTYAGTVLPLKELALKHGFFLFEDRKFADIGNTVKHQYRCHRIAEWSD ITNAHGVPGTGIIAGLRAGAEETVSEQKKEDVSDYENSQYKEFLVPS PNEKLARGLLMLAELSCKGSLATGEYSKQTIELARSDPEFVVGFIAQNRPKGDSEDWLI LTPGVGLDDKGDALGQQYRTVEDVMSTGTDIIIVGRGLYGQNRDPIEEAKRYQKAGWE AYQKINC

[0196] The DNA sequence of Y. lipolytica URA3 (GenBank #: AJ306421 ) is shown, below, as SEQ ID NO: 37:

[0197] ATGCCCTCCTACGAAGCTCGAGCTAACGTCCACAAGTCCGCCTTTGGCG CTCGAGTGCTCAAGCTCGTGGCAGCCAAGAAAACCAAGCTGTGTGCTTCTCTGGAT GTTACCACCACCAAGGAGCTCATTGAGCTTGCCGATAAGGTCGGACCTTATGTGTG CATGATGAAAACCCATATCGACATCATTGACGACTTCACCTACGCCGGCACTGTGC TCCCCCTCAAGGAACTTGCTCTTAAGCACGGTTTCTTCCTGTTCGAGGACAGAAAG TTCGCAGATATTGGCAACACTGTCAAGCACCAGTACCGGTGTCACCGAATCGCCGA GTGGTCCGATATCACCAACGCCCACGGTGTACCCGGAACCGGAATCATTGCTGGC CTGCGAGCTGGTGCCGAGGAAACTGTCTCTGAACAGAAGAAGGAGGACGTCTCTG ACTACGAGAACTCCCAGTACAAGGAGTTCCTAGTCCCCTCTCCCAACGAGAAGCTG GCCAGAGGTCTGCTCATGCTGGCCGAGCTGTCTTGCAAGGGCTCTCTGGCCACTG GCGAGTACTCCAAGCAGACCATTGAGCTTGCCCGATCCGACCCCGAGTTTGTGGT TGGCTTCATTGCCCAGAACCGACCTAAGGGCGACTCTGAGGACTGGCTTATTCTGA CCCCCGGGGTGGGTCTTGACGACAAGGGAGACGCTCTCGGACAGCAGTACCGAA CTGTTGAGGATGTCATGTCTACCGGAACGGATATCATAATTGTCGGCCGAGGTCTG TACGGCCAGAACCGAGATCCTATTGAGGAGGCCAAGCGATACCAGAAGGCTGGCT GGGAGGCTTACCAGAAGATTAACTGTTAG

[0198] The DNA sequence of the Sph\ and Asci large fragment of plasmid pY-P3 is shown, below, as SEQ ID NO: 38:

[0199] CCATGCATTCCGACAGCAGCGACTGGGCACCATGATCAAGCGAAACAC CTTCCCCCAGCTGCCCTGGCAAACCATCAAGAACCCTACTTTCATCAAGTGCAAGA ACGGTTCTACTCTTCTCACCTCCGGTGTCTACGGCTGGTGCCGAAAGCCTAACTAC ACCGCTGATTTCATCATGTGCCTCACCTGGGCTCTCATGTGCGGTGTTGCTTCTCC CCTGCCTTACTTCTACCCGGTCTTCTTCTTCCTGGTGCTCATCCACCGAGCTTACC GAGACTTTGAGCGACTGGAGCGAAAGTACGGTGAGGACTACCAGGAGTTCAAGCGAGAGGTCCCTTGGATCTTCATCCCTTATGTTTTCTAAACGATAAGCTTAGTGAGCGAATGGTGAGGTTACTTAATTGAGTGGCCAGCCTATGGGATTGTATAACAGACAGTCAATATATTACTGAAAAGACTGAACAGCCAGACGGAGTGAGGTTGTGAGTGAATCGTAGAGGGCGGCTATTACAGCAAGTCTACTCTACAGTGTACTAACACAGCAGAGAACAAATACAGGTGTGCATTCGGCTATCTGAGAATTAGTTGGAGAGCTCGAGACCCTCGGCGATAAACTGCTCCTCGGTTTTGTGTCCATACTTGTACGGACCATTGTAATGGGGCAAGTCGTTGAGTTCTCGTCGTCCGACGTTCAGAGCACAGAAACCAATGTAATCAATGTAGCAGAGATGGTTCTGCAAAAGATTGATTTGTGCGAGCAGGTTAATTAAGTACGATGAATGAGGATGAGAGTTCTGTTTCAGGTGACTGGCTCTCCACTCTCTCCAAGGTTTGAGAAAGTAACCTGTTCACGTGGGCTTTCGGTAAAACCTGTCCATTTTCGTCTTACGCCTGGTTCTACTTTGATCAGAAATATTTCGATCTAAGGCATTGGTCATGTAAGTAACAGTAAGTGCTGTACAAGTACATACAGTACGGGTACTGGTAGATACAAGTGGCAATAACATGTATGTGTATCTTTATCGGTGTACCAATAGCCACTAGGAGGCTCCTCTACTTATGTTGGATACGGTGCAAGTATCCGTACTAGCACTACCTATGCCTCCAGCTACGAGTGGTCTTCAAGATATCCCGTGAGATCACACCTTCAGTATGTATGCTCTACATGATGTACAAGTTACTCTGTCATAAATCATTTGTGTACAAATGAGAGCGCTGTATTCAGTCCCCATCTTCGTTCTTCGTGTCTTTCGTGAATGAGCCTCTAGAGAGCCCAATCAAAAGATGAATAGACCACAGCACTAAAGGAGGTCAGCTCCTGCACTTGGTTCATCACGTCAATGTACTCGTTTTCCACGCCCGGCGATCCCTGGGCCATGCCTCCAGCCTGTCGAGCCATGTTGGCAAGAATGGAGGGCAGCTTGACCTTTGCAGTAGGCAGCACCACAGGATAAGGCGAACCCGAATCAGCAGGTGTGGCTCCAAACGTGACAGCCAGCTTGTCCATGAACACAGAGAGACCGGAGTGAACAAGTCGCTCAATCACCTCTGCGGCATTAGGGGAGTCGATGAAGTCGGCAGTGTCGTCCAGAAGCCGTCGGAGGGTAGGTCCTACAATGGGGGCAGCAGTATCGCTGGGGTTCTGGGCAAGCACGTACTCCTCCTGCTCCCGTGGTGGCAGCAGCTGGCTCAGAAAGTTGAGTGGCGACGAGGGATTGAGAGGTCGATCAATGAGCATCTGTGTTCGTCCAATGAGTTCCGACAACTCGTCAAATCCAAGTTCGGCCTTGGGTGACACGGGGTCCCACACTCGACGAACCGCTTCCTCCACTCGCTCGCTCAGCGACACCCATCCCTCGTTCAGCAGCCACCACGAAAAGGTAAGGTACTGTTCGTCCAGATCGGGATCGACAATAGTTTCTCCGAAGCTGGACGAGCCTGAAAGGTCGCCGAAATAGCCCATATCTGCAAGATCACCAAACGAGGGCGCCACTCGACCCTCGGCATTACCCTCTCGGCCCTGCTGCGCCAGAGCGACAACACTGTTGACGTAGTTTCGTCGTCCTAGAATGTTGAGCTGCAAACGCGTGAAGAAAATAAGAAGTGCATCTGCATAGACAAGAGAAAACGCTCGGGAAATGGTCGTTCGCTTGAGATCCTGCCACAACTGTGTTTTCGACTTCTCGCCCTCCTCTGTGGCGCCACTTGTCATGGTGGGCACGGTTTGTGCGGTAGCTGAGGACGAGGTGGAGCTCTCGAGAGCCAAAACCCGGTCTGTGGGGCGTCTCTTGCTCTGTAACTCTGCCTTGATCTGGTCGACGGGGTAACGCTCCATTACCGGTGTCGTCAAGCTGGATAGCAGAGCCATGATTGTAAAATCTGCATGCTGCTGGTTCTGTTCAAATCGGCGCCGGAGATTCTGTTTAGCGGTTCGCTCCGAGCTCAAACGACCCTGGATCTCGAAGAACTTTTTCTTCACATAGTCGATAAACAGGTAGGAACTCAGCGCCACACCTACCAGTGCCAGCACCTTTTTCTGGTGCCGTCTGAAGAAATCCATGGTGGTGTAGTCAGAGGTGTAGATGTGGAAGCAGTTTAGCTGACTGTGATAAACTCTAATAGTGCGGGGTGTGGGGCACGGGGGTAGATGATTGAAGACGGTGCATAGGAGTTGGAAGGCCGAAGAACTGACAATCACATAACTTACAGCCATACAACTATGTGCTACTTTTTTTTGGACGATTTATGTCACTTTTCATACATAAACACTTGACCTCGGTTAATGTGGGGTGCATGTCTTGTGCTCTTGTATACAAGAGAGCGCCGGAAATATTGAACAGAGTTGTATTAGAACATCACGAATACTAGCAATTTCACCAGGTATTTACGTGATCGTGTCAATTCACATGAGCACTTGAATATTTTTTGCCACAATAATGCTACGATCCTAATAATATACAATGGGCTTCTCAACAGATGTTGCAAGTGTATGGGATCTGTGACTTTTAGACGAAACACGGGTGAGTGCATAGAAGCCTAAAAATTGGGTCCGGATGGTACAGTACAGTAAATACAAGGATGTTCTCCAAGGGCTTGATCTGAAGAGATGAGGAGGCAATGAAGAGTTATTACGACCTAATTTGCACGTTTCAGTTTTATGGTGCCTGTATGAAAGTCTCGCCGTTGCGACGAACTTGTGACTGATGAATAATCACATCACAATGGCACACCCATTCGGATAATACTACATTAAATGAATGTATATGCAGAAAAAGAAAAAAAAACAGTCCATCATTTTTCAGCTCCCACATCTCCGTGTGGACATGGGGAAACTTGGTTGGATAATTTTAGTCTGCGGAATTCTCTCTCTTGAGCTTTTCCATAACAAGTTCTTCTGCCTCCAGGAAGTCCATGGGTGGTTTGATCATGGTTTTGGTGTAGTGGTAGTGCAGTGGTGGTATTGTGACTGGGGATGTAGTTGAGAATAAGTCATACACAAGTCAGCTTTCTTCGAGCCTCATATAAGTATAAGTAGTTCAACGTATTAGCACTGTACCCAGCATCTCCGTATCGAGAAACACAACAACATGCCCCATTGGACAGATCATGCGGATACACAGGTTGTGCAGTATCATACATACTCGATCAGACAGGTCGTCTGACCATCATACAAGCTGAACAAGCGCTCCATACTTGCACGCTCTCTATATACACAGTTAAATTACATATCCATAGTCTAACCTCTAACAGTTAATCTTCTGGTAAGCCTCCCAGCCAGCCTTCTGGTATCGCTTGGCCTCCTCAATAGGATCTCGGTTCTGGCCGTACAGACCTCGGCCGACAATTATGATATCCGTTCCGGTAGACATGACATCCTCAACAGTTCGGTACTGCTGTCCGAGAGCGTCTCCCTTGTCGTCAAGACCCACCCCGGGGGTCAGAATAAGCCAGTCCTCAGAGTCGCCCTTAGGTCGGTTCTGGGCAATGAAGCCAACCACAAACTCGGGGTCGGATCGGGCAAGCTCAATGGTCTGCTTGGAGTACTCGCCAGTGGCCAGAGAGCCCTTGCAAGACAGCTCGGCCAGCATGAGCAGACCTCTGGCCAGCTTCTCGTTGGGAGAGGGGACTAGGAACTCCTTGTACTGGGAGTTCTCGTAGTCAGAGACGTCCTCCTTCTTCTGTTCAGAGACAGTTTCCTCGGCACCAGCTCGCAGGCCAGCAATGATTCCGGTTCCGGGTACACCGTGGGCGTTGGTGATATCGGACCACTCGGCGATTCGGTGACACCGGTACTGGTGCTTGACAGTGTTGCCAATATCTGCGAACTTTCTGTCCTCGAACAGGAAGAAACCGTGCTTAAGAGCAAGTTCCTTGAGGGGGAGCACAGTGCCGGCGTAGGTGAAGTCGTCAATGATGTCGATATGGGTTTTGATCATGCACACATAAGGTCCGACCTTATCGGCAAGCTCAATGAGCTCCTTGGTGGTGGTAACATCCAGAGAAGCACACAGGTTGGTTTTCTTGGCTGCCACGAGCTTGAGCACTCGAGCGGCAAAGGCGGACTTGTGGACGTTAGCTCGAGCTTCGTAGGAGGGCATTTTGGTGGTGAAGAGGAGACTGAAATAAATTTAGTCTGCAGAACTTTTTATCGGAACCTTATCTGGGGCAGTGAAGTATATGTTATGGTAATAGTTACGAGTTAGTTGAACTTATAGATAGACTGGACTATACGGCTATCGGTCCAAATTAGAAAGAACGTCAATGGCTCTCTGGGCGTCGCCTTTGCCGACAAAAATGTGATCATGATGAAAGCCAGCAATGACGTTGCAGCTGATATTGTTGTCGGCCAACCGCGCCGAAAACGCAGCTGTCAGACCCACAGCCTCCAACGAAGAATGTATCGTCAAAGTGATCCAAGCACACTCATAGTTGGAGTCGTACTCCAAAGGCGGCAATGACGAGTCAGACAGATACTCGTCGACCTTTTCCTTGGGAACCACCACCGTCAGCCCTTCTGACTCACGTATTGTAGCCACCGACACAGGCAACAGTCCGTGGATAGCAGAATATGTCTTGTCGGTCCATTTCTCACCAACTTTAGGCGTCAAGTGAATGTTGCAGAAGAAGTATGTGCCTTCATTGAGAATCGGTGTTGCTGATTTCAATAAAGTCTTGAGATCAGTTTGGCCAGTCATGTTGTGGGGGGTAATTGGATTGAGTTATCGCCTACAGTCTGTACAGGTATACTCGCTGCCCACTTTATACTTTTTGATTCCGCTGCACTTGAAGCAATGTCGTTTACCAAAAGTGAGAATGCTCCACAGAACACACCCCAGGGTATGGTTGAGCAAAAAATAAACACTCCGATACGGGGAATCGAACCCCGGTCTCCACGGTTCTCAAGAAGTATTCTTGATGAGAGCGTATCGATGAGCCTAAAATGAACCCGAGTATATCTCATAAAATTCTCGGTGAGAGGTCTGTGACTGTCAGTACAAGGTGCCTTCATTATGCCCTCAACCTTACCATACCTCACTGAATGTAGTGTACCTCTAAAAATGAAATACAGTGCCAAAAGCCAAGGCACTGAGCTCGTCTAACGGACTTGATATACAACCAATTAAAACAAATGAAAAGAAATACAGTTCTTTGTATCATTTGTAACAATTACCCTGTACAAACTAAGGTATTGAAATCCCACAATATTCCCAAAGTCCACCCCTTTCCAAATTGTCATGCCTACAACTCATATACCAAGCACTAACCTACCGTTTAAATGTAACGAAACTGAAATTTGACCAGATATTGTGTCCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAAGCTTCCACACAACGTACGTTGATTGAGGTGGAGCCAGATGGGCTATTGTTTCATATATAGACTGGCAGCCACCTCTTTGGCCCAGCATGTTTGTATACCTGGAAGGGAAAACTAAAGAAGCTGGCTAGTTTAGTTTGATTATTATAGTAGATGTCCTAATCACTAGAGATTAGAATGTCTTGGCGATGATTAGTCGTCGTCCCCTGTATCATGTCTAGACCAACTGTGTCATGAAGTTGGTGCTGGTGTTTTACCTGTGTACTACAAGTAGGTGTCCTAGATCTAGTGTACAGAGCCGTTTAGACCCATGTGGACTTCACCATTAACGATGGAAAATGTTCATTATATGACAGTATATTACAATGGACTTGCTCCATTTCTTCCTTGCATCACATGTTCTCCACCTCCATAGTTGATCAACACATCATAGTAGCTAAGGCTGCTGCTCTCCCACTACAGTCCACCACAAGTTAAGTAGCACCGTCAGTACAGCTAAAAGTACACGTCTAGTACGTTTCATAACTAGTCAAGTAGCCCCTATTACAGATATCAGCACTATCACGCACGAGTTTTTCTCTGTGCTATCTAATCAACTTGCCAAGTATTCGGAGAAGATACACTTTCTTGGCATCAGGTATACGAGGGAGCCTATCAGATGAAAAAGGGTATATTGGATCCATTCATATCCACCTACACGTTGTGATAATCTCCTCATTCACGTGATTCATTTCGTGACACTAGTTTCTCACTTTCCCGCCCGCACCTATAGTCAACTTGGCGGACACGCTACTTGTAGCTGACGTTGATTTATAGACCCAATCAAAGCGGGTTATCGGTCAGGTAGCACTTATCATTCATCGTTCATACTACGATGAGCAATCTCGGGCATGTCCGGAAAAGTGTCGGGCGCGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGGGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACGGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGCCCGACGTC

[0200] The DNA sequence of the 1 ,435 bp PGR fragment is shown, below, as SEQID NO: 39:

[0201] GCTCTCTTGTATACAAG AGCAC AAG ACATGCACCCCACATTAACCG AGGTCAAGTGTTTATGTATGAAAAGTGACATAAATCGTCCAAAAAAAAGTAGCACATAGTTGTATGGCTGTAAGTTATGTGATTGTCAGTTCTTCGGCCTTCCAACTCCTATGCACCGTCTTCAATCATCTACCCCCGTGCCCCACACCCCGCACTATTAGAGTTTATCACAGTCAGCTAAACTGCTTGCACATCTACACCTCTGACTACACCACCATGGATTTCTTCAGACGGCACCAGAAAAAGGTGCTGGCACTGGTAGGTGTGGCGCTGAGTTCCTACCTGTTTATCGACTATGTGAAGAAAAAGTTCTTCGAGATCCAGGGTCGTTTGAGCTCGGAGCGAACCGCTAAACAGAATCTCCGGCGCCGATTTGAACAGAACCAGCAGGATGCAGATTTTACAATCATGGCTCTGCTATCCAGCTTGACGACACCGGTAATGGAGCGTTACCCCGTCGACCAGATCAAGGCAGAGTTACAGAGCAAGAGACGCCCCACAGACCGGGTTTTGGCTCTCGAGAGCTCCACCTCGTCCTCAGCTACCGCACAAACCGTGCCCACCATGACAAGTGGCGCCACAGAGGAGGGCGAGAAGTCGAAAACACAGTTGTGGCAGGATCTCAAGCGAACGACCATTTCCCGAGCGTTTTCTCTTGTCTATGCAGATGCACTTCTTATTTTCTTCACGCGTTTGCAGCTCAACATTCTAGGACGACGAAACTACGTCAAC AGTGTTGTCGCTCTGGCGCAGCAGGGCCGAGAGGGTAATGCCGAGGGTCGAGTG GCGCCCTCGTTTGGTGATCTTGCAGATATGGGCTATTTCGGCGACCTTTCAGGCTC GTCCAGCTTCGGAGAAACTATTGTCGATCCCGATCTGGACGAACAGTACCTTACCT TTTCGTGGTGGCTGCTGAACGAGGGATGGGTGTCGCTGAGCGAGCGAGTGGAGG AAGCGGTTCGTCGAGTGTGGGACCCCGTGTCACCCAAGGCCGAACTTGGATTTGA CGAGTTGTCGGAACTCATTGGACGAACACAGATGCTCATTGATCGACCTCTCAATC CCTCGTCGCCACTCAACTTTCTGAGCCAGCTGCTGCCACCACGGGAGCAGGAGGA GTACGTGCTTGCCCAGAACCCCAGCGATACTGCTGCCCCCATTGTAGGACCTACC CTCCGACGGCTTCTGGACGAGACTGCCGACTTCATCGAGTCCCCTAATGCCGCAG AGGTGATTGAGCGACTTGTTCACTCCGGTCTCTCTGTGTTCATGGACAAGCTGGCT GTCACGTTTGGAGCCACACCTGCTGATTCGGGTTCGCCTTATCCTGTGGTGCTGCC TACTGCAAAGGTCAAGCTGCCCTCCATTCTTGCCAACATGGCTCGACAGEXAMPLE 8: Analyses of beta-oxidation capabilities by strains with deletion of one, two or three pex genes

[0202] Fatty acid p-oxidation is the process in which fatty acids are broken down to produce energy. Fatty acid p-oxidation capability can be analyzed by cells growing on YPO plate.

[0203] YPO Plate. The YPO plate consists of 0.1% yeast extract, 2% peptone, 0.2% oleic acid, pH 7, in which oleic acid is the sole carbon source.

[0204] Strains that can degrade oleate by beta-oxidation can grow on YPO plate. If beta-oxidation is blocked, there is no growth of cells on YPO plate. If beta-oxidation is partially blocked, there is slow or delayed growth cells on YPO plate.

[0205] If beta-oxidation is not affected, cells will grow normally on YPO plate.

[0206] The results of YPO plate assay are shown on Figures 2A-C.

[0207] Strain AH291 (MATA, dgatl -, dgat2-, ura3-) with no PEX gene deletion showed normal growth on YPO plate. All pex gene deletion strains showed (1 ) almost no growth such as strain AH256 (pex3-, pex5-) and strain AH294 (pex3-, pex5-, pex20-) and (2) delayed growth as strain AH246 (pex3-), AH292 (pex20-) and AH293 (pex3-, pex20-) on YPO plate.

[0208] Strains AH246, AH292, and AH293 had some degree of growth on YPO plate at 72 hours, but they significantly showed >80% less growth than the control AH291 strain.

[0209] These results demonstrated that beta-oxidation was blocked or partially blocked in strains with deleting pex genes. Amongst the three deleted genes, the pex5deletion had the most effect on beta-oxidation capabilities when comparing its betaoxidation abilities with pex3, or pex20.

[0210] The range of recited numerical values disclosed in the specification includes values, e.g., + / — 5-10% of the recited value, that a person of ordinary skill in the art would consider equivalent to the recited value, e.g., having the same function or result.

[0211] The claims are not limited by the preferred embodiments and examples but will cover many modifications and equivalents consistent with the written description as a whole.

Claims

WHAT IS CLAIMED IS:

1. A modified yeast cell having a reduction of beta-oxidation activity compared to beta-oxidation activity of a non-modified parental yeast cell, wherein the modified cell comprises a genetic modification reducing or eliminating the expression, activity and / or function of one or more native peroxisome biogenesis factor proteins (PEX) encoded by one or more endogenous peroxisome biogenesis factor genes (PEX .

2. The modified yeast cell of claim 1 , wherein the genetic modification reducing or eliminating the expression, activity and / or function of one or more native PEX proteins comprises disrupting, partially deleting, completely deleting, mutating and / or down-regulating the one or more endogenous PEX genes.

3. The modified yeast cell of claim 1 or 2, wherein the one or more native PEX proteins are selected from group consisting of a PEX3 protein, a PEX5 protein and a PEX 20 protein.

4. The modified yeast cell of any one of claims 1 -3, wherein the one or more genes encoding the native PEX proteins comprises a PEX3 ger\e.

5. The modified yeast cell of any one of claims 1 -4, wherein the one or more genes encoding the native PEX proteins comprises a PEX5 gene.

6. The modified yeast cell of any one of claims 1 -5, wherein the one or more genes encoding the native PEX proteins comprises a PEX20 gene.

7. The modified yeast cell of any one of claims 1 -6, wherein the genetic modification which eliminates or reduces the activity and / or function of the one more native PEX proteins comprises disrupting, partially deleting and / or mutating a subsequence of a PEX gene encoding a PEX protein ATP binding site, a PEX protein transmembrane (TM) domain or a PEX protein zinc-finger domain or protein-protein interaction domain, or combinations thereof.

8. The modified yeast cell of any one of claims 1 -7, wherein the modified yeast cell is Yarrowia lipolytica.

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