# Patent

## Title
Ammonium transporter promoters for gene expression in oleaginous yeast

## Abstract

The promoter region associated with the Yarrowia lipolytica ammonium transporter (yat1) gene has been found to be particularly effective for the expression of heterologous genes in oleaginous yeast. The promoter regions of the instant invention have been shown to be advantageously inducible under oleaginous conditions (i.e., nitrogen limitation) and are useful to drive expression of genes involved in the production of ω-3 and ω-6 fatty acids.

## Background
This application is a Continuation-In-Part of U.S. patent application Ser. No. 11/185,301, filed Jul. 20, 2005, now abandoned, and claims the benefit of U.S. Provisional Application No. 60/624,812, filed Nov. 4, 2004, the disclosures of which are hereby incorporated by reference in their entirety.


FIELD OF THE INVENTION
This invention is in the field of biotechnology. More specifically, this invention pertains to promoter regions isolated from Yarrowia lipolytica that are useful for gene expression in oleaginous yeast.
BACKGROUND OF THE INVENTION
Oleaginous yeast are defined as those organisms that are naturally capable of oil synthesis and accumulation, wherein oil accumulation ranges from at least about 25% up to about 80% of the cellular dry weight. Genera typically identified as oleaginous yeast include, but are not limited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. More specifically, illustrative oil-synthesizing yeast include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R. graminis and Yarrowia lipolytica (formerly classified as Candida lipolytica).
The technology for growing oleaginous yeast with high oil content is well developed (for example, see EP 0 005 277B1; Ratledge, C., Prog. Ind. Microbiol., 16:119-206 (1982)). And, these organisms have been commercially used for a variety of purposes in the past. For example, various strains of Yarrowa lipolytica have historically been used for the manufacture and production of: isocitrate lyase, lipases, polyhydroxy-alkanoates, citric acid, erythritol, 2-oxoglutaric acid, γ-decalactone, γ-dodecalactone and pyruvic acid. More recently, however, the natural abilities of oleaginous yeast have been enhanced by advances in genetic engineering, resulting in organisms capable of producing polyunsaturated fatty acids [“PUFAs”]. Specifically, Zhu et al. have demonstrated that Y. lipolytica can be engineered for production of ω-3 and ω-6 fatty acids, by introducing and expressing genes encoding the ω-3/ω-6 biosynthetic pathway (see U.S. Pat. No. 7,238,482, U.S. Pat. Appl. Pub. No. 2006-0115881-A1 and U.S. Pat. Appl. Pub. No. 2009-0093543-A1).
Recombinant production of any heterologous protein is generally accomplished by constructing an expression cassette in which the DNA coding for the protein of interest is placed under the control of appropriate regulatory sequences (i.e., promoters) suitable for the host cell. The expression cassette is then introduced into the host cell, usually by plasmid-mediated transformation or targeted integration into the host genome, and production of the heterologous protein is achieved by culturing the transformed host cell under conditions necessary for the proper function of the promoter contained within the expression cassette. Thus, the development of new host cells such as oleaginous yeast for recombinant production of proteins generally requires the availability of promoters that are suitable for controlling the expression of a protein of interest in the host cell.
A variety of strong promoters have been isolated from Yarrowia lipolytica that are useful for heterologous gene expression in yeast. For example, U.S. Pat. No. 4,937,189 and EP220864 (Davidow et al.) disclose the sequence of the XPR2 gene (which encodes an inducible alkaline extracellular protease) and upstream promoter region for use in expression of heterologous proteins. U.S. Pat. No. 6,265,185 (Muller et al.) describes promoters for the translation elongation factor EF1-α [“TEF”] protein and ribosomal protein S7 that are suitable for expression cloning in yeast and heterologous expression of proteins. These promoters were improved relative to the XPR2 promoter, when tested for yeast promoter activity on growth plates (Example 9, U.S. Pat. No. 6,265,185) and based on their activity in the pH range of 4-11. U.S. Pat. No. 7,259,255 and U.S. Pat. No. 7,459,546 describe regulatory sequences (e.g., promoters, introns) of the glyceraldehyde-3-phosphate dehydrogenase (gpd) and phosphoglycerate mutase (gpm) genes; U.S. Pat. No. 7,202,356 describes regulatory sequences (e.g., promoters, introns) of the fructose-bisphosphate aldolase (fba) gene; and, U.S. Pat. No. 7,264,949 describes promoters of the glycerol-3-phosphate O-acyltransferase (gpat) gene. Similarly, Juretzek et al. (Biotech. Bioprocess Eng., 5:320-326 (2000)) compares the glycerol-3-phosphate dehydrogenase (G3P), isocitrate lyase (ICL1), 3-oxo-acyl-CoA thiolase (POT1) and acyl-CoA oxidase (POX1, POX2 and POX5) promoters with respect to their regulation and activities during growth on different carbon sources.
Despite the utility of these known promoters, however, there is a need for new improved yeast promoters for metabolic engineering of yeast (oleaginous and non-oleaginous) and for controlling the expression of heterologous genes in yeast. Furthermore, possession of a suite of promoters that are regulatable under a variety of natural growth and induction conditions in yeast will play an important role in industrial settings, wherein it is desirable to express heterologous polypeptides in commercial quantities in said hosts for economical production of those polypeptides. Thus, it is an object herein to provide such promoters that will be useful for gene expression in a variety of yeast cultures, and preferably in Yarrowia sp. cultures and other oleaginous yeast. Specifically, YAT1 promoters from Yarrowia lipolytica, have been identified that are responsible for driving expression of the gene (yat1) encoding an ammonium transporter (YAT1). Advantageously, the promoters are useful for regulated expression of heterologous genes in Yarrowia, have improved activity with respect to the TEF promoter and are inducible under oleaginous conditions (i.e., nitrogen limitation).
SUMMARY OF THE INVENTION
In a first embodiment, the invention concerns a recombinant expression cassette comprising at least one coding region of interest expressible in an oleaginous yeast cell operably linked to an isolated nucleic acid fragment comprising a promoter region of a Yarrowia yat 1 gene, said promoter region consisting essentially of a nucleotide sequence as set forth in SEQ ID NO:25 wherein said sequence can be unmodified or modified, wherein the modification is selected from the group consisting of:

    
    
        a) a truncation of one or more consecutive nucleotides occurring at the 5′ end ranging from nucleotide 1 up to and including nucleotide 102;
        b) a mutation at nucleotide 414 in which deoxycytidine is replaced by deoxythymidine, deoxyadenosine, or deoxyguanosine;
        c) an insertion of two deoxycytidines at nucleotide 776;
        d) a truncation of one or more consecutive nucleotides occurring at the 5′ end ranging from nucleotide 1 up to and including nucleotide 102 and a mutation at nucleotide 414 in which deoxycytidine is replaced by deoxythymidine, deoxyadenosine, or deoxyguanosine;
        e) a truncation of one or more consecutive nucleotides occurring at the 5′ end ranging from nucleotide 1 up to and including nucleotide 102 and an insertion of two deoxycytidines at nucleotide 776;
        f) a mutation at nucleotide 414 in which deoxycytidine is replaced by deoxythymidine, deoxyadenosine, or deoxyguanosine and an insertion of two deoxycytidines at nucleotide 776;
        g) a truncation of one or more consecutive nucleotides occurring at the 5′ end ranging from nucleotide 1 up to and including nucleotide 102, a mutation at nucleotide 414 in which deoxycytidine is replaced by deoxythymidine, deoxyadenosine, or deoxyguanosine, and an insertion of two deoxycytidines at nucleotide 776.
    
    


In a second embodiment, the recombinant expression cassette of the invention further comprises a restriction enzyme site inserted upstream and adjacent to the promoter region.
In a third embodiment, the recombinant expression cassette of the invention which comprises a promoter region of a Yarrowia yat 1 gene wherein the Yarrowia yat 1 gene is isolated from Yarrowia lipolytica. 
In a fourth embodiment, the recombinant expression cassette of the invention comprises a promoter region that consists essentially of a nucleotide sequence selected from the group consisting of: SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.
In a fifth embodiment, the invention concerns a method for expressing at least one coding region of interest in an oleaginous yeast cell which comprises:

    
    
        a) transforming an oleaginous yeast cell with the recombinant expression cassette of any of claims 1-4; and,
        b) growing the transformed oleaginous yeast cell of step (a) under conditions whereby the coding region of interest is expressed in the transformed oleaginous yeast cell.
    
    


Specifically, the oleaginous yeast can be a member of a genus selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon Lipomyces and Saccharomyces having the property of oleaginy. More specifically, the oleaginous yeast can be Yarrowia lipolytica. 
In a sixth embodiment, the method of the invention relates to expression of at least one coding region of interest wherein the coding region of interest encodes a polypeptide selected from the group consisting of: desaturases, elongases, acyltransferases, aminopeptidases, amylases, carbohydrases, carboxypeptidases, catalyases, cellulases, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, esterases, α-galactosidases, galactosidases, glucoamylases, α-glucosidases, β-glucanases, β-glucosidases, invertases, laccases, lipases, mannosidases, mutanases, oxidases, pectinolytic enzymes, peroxidases, phospholipases, phytases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transglutaminases and xylanases. Specifically, the desaturase is selected from the group consisting of: Δ9 desaturase, Δ12 desaturase, Δ6 desaturase, Δ5 desaturase, Δ17 desaturase, Δ15 desaturase, Δ8 desaturase and Δ4 desaturase; and, the elongase is selected from the group consisting of: Δ9 elongase, C14/16 elongase, C16/18 elongase, C18/20 elongase and/or C20/22 elongase.
In a seventh embodiment, the method of the invention can be practiced wherein the transformed oleaginous yeast cell is grown under conditions of nitrogen limitation.
In an eighth embodiment, the method of the invention relates to expression of at least one coding region of interest wherein the coding region of interest encodes at least one enzyme selected from the group consisting of an enzyme of the ω-3/ω-6 fatty acid biosynthetic pathway, an enzyme of the neutral lipid biosynthetic pathway and an enzyme of the phospholipid biosynthetic pathway. Specifically, the at least one enzyme of the ω-3 or ω-6 fatty acid produces a polyunsaturated fatty acid product selected from the group consisting of: linoleic acid, α-linolenic acid, γ-linolenic acid, stearidonic acid, dihomo-γ-linolenic acid, eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid, ω-6 docosapentaenoic acid, ω-3 docosapentaenoic acid, docosahexaenoic acid, eicosadienoic acid and eicosatrienoic acid.
Biological Deposits
The following biological materials have been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, and bear the following designations, accession numbers and dates of deposit.












Biological Material
Accession No.
Date of Deposit







Yarrowia lipolytica Y4128
ATCC PTA-8614
Aug. 23, 2007









The biological materials listed above were deposited under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The listed deposit will be maintained in the indicated international depository for at least 30 years and will be made available to the public upon the grant of a patent disclosing it. The availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.



BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS
FIG. 1 graphically represents the relationship between SEQ ID NOs:1, 2, 3, 24, 25, 30, 33 and 35, each of which relates to the yat1 ammonium transporter in Y. lipolytica. 
FIG. 2 illustrates the ω-3/ω-6 fatty acid biosynthetic pathway.
FIG. 3 provides plasmid maps for the following: (A) pY5-30; (B) pDMW214; (C) pYGPAT-GUS; and (D) pYAT-GUS, respectively.
FIG. 4A, FIG. 4B and FIG. 4C illustrate the relative promoter activities of YAT1, TEF, GPAT and FBAIN in Y. lipolytica grown in various media as determined by histochemical staining.
FIG. 5 provides a plasmid map for pYAT-GUS1.
FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, FIG. 6G and FIG. 6H, when viewed together, show an alignment of twelve YAT1 promoters. Eleven of these promoters are derived from the wildtype YAT1 promoter (SEQ ID NO:25; “775 by wt YAT1”), which corresponds to the −775 to −1 region upstream of the yat1 gene. Specifically, aligned are the 777 by YAT1-CC promoter (SEQ ID NO:26), the 783 by YAT1-CC-SalI promoter (SEQ ID NO:27), the 777 by YAT1-CC-NcoI* promoter (SEQ ID NO:28), the 783 by YAT1-CC-NcoI*-SalI promoter (SEQ ID NO:29), the 751 by YAT1-CC-NcoI*-26 promoter (SEQ ID NO:30), the 757 by YAT1-CC-NcoI*-26-ClaI promoter (SEQ ID NO:31), the 759 by YAT1-CC-NcoI*-26-SwaI promoter (SEQ ID NO:32), the 729 by YAT1-CC-NcoI*-48 promoter (SEQ ID NO:33), the 737 by YAT1-CC-NcoI*-48-PmeI promoter (SEQ ID NO:34), the 675 by YAT1-CC-NcoI*-102 promoter (SEQ ID NO:35) and the 681 bp YAT1-CC-NcoI*-102-EcoRI promoter (SEQ ID NO:36).
FIG. 7 diagrams the development of Yarrowia lipolytica strain Y4259, producing greater than 46.5% EPA in the total lipid fraction and identifies various YAT1 promoters used within chimeric genes in specific expression cassettes and constructs.



The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions, which form a part of this application.
The following sequences comply with 37 C.F.R. §1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
SEQ ID NOs:1-45 correspond to ORFs (i.e., encoding genes or proteins), primers, promoters and plasmids, as identified in Table 1.







TABLE 1







Summary Of Nucleotide And Protein SEQ ID Numbers










Nucleotide
Protein SEQ


Description
SEQ ID NO
ID NO





Yarrowia lipolytica ammonium
 1 (1461 bp)
 2 (486 AA)


transporter (yat1) (GenBank Accession


No. XM_504457)


YAT1 promoter fragment
 3 (778 bp)
—


BD-Clontech's Creator Smart ® cDNA
 4
—


library kit primer


SMART IV oligonucleotide


BD-Clontech's Creator Smart ®
 5
—


cDNA library kit


primer CDSIII/3′ PCR


BD-Clontech's Creator Smart ® cDNA
 6
—


library kit primer 5′-PCR


M13 forward primer
 7
—


Primer 27203-F
 8
—


Primer 27203-R
 9
—


Plasmid pY5-30
10 (8953 bp)
—


FBAIN promoter
11 (973 bp)
—


GPAT promoter
12 (1130 bp)
—


Plasmid pKUNF12T6E
13 (12,649 bp)
—


Synthetic elongase gene derived
14 (957 bp)
15 (318 AA)


from Mortierella alpina, codon-optimized


for expression in Yarrowia lipolytica


Synthetic Δ6 desaturase, derived
16 (1374 bp)
17 (457 AA)


from Mortierella alpina, codon-optimized


for expression in Yarrowia lipolytica


Fusarium moniliforme Δ12 desaturase
19 (1434 bp)
20 (477 AA)


Synthetic elongase gene derived from
21 (819 bp)
22 (272 AA)


Thraustochytrium aureum,


codon-optimized for


expression in Yarrowia lipolytica


Plasmid pDMW232
23 (10,945 bp)
—


Yarrowia lipolytica yat1 gene: −775
24 (2236 bp)
—


to +1461 region


Wildtype YAT1 promoter,
25 (775 bp)
—


corresponding to the −775 to −1


region upstream of the yat1 gene


Modified YAT1-CC promoter
26 (777 bp)
—


Modified YAT1-CC-Sa/I promoter
27 (783 bp)
—


Modified YAT1-CC-Ncol* promoter
28 (777 bp)
—


Modified YAT1-CC-Ncol*-Sa/I promoter
29 (783 bp)
—


Modified YAT1-CC-Ncol*-26 promoter
30 (751 bp)
—


Modified YAT1-CC-Ncol*-26-C/aI
31 (757 bp)
—


promoter


Modified YAT1-CC-Ncol*-26-SwaI
32 (759 bp)
—


promoter


Modified YAT1-CC-Ncol*-48 promoter
33 (729 bp)
—


Modified YAT1-CC-Ncol*-48-PmeI
34 (737 bp)
—


promoter


Modified YAT1-CC-Ncol*-102 promoter
35 (675 bp)
—


Modified YAT1-CC-Ncol*-102-EcoRI
36 (681 bp)
—


promoter


Y. lipolytica consensus sequence
37
—


of translation initiation site


Plasmid pZKLeuN-29E3
38 (14,688 bp)
—


Plasmid pKO2UF8289
39 (15,337 bp)
—


Plasmid pZKSL-555R
40 (13,707 bp)
—


Plasmid pZP3-Pa777U
41 (13,066 bp)
—


Plasmid pZP2-2988
42 (15,743 bp)
—


Plasmid pZKL2-5U89GC
43 (15,812 bp)
—


Plasmid pZKL1-2SP98C
44 (15,877 bp)
—


Plasmid pYAT-GUS1
45 (6645 bp)
—









DETAILED DESCRIPTION OF THE INVENTION
All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.
In this disclosure, a number of terms and abbreviations are used. The following definitions are provided.
“Ammonium transporter” is abbreviated “YAT”.
“Open reading frame” is abbreviated “ORF”.
“Polymerase chain reaction” is abbreviated “PCR”.
“Polyunsaturated fatty acid(s)” is abbreviated “PUFA(s)”.
The term “oleaginous” refers to those organisms that tend to store their energy source in the form of oil (Weete, In: Fungal Lipid Biochemistry, 2nd Ed., Plenum, 1980). The term “oleaginous yeast” refers to those microorganisms classified as yeasts that can make oil. Generally, the cellular oil content of oleaginous microorganisms follows a sigmoid curve, wherein the concentration of lipid increases until it reaches a maximum at the late logarithmic or early stationary growth phase and then gradually decreases during the late stationary and death phases (Yongmanitchai and Ward, Appl. Environ. Microbiol., 57:419-25 (1991)). It is not uncommon for oleaginous microorganisms to accumulate in excess of about 25% of their dry cell weight as oil. Examples of oleaginous yeast include, but are no means limited to, the following genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. In alternate embodiments, a non-oleaginous organism can be genetically modified to become oleaginous, wherein the organism obtains the ability to accumulate in excess of about 25% of their dry cell weight as oil by means of genetic engineering, e.g., yeast such as Saccharomyces cerevisiae. 
The term “ammonium transporter” refers to a family of transporters whose physiological role within a cell is to scavenge external ammonium for use as a nitrogen source (and, under some circumstances, to incorporate ammonium that leaks out of cells), based on studies of the MEP family of ammonium transporters in Saccharomyces cerevisiae (A.M. Marini et al., EMBO J., 13(15):3456-3463 (1994); Mol Cell Biol., 17(8):4282-93 (1997)). In general, the proteins are subject to nitrogen control and each has different kinetic properties, specificities and regulation; for example, the three S. cerevisiae isozymes are characterized as follows: MEP1, a low affinity/high capacity ammonia transporter (Km, 5-10 μM); MEP2, a high affinity/low capacity ammonia transporter (Km, 1-2 μM); and MEP3, a low affinity ammonia transporter (Km, 1.4-2.1 mM). Other known members of this family include: the high affinity ammonium transporter (amt1), the ammonium and methylammonium transport system, the putative ammonium transporter amtB and nrgA.
As used herein, the term “YAT1” refers to an ammonium transporter enzyme (TC 2.A.49) encoded by the yat1 gene and isolated from Yarrowia lipolytica (GenBank Accession No. XM—504457). The sequence disclosed therein (/locus_tag=“YALI0E27203g”) was identified as a hypothetical protein having similarity “to sp|P41948 Saccharomyces cerevisiae YNL142w MEP2, hypothetical start”. The Y. lipolytica yat1 gene is presented herein as SEQ ID NO:1, while the corresponding YAT1 protein is provided herein as SEQ ID NO:2. A 2236 by DNA contig comprising the 5′ upstream region and the Y. lipolytica yat1 gene are set forth in SEQ ID NO:24.
“Promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
The term “YAT1 promoter” or “YAT1 promoter region” refers to the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of yat1 and that is necessary for expression. An example of a suitable YAT1 promoter region is provided as SEQ ID NO:25, corresponding to the −775 to −1 region upstream of the yat1 gene, but this is not intended to be limiting in nature. One skilled in the art will recognize that since the exact boundaries of the YAT1 promoter sequence have not been completely defined, DNA fragments of increased or diminished length may have identical promoter activity. Thus, for example, it is expected that a promoter region comprising at least the −673 to −1 region upstream of the yat1 gene will also be functional as a suitable YAT1 promoter, as will a promoter region comprising at least the −727 to −1 region and a promoter region comprising at least the −749 to −1 region upstream of the yat1 gene.
The term “mutant promoter” or “modified promoter” is defined herein as a promoter having a nucleotide sequence comprising a substitution, deletion, and/or insertion of one or more nucleotides relative to the parent promoter, wherein the modified promoter has more promoter activity, less promoter activity or equivalent promoter activity when compared to the corresponding parent promoter. The term “modified promoter” will encompass natural variants and in vitro generated variants obtained using methods well known in the art (e.g., classical mutagenesis, site-directed mutagenesis and “DNA shuffling”). Non-limiting examples of mutant promoters derived from the wildtype YAT1 promoter sequence of SEQ ID NO:25 include: 1) promoters comprising an insertion of two deoxycytidines [“CC”] at by +776 with respect to SEQ ID NO:25; 2) promoters comprising a deoxycytidine [“C”] to deoxyadenosine [“A”], deoxyguanosine [“G”], or deoxythymidine [“T”] mutation at +414 with respect to SEQ ID NO:25; and 3) promoters comprising a deoxyguanosine [“G”] to deoxythymidine [“T”] mutation at +100 with respect to SEQ ID NO:25.
Although mutant YAT1 promoters are different than wildtype YAT1 promoters in sequence (and may be different in promoter activity), the term “YAT1 promoter” will be applied throughout the specification to refer to either mutant or wildtype YAT1 promoters that are derived or identified from the 5′ upstream region of the yat1 gene, unless specifically stated to be otherwise.
The term “GPAT” refers to a glycerol-3-phosphate O-acyltransferase enzyme (E.C. 2.3.1.15) encoded by the gpat gene and which converts acyl-CoA and sn-glycerol 3-phosphate to CoA and 1-acyl-sn-glycerol 3-phosphate (the first step of phospholipid biosynthesis). The term “GPAT promoter” or “GPAT promoter region” refers to the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of gpat and that is necessary for expression. One example of a suitable GPAT promoter region is provided as SEQ ID NO:12, but this is not intended to be limiting in nature (see U.S. Pat. No. 7,264,949).
The term “FBA1” refers to a fructose-bisphosphate aldolase enzyme (E.C. 4.1.2.13) encoded by the fba1 gene and which converts D-fructose 1,6-bisphosphate into glycerone phosphate and D-glyceraldehyde 3-phosphate. The term “FBAIN promoter” or “FBAIN promoter region” refers to the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of fba1 and that is necessary for expression, plus a portion of 5′ coding region comprising an intron of the fba1 gene. An example of a suitable FBAIN promoter region is provided as SEQ ID NO:11, but this is not intended to be limiting in nature (see U.S. Pat. No. 7,202,356).
The term “promoter activity” will refer to an assessment of the transcriptional efficiency of a promoter. This may, for instance, be determined directly by measurement of the amount of mRNA transcription from the promoter (e.g., by Northern blotting or primer extension methods) or indirectly by measuring the amount of gene product expressed from the promoter, as for example by histochemical means.
The term “conditions of nitrogen limitation” refers to a medium having a low concentration of nitrogen, wherein the nitrogen may be supplied from an inorganic (e.g., (NH4)2SO4) or organic (e.g., urea or glutamate) source, or a medium having no nitrogen source. Although one skilled in the art will be able to determine an appropriate low concentration of nitrogen suitable to induce the YAT1 promoters of the present disclosure, in one embodiment a preferred medium would be one characterized as having a high carbon to nitrogen (i.e., C:N) ratio and about 0.1% or less ammonium sulfate or other suitable ammonium salts.
The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”, “nucleic acid fragment” and “isolated nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (usually found in their 5′-monophosphate form) are referred to by a single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.
The term “genetic construct” is a non-limiting term meaning any contiguous series of nucleic acids capable of being expressed in a host organism. A genetic construct may include but is not limited to an open reading frame [“ORF”], an ORF operably linked to regulatory sequences, or a wildtype or mutant gene. Genetic constructs may encode polypeptides or be nucleic acid fragments or molecules that are oriented for antisense expression.
A nucleic acid molecule is “hybridizable” to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), which is hereby incorporated herein by reference, particularly Chapter 11 and Table 11.1. The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringent conditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.
Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.
A “substantial portion” of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to identify putatively a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation, such as in situ hybridization of bacterial colonies or bacteriophage plaques. In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid molecule comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid molecule comprising the sequence.
As used herein, the terms “homology” and “homologous” are used interchangeably. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment.
Moreover, the skilled artisan recognizes that homologous nucleic acid sequences are also defined by their ability to hybridize, under moderately stringent conditions, e.g., 0.5×SSC, 0.1% SDS, 60° C., with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent thereto. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, N.Y. (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).
Described herein are nucleotide sequences encoding a particular microbial promoter region. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the disclosure comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
The term “oligonucleotide” refers to a nucleic acid, generally of at least 14 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule. In one embodiment, a labeled oligonucleotide can be used as a “probe” to detect the presence of a nucleic acid. Thus, the term “probe” refers to a single-stranded nucleic acid molecule that can base pair with a complementary single-stranded target nucleic acid to form a double-stranded molecule. The term “label” will refer to any conventional molecule which can be readily attached to mRNA or DNA and which can produce a detectable signal, the intensity of which indicates the relative amount of hybridization of the labeled probe to the DNA fragment.
The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another.
For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the disclosure also includes isolated nucleic acid molecules that are complementary to the complete sequences as reported in the accompanying Sequence Listing, as well as those substantially similar nucleic acid sequences.
The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. “Percent identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Percent identity” and “percent similarity” can be readily calculated by known methods, including but not limited to those described in: 1) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).
Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the MegAlignTM program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the Clustal method of alignment” which encompasses several varieties of the algorithm including the “Clustal V method of alignment” and the “Clustal W method of alignment” (described by Higgins and Sharp, CABIOS, 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191(1992)) and found in the MegAlignTM (version 8.0.2) program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). After alignment of the sequences using either Clustal program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the program.
For multiple alignments using the Clustal V method of alignment, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. Default parameters for multiple alignment using the Clustal W method of alignment correspond to GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergent Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB.
It is well understood by one skilled in the art that various measures of sequence percent identity are useful in identifying polynucleotides and polypeptides, from other species, wherein such polynucleotides and polypeptides have the same or similar function or activity. Suitable promoter regions (isolated polynucleotides of the present invention) encode promoter regions that are at least about 70% identical, preferably at least about 75% identical, and more preferably at least about 80% identical to the nucleotide sequences reported herein. Preferred nucleic acid molecules are about 85% identical to the nucleotide sequences reported herein, more preferred nucleic acid molecules are at least about 90% identical, and most preferred are nucleic acid molecules at least about 95% identical to the nucleotide sequences reported herein. Suitable promoter regions not only have the above homologies but typically are at least 50 nucleotides in length, more preferably at least 100 nucleotides in length, more preferably at least 250 nucleotides in length, and more preferably at least 500 nucleotides in length.
“Codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
“Synthetic genes” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments that are then enzymatically assembled to construct the entire gene. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell, where sequence information is available. For example, the codon usage profile for Yarrowia lipolytica is provided in U.S. Pat. No. 7,125,672.
“Gene” refers to a nucleic acid molecule that expresses a specific protein, and which may refer to the coding region alone or may include regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Chimeric genes of the present disclosure will typically comprise a YAT1 promoter region operably linked to a coding region of interest. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure. A “codon-optimized gene” is a gene having its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.
“Coding sequence” refers to a DNA sequence that codes for a specific amino acid sequence.
“Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, enhancers, silencers, 5′ untranslated leader sequence (e.g., between the transcription start site and the translation initiation codon), introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.
The term “3′ non-coding sequences” or “transcription terminator” refers to DNA sequences located downstream of a coding sequence. This includes polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The 3′ region can influence the transcription, RNA processing or stability, or translation of the associated coding sequence.
“RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA” or “mRNA” refers to the RNA that is without introns and that can be translated into protein by the cell. “cDNA” refers to a double-stranded DNA that is complementary to, and derived from, mRNA. “Sense” RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell. “Antisense RNA” refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065; Intl. App. Pub. No. WO 99/28508).
The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid molecule so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence. That is, the coding sequence is under the transcriptional control of the promoter. Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a coding sequence. Expression may also refer to translation of mRNA into a polypeptide.
“Transformation” refers to the transfer of a nucleic acid molecule into a host organism, resulting in genetically stable inheritance. The nucleic acid molecule may be a plasmid that replicates autonomously, for example; or, it may integrate into the genome of the host organism. Host organisms containing the transformed nucleic acid molecules are referred to as “transgenic”, “recombinant” or “transformed” organisms or “transformants”.
The term “recombinant” refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
The terms “plasmid” and “vector” refer to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing an expression cassette(s) into a cell.
“Expression cassette” refers to a fragment of DNA comprising the coding sequence of a selected gene and regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence that are required for expression of the selected gene product. Thus, an expression cassette is typically composed of: 1) a promoter sequence; 2) a coding sequence (i.e., ORF); and, 3) a 3′ untranslated region (i.e., a terminator) that, in eukaryotes, usually contains a polyadenylation site. The expression cassette(s) is usually included within a vector, to facilitate cloning and transformation. Different expression cassettes can be transformed into different organisms including bacteria, yeast, plants and mammalian cells, as long as the correct regulatory sequences are used for each host.
The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and, 5) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Suhai, Sandor, Ed. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters that originally load with the software when first initialized.
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience, Hoboken, N.J. (1987).
Ammonium transporters are a family of transporters whose physiological role within a cell is to scavenge external ammonium for use as a nitrogen source (and, under some circumstances, to incorporate ammonium that leaks out of cells). In general, the proteins are subject to nitrogen control and each have different kinetic properties, specificities and regulation; for example, the three S. cerevisiae MEP1, MEP2, and MEP3 isozymes have been extensively characterized by A. M. Marini et al. (EMBO J., 13(15): 3456-3463 (1994); Mol Cell Biol., 17(8):4282-93 (1997)) as a low affinity/high capacity ammonia transporter, a high affinity/low capacity ammonia transporter, and a low affinity ammonia transporter, respectively.
A cDNA library was constructed from Yarrowia lipolytica cells grown in an oleaginous medium containing no nitrogen source for 4 hrs (i.e., oleaginous conditions that promoter oil production). The relative abundance of each species of cDNA was then examined to identify those genes that were highly expressed under the oleaginous conditions. One of the genes that appeared multiple times (16/9984 or 0.16%) was GenBank Accession No. XM—504457, locus_tag=“YALI0E27203g”. This gene was annotated as a hypothetical protein having similarity “to sp|P41948 Saccharomyces cerevisiae YNL142w MEP2 [the high affinity, low capacity ammonia transporter; GenBank Accession No. X83608], hypothetical start”.
Based on sequence comparison to the MEP1, MEP2 and MEP3 genes (supra), it was hypothesized that the Y. lipolytica ORF likely encoded an ammonium transporter and designated the gene as the “yat1” gene (SEQ ID NOs:1 and 2). The “YAT1 promoter” was identified as the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of yat1 and that is necessary for expression. Accordingly, this putative YAT1 promoter region will be useful for driving expression of any suitable coding region of interest in a transformed yeast cell. In general, a promoter useful in an oleaginous yeast should meet the following criteria:

    
    
        1) Strength. A strong yeast promoter is a necessary premise for a high expression level, and the low copy number of the ars18 (Fournier, P. et al., Yeast, 7:25-36 (1991)) based expression vectors or chimeric genes integrated into the genome makes this demand even more important when Y. lipolytica is used as the host organism.
        2) Activity in a medium suitable for expression of the coding region of interest, and high enzymatic activity of that coding region of interest.
        3) pH Tolerance. If the coding region of interest is known to be produced only in e.g., an acidic environment, then the promoter operably linked to said coding region of interest must function at the appropriate pH. pH tolerance is of course limited by the tolerance of the host organism.
        4) Inducibility. A tightly regulated yeast promoter makes it possible to separate the growth stage from the expression stage, thereby enabling expression of products that are known to inhibit cell growth.
        5) Activity in the stationary phase of growth in oleaginous yeast hosts for accumulation of PUFAs.
    
    


Additionally, it is preferable for novel yeast promoters to possess differences in activity with respect to the known Yarrowia lipolytica TEF (U.S. Pat. No. 6,265,185), XPR2 (U.S. Pat. No. 4,937,189; EP220864; EP832258), GPD and GPM (U.S. Pat. No. 7,259,255 and U.S. Pat. No. 7,459,546), FBA, FBAIN and FBAINm (U.S. Pat. No. 7,202,356) and GPAT (U.S. Pat. No. 7,264,949) promoters and/or the G3P, ICL1, POT1, POX1, POX2 and POX5 promoters (Juretzek et al., Biotech. Bioprocess Eng., 5:320-326 (2000)).
A comparative study of the TEF, FBAIN and GPAT promoters and the 775 by YAT1 promoter described herein as SEQ ID NO:25, corresponding to the −775 to −1 region upstream of the yat1 gene, is provided in Examples 8 and 9. As shown in Table 5 of Example 9, the yeast promoter has improved activity compared to the TEF promoter (i.e., 1.3:1 nmoles of 4-methylumbelliferone per minute per mg of total protein) under conditions where nitrogen is not limiting (i.e., minimal medium containing ammonium sulfate as the nitrogen source). Furthermore, under conditions of nitrogen limitation, the YAT1 promoter is significantly induced such that the activity is  ̃20-28 times greater than that of TEF; thus, when grown in such medium, the activity of the YAT1 promoter is comparable to that of the strong FBAIN promoter. Based on these results, the Applicants characterize the YAT1 promoter of SEQ ID NO:25 as the first promoter identified within Yarrowia that is inducible under oleaginous conditions (i.e., nitrogen limitation).
An example of a suitable YAT1 promoter region is provided SEQ ID NO:25 (comprising the −775 to −1 region of the Y. lipolytica yat1 gene [wherein the ‘A’ position of the ‘ATG’ translation initiation codon is designated as +1]), but this is not intended to be limiting in nature. One skilled in the art will recognize that since the exact boundaries of the YAT1 promoter sequence have not been completely defined, DNA fragments of increased or diminished length may have identical promoter activity. For example, Example 10 describes a variety of “truncated” YAT1 promoters when compared to the wildtype promoter of SEQ ID NO:25, wherein the truncation is of one or more consecutive nucleotides occurring at the 5′ end ranging from nucleotide 1 up to and including nucleotide 102. Thus, for example, the YAT1-CC-NcoI*-26 promoter of SEQ ID NO:30 comprises the −749 to −1 region upstream of the yat1 gene (i.e., nucleotides +1 to +26 of SEQ ID NO:25 were deleted), the YAT1-CC-NcoI*-48 promoter of SEQ ID NO:33 comprises the −727 to −1 region upstream of the yat1 gene (i.e., nucleotides +1 to +48 of SEQ ID NO:25 were deleted) and the YAT1-CC-NcoI*-102 promoter of SEQ ID NO:35 comprises the −673 to −1 region upstream of the yat1 gene (i.e., nucleotides +1 to +97 of SEQ ID NO:25 were deleted). In all cases, these “truncated” YAT1 promoters were found to be functional, with activity comparable to that of the wildtype YAT1 promoter of SEQ ID NO:25.
Accordingly, the term “YAT1 promoter” as used herein applies to any and all YAT1 promoters that are identified or created via truncation from the 5′ upstream region of the yat1 gene, unless a specific SEQ ID NO is referenced.
Thus, as one of skill in the art will appreciate, suitable YAT1 promoters will correspond to the −775 to −1 region, −774 to −1 region, −773 to −1 region, −772 to −1 region, −771 to −1 region, −770 to −1 region, −769 to −1 region, −768 to −1 region, −767 to −1 region, −766 to −1 region, −765 to −1 region, −764 to −1 region, −763 to −1 region, −762 to −1 region, −761 to −1 region, −760 to −1 region, −759 to −1 region, −758 to −1 region, −757 to −1 region, −756 to −1 region, −755 to −1 region, −754 to −1 region, −753 to −1 region, −752 to −1 region, −751 to −1 region, −750 to −1 region, −749 to −1 region, −748 to −1 region, −747 to −1 region, −746 to −1 region, −745 to −1 region, −744 to −1 region, −743 to −1 region, −742 to −1 region, −741 to −1 region, −740 to −1 region, −739 to −1 region, −738 to −1 region, −737 to −1 region, −736 to −1 region, −735 to −1 region, −734 to −1 region, −733 to −1 region, −732 to −1 region, −731 to −1 region, −730 to −1 region, −729 to −1 region, −728 to −1 region, −727 to −1 region, −726 to −1 region, −725 to −1 region, −724 to −1 region, −723 to −1 region, −722 to −1 region, −721 to −1 region, −720 to −1 region, −719 to −1 region, −718 to −1 region, −717 to −1 region, −716 to −1 region, −715 to −1 region, −714 to −1 region, −713 to −1 region, −712 to −1 region, −711 to −1 region, −710 to −1 region, −709 to −1 region, −708 to −1 region, −707 to −1 region, −706 to −1 region, −705 to −1 region, −704 to −1 region, −703 to −1 region, −702 to −1 region, −701 to −1 region, −700 to −1 region, −699 to −1 region, −698 to −1 region, −697 to −1 region, −696 to −1 region, −695 to −1 region, −694 to −1 region, −693 to −1 region, −692 to −1 region, −691 to −1 region, −690 to −1 region, −689 to −1 region, −688 to −1 region, −687 to −1 region, −686 to −1 region, −685 to −1 region, −684 to −1 region, −683 to −1 region, −682 to −1 region, −681 to −1 region, −680 to −1 region, −679 to −1 region, −678 to −1 region, −677 to −1 region, −676 to −1 region, −675 to −1 region, −674 to −1 region and −673 to −1 region upstream of the yat1 gene (see, SEQ ID NO:24 and SEQ ID NO:25).
In an alternate embodiment, the YAT1 promoter may comprise nucleotides −500 to −1 upstream of the yat1 gene (i.e., nucleotides 276-775 of SEQ ID NO:25), thereby permitting relatively strong promoter activity; in another embodiment, the −100 to −1 region upstream of the yat1 gene (i.e., nucleotides 676-775 of SEQ ID NO:25) should be sufficient for basal activity of the promoter. Likewise, the promoter region of the invention may comprise additional nucleotides to those specified above. For example, the promoter sequences of the invention may be constructed on the basis of the −1000 to −1 region of the yat1 gene (e.g., nucleotide bases 3,222,879 to 3,223,879 of GenBank Accession No. CR382131, comprising the complete nucleotide sequence of chromosome E of strain CLIB99 of Y. lipolytica).
In addition to truncations which reduce the total length of the YAT1 promoter, from that described in SEQ ID NO:25, one of skill in the art will understand that mutant promoters may also be constructed, wherein the DNA sequence of the promoter has one or more nucleotide substitutions (i.e., deletions, insertions, or addition of one or more nucleotides in the sequence) which do not effect (in particular impair) the yeast promoter activity. Regions that can be modified without significantly affecting the yeast promoter activity can be identified by deletion studies. A mutant promoter has at least about 20%, preferably at least about 40%, more preferably at least about 60%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 100%, more preferably at least about 200%, more preferably at least about 300% and most preferably at least about 400% of the promoter activity of the YAT1 promoter region described herein as SEQ ID NO:25.
Methods for mutagenesis are well known in the art and suitable for the generation of mutant promoters. For example, in vitro mutagenesis and selection, PCR based random mutagenesis, site-directed mutagenesis, chemical synthesis of a mutated DNA fragment or other means can be employed to obtain mutations of the naturally occurring promoter of e.g., SEQ ID NO:25 (wherein such mutations may include deletions, insertions and point mutations, or combinations thereof). This would permit production of a putative promoter having a more desirable level of promoter activity in the host cell. Or, if desired, the regions of a nucleotide of interest important for promoter activity can be determined through routine mutagenesis, expression of the resulting mutant promoters and determination of their activities. An overview of these techniques is described in U.S. Pat. No. 7,259,255. All such mutant promoters that are derived from the instant YAT1 promoter described herein are within the scope of the present disclosure.
Non-limiting examples of modified or “mutated” YAT1 promoters, when compared to the wildtype promoter of SEQ ID NO:25, are described in Example 10 and their use is demonstrated in Example 11. Specifically, the YAT1-CC promoter of SEQ ID NO:26 is identical to the YAT1 promoter of SEQ ID NO:25, with the exception of two deoxycytidines [“CC”] inserted at nucleotide 776 (thereby creating a preferred consensus sequence around the translation initiation site of operably linked genes, for optimal gene expression). SEQ ID NO:27, comprising the YAT1-CC-SalI promoter, is identical to SEQ ID NO:26, although a 6 by SalI restriction enzyme [“RE”] site is inserted upstream and adjacent to the promoter region, for cloning convenience. One of skill in the art will recognize that the particular RE site to be inserted upstream and adjacent to the promoter region will not affect the activity of the promoter. Similarly, the YAT1-CC-NcoI* promoter of SEQ ID NO:28 tolerates a mutation at nucleotide 414 in which a C nucleotide is replaced by a deoxythymidine [“T”] (thereby removing an internal NcoI RE site from the promoter fragment) and a CC insertion at nucleotide 776, with respect to SEQ ID NO:25. It is assumed that the C nucleotide at position 414 could also be substituted with a deoxyadenosine [“A”] or a deoxyguanosine [“G”]. In all cases, these “mutated” YAT1 promoters were found to be functional, with activity comparable to that of the wildtype YAT1 promoter of SEQ ID NO:25. Furthermore, it is also demonstrated that these mutations could be combined with the truncations previously described. Furthermore, the term “YAT1 promoter” also encompasses any and all YAT1 promoters that are identified or created via mutation from the 5′ upstream region of the yat1 gene, unless a specific SEQ ID NO is referenced.
Promoter activity is typically measured against the activity of the wild type promoter under similar conditions. Promoter activity is generally measured as a function of gene expression and may be determined in a variety of ways including gene expression profiling, measurement of the level of RNA and/or protein expression, or the measurement of reporter activity where reporter gene fusions have been created.
It will be appreciated by a person of skill in the art that the promoter regions of the present disclosure have homologs in a variety of yeast species; and, the use of the promoters for regulated, heterologous gene expression are not limited to those promoters derived from Yarrowia lipolytica, but extend to homologs in other yeast species. For example, encompassed herein are homologs derived from oleaginous genera including, but not limited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces; examples of preferred species within these genera include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus and R. graminis. 
Homology typically is measured using sequence analysis software, wherein the term “sequence analysis software” refers to any computer algorithm or software program (commercially available or independently developed) that is useful for the analysis of nucleotide or amino acid sequences. In general, such computer software matches similar sequences by assigning degrees of homology to various substitutions, deletions and other modifications.
As is well known in the art, isolation of homologous promoter regions using sequence-dependent protocols is readily possible using various techniques; and, these techniques can rely on either the direct identification of a promoter having homology to the YAT1 promoter described herein or the indirect identification of a promoter by initial identification of a gene having significant homology to the yat1 gene and then analysis of the 5′ upstream sequence of the homologous gene. Examples of sequence-dependent protocols include, but are not limited to: 1) methods of nucleic acid hybridization; 2) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies such as polymerase chain reaction [“PCR”] (U.S. Pat. No. 4,683,202); ligase chain reaction [“LCR”] (Tabor, S. et al., Proc. Acad. Sci. U.S.A., 82:1074 (1985)); or strand displacement amplification [“SDA”] (Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)); and 3) methods of library construction and screening by complementation.
For example, putative promoter regions to those of the instant disclosure could be isolated by using all or a portion of the YAT1 nucleic acid molecules (e.g., corresponding to the promoter, gene or a combination thereof) as DNA hybridization probes to screen libraries from any desired microbe using methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan (e.g., random primers DNA labeling, nick translation, or end-labeling techniques), or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of (or full-length of) the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length DNA fragments under conditions of appropriate stringency.
Typically, in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PCR primer design are common and well known in the art (Thein and Wallace, “The use of oligonucleotides as specific hybridization probes in the Diagnosis of Genetic Disorders”, in Human Genetic Diseases: A Practical Approach, K. E. Davis (Ed.), (1986) pp 33-50 IRL: Herndon, Va.; and Rychlik, W., In Methods in Molecular Biology, White, B. A. (Ed.), (1993) Vol. 15, pp 31-39, PCR Protocols: Current Methods and Applications. Humania: Totowa, N.J.).
Generally two short segments of the instant sequences may be used in PCR protocols to amplify longer nucleic acid molecules encoding homologous polynucleotides from DNA or RNA. The PCR may also be performed on a library of cloned nucleic acid molecules wherein the sequence of one primer is derived from the instant nucleic acid molecules, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding microbial genes.
Alternatively, the instant sequences may be employed as hybridization reagents for the identification of homologs. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the nucleotide sequence of interest, and a specific hybridization method. Probes are typically single-stranded nucleic acid sequences that are complementary to the nucleic acid sequences to be detected. Probes are “hybridizable” to the nucleic acid sequence to be detected. The probe length can vary from 5 bases to tens of thousands of bases, and will depend upon the specific test to be done. Typically a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.
Hybridization methods are well defined. Typically the probe and sample must be mixed under conditions that will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed. Optionally, a chaotropic agent may be added. The chaotropic agent stabilizes nucleic acids by inhibiting nuclease activity. Furthermore, the chaotropic agent allows sensitive and stringent hybridization of short oligonucleotide probes at room temperature (Van Ness and Chen, Nucl. Acids Res., 19:5143-5151 (1991)). Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide and cesium trifluoroacetate, among others. Typically, the chaotropic agent will be present at a final concentration of about 3 M. If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v) [“by volume”].
Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent. A common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal), polyvinylpyrrolidone (about 250-500 kdal) and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to 2% wt/vol [“weight by volume”] glycine. Other additives may also be included, such as volume exclusion agents that include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate) and anionic saccharidic polymers, such as dextran sulfate.
Transcription initiation control regions or promoter regions that are useful to drive expression of a coding gene of interest in the desired host cell are selected from those derived from the upstream portion of the yat1 gene (SEQ ID NO:1). The promoter regions may be identified from the upstream sequences of yat1 and its homologs and isolated according to common methods (Maniatis, supra). Once a promoter region is identified and isolated (e.g., SEQ ID NO:25), it may be operably linked to a coding region of interest to be expressed in a suitable expression cassette. These chimeric genes may then be expressed in natural host cells and heterologous host cells, particularly in the cells of oleaginous yeast hosts. Thus, one aspect of the present disclosure provides a recombinant expression cassette comprising a YAT1 yeast promoter.
In a further aspect, provided herein is a method of expressing at least one coding region of interest in a transformed yeast, wherein a transformed yeast is provided having a chimeric gene comprising: (i) a promoter region of a Yarrowia yat1 gene; and, (ii) a coding region of interest expressible in the yeast, wherein the promoter region is operably linked to the coding region of interest to create a recombinant expression cassette; and the transformed yeast is grown under conditions wherein the chimeric gene is expressed. The polypeptide so produced can optionally be recovered from the culture.
Microbial expression systems and expression vectors are well known to those skilled in the art. Any of these could be used to construct chimeric genes comprising a promoter region derived from the yat1 gene for production of any specific coding region of interest suitable for regulated expression in a desirable yeast host cell. These chimeric genes could then be introduced into appropriate microorganisms by integration via transformation to provide expression of the enzymes upon induction. Alternatively, the promoters can be cloned into a plasmid that is capable of transforming and replicating itself in the preferred yeast. The coding region of interest to be expressed can then be cloned downstream from the promoter. Once the recombinant host is established, regulated gene expression can be accomplished by growing the cells under suitable conditions (infra).
Useful chimeric genes will include the promoter region of the yat1 gene as defined herein or a mutant promoter thereof, operably linked to a suitable coding region of interest to be expressed in a preferred host cell.
Coding regions of interest to be expressed in the recombinant yeast host may be either endogenous to the host or heterologous and must be compatible with the host organism. Genes encoding proteins of commercial value are particularly suitable for expression. For example, suitable coding regions of interest may include (but are not limited to) those encoding viral, bacterial, fungal, plant, insect, or vertebrate coding regions of interest, including mammalian polypeptides. Further, these coding regions of interest may be, for example, structural proteins, enzymes such as oxidoreductases, transferases, hydrolyases, lyases, isomerases or ligases, or peptides. A non-limiting list includes genes encoding enzymes such as acyltransferases, aminopeptidases, amylases, carbohydrases, carboxypeptidases, catalyases, cellulases, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, esterases, α-galactosidases, β-glucanases, β-galactosidases, glucoamylases, α-glucosidases, β-glucosidases, invertases, laccases, lipases, mannosidases, mutanases, oxidases, pectinolytic enzymes, peroxidases, phospholipases, phytases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transglutaminases, or xylanases.
In some embodiments, coding regions of the enzymes involved in the production of microbial oils, including ω-6 and ω-3 fatty acids, are preferred. These coding regions include desaturases and elongases (e.g., see U.S. Pat. No. 7,238,482 and U.S. Pat. Appl. Pub. No. 2009-0093543-A1 for a partial review of available genes in GenBank and/or the patent literature and considerations for choosing a specific polypeptide having desaturase or elongase activity).
Alternately, coding regions of the enzymes involved in the production of neutral lipids such as TAGs and phospholipids are preferred, These coding regions include various acyltransferases such as diacylglycerol cholinephosphotransferases, acyl-CoA:lysophospholipid acyltransferases (e.g., lysophosphatidic acid acyltransferases, lysophosphatidylcholine acyltransferases lysophosphatidylethanolamine acyltransferases, lysophosphatidylserine acyltransferases, lysophosphatidylglycerol acyltransferases and lysophosphatidylinositol acyltransferases), diacylglycerol acyltransferases and phospholipid:diacylglycerol acyltransferases, for example.
Vectors or DNA cassettes useful for the transformation of suitable host cells are well known in the art. In general, the choice of sequences included in the construct depends upon the desired expression products (supra), the nature of the host cell, and the proposed means of separating transformed cells versus non-transformed cells. The skilled artisan is aware of the genetic elements that must be present on the plasmid vector to successfully transform, select and propagate host cells containing the chimeric gene. Typically, however, the vector or cassette contains sequences directing transcription and translation of the relevant gene(s), a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the gene that controls transcriptional initiation, i.e., a promoter, the gene coding sequence, and a region 3′ of the DNA fragment that controls transcriptional termination, i.e., a terminator. It is most preferred when both control regions are derived from genes from the transformed host cell, although they need not be derived from genes native to the production host.
Nucleotide sequences surrounding the translational initiation codon ‘ATG’ have been found to affect expression in yeast cells. If the desired polypeptide is poorly expressed in yeast, the nucleotide sequences of exogenous genes can be modified to include an efficient yeast translation initiation sequence motif to obtain optimal gene expression. For expression in yeast, this can be done by site-directed mutagenesis of an inefficiently expressed gene to include the favored translation initiation motif.
3′ non-coding sequences encoding transcription termination regions may be provided in a recombinant construct and may be from the 3′ region of the gene from which the initiation region was obtained or from a different gene. A large number of termination regions are known and function satisfactorily in a variety of hosts when utilized both in the same and different genera and species from which they were derived. Termination regions may also be derived from various genes native to the preferred hosts. The termination region is usually selected more for convenience rather than for any particular property.
Particularly useful termination regions for use in yeast are derived from a yeast gene, particularly Saccharomyces, Schizosaccharomyces, Candida, Yarrowia or Kluyveromyces. The 3′-regions of mammalian genes encoding γ-interferon and α-2 interferon are also known to function in yeast. The 3′-region can also be synthetic, as one of skill in the art can utilize available information to design and synthesize a 3′-region sequence that functions as a transcription terminator. A termination region may be unnecessary, but is highly preferred.
The vector may also comprise a selectable and/or scorable marker, in addition to the regulatory elements described above. Preferably, the marker gene is an antibiotic resistance gene such that treating cells with the antibiotic results in growth inhibition, or death, of untransformed cells and uninhibited growth of transformed cells. For selection of yeast transformants, any marker that functions in yeast is useful with resistance to kanamycin, hygromycin and the amino glycoside G418 and the ability to grow on media lacking uracil, lysine, histine or leucine being particularly useful.
Merely inserting a chimeric gene into a cloning vector does not ensure its expression at the desired rate, concentration, amount, etc. In response to the need for a high expression rate, many specialized expression vectors have been created by manipulating a number of different genetic elements that control aspects of transcription, RNA stability, translation, protein stability and location, oxygen limitation and secretion from the host cell. Some of the manipulated features include: the nature of the relevant transcriptional promoter and terminator sequences, whether the gene is plasmid-borne or integrated into the genome of the host cell and the number of copies of the cloned gene [e.g., additional copies a particular coding region of interest (operably linked to the promoter of the instant invention) may be introduced into the host to increase expression], the final cellular location of the synthesized protein, the efficiency of translation and correct folding of the protein in the host organism, the intrinsic stability of the mRNA and protein of the cloned gene within the host cell [e.g., expression of the coding region of interest can be increased by removing/deleting destabilizing sequences from either the mRNA or the encoded protein or by adding stabilizing sequences to the mRNA (U.S. Pat. No. 4,910,141)], and the codon usage within the cloned gene, such that its frequency approaches the frequency of preferred codon usage of the host cell [e.g. translational efficiency of the encoded mRNAs can be increased by replacement of codons in the native gene with those for optimal gene expression in the selected host microorganism, to thereby substantially enhance the expression of the foreign gene encoding the polypeptide]. Each of these may be used in the methods and host cells described herein to further optimize expression of a recombinant expression cassette comprising a promoter region of the yat1 gene.
After a recombinant construct is created comprising at least one chimeric gene comprising a YAT1 promoter, a suitable ORF and a terminator, it is placed in a plasmid vector capable of autonomous replication in the host cell or is directly integrated into the genome of the host cell. Integration of expression cassettes can occur randomly within the host genome or can be targeted through the use of constructs containing regions of homology with the host genome sufficient to target recombination with the host locus. Where constructs are targeted to an endogenous locus, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus.
Where two or more genes are expressed from separate replicating vectors, each vector may have a different means of selection and should lack homology to the other construct(s) to maintain stable expression and prevent reassortment of elements among constructs. Judicious choice of regulatory regions, selection means and method of propagation of the introduced construct(s) can be experimentally determined so that all introduced genes are expressed at the necessary levels to provide for synthesis of the desired products.
Constructs comprising a coding region of interest may be introduced into a host cell by any standard technique. These techniques include transformation, e.g., lithium acetate transformation (Methods in Enzymology, 194:186-187 (1991)), biolistic impact, electroporation, microinjection, vacuum filtration or any other method that introduces the gene of interest into the host cell. More specific teachings applicable for oleaginous yeast (i.e., Yarrowia lipolytica) include U.S. Pat. No. 4,880,741 and U.S. Pat. No. 5,071,764 and Chen, D. C. et al. (Appl Microbiol Biotechnol., 48(2):232-235 (1997)).
For convenience, a host cell that has been manipulated by any method to take up a DNA sequence, for example, in an expression cassette, is referred to herein as “transformed” or “recombinant” or “transformant”. The transformed host will have at least one copy of the expression cassette and may have two or more, depending upon whether the gene is integrated into the genome, amplified, or is present on an extrachromosomal element having multiple copy numbers.
The transformed host cell can be identified by selection for a marker contained on the introduced construct. Alternatively, a separate marker construct may be co-transformed with the desired construct, as many transformation techniques introduce many DNA molecules into host cells.
Typically, transformed hosts are selected for their ability to grow on selective media, which may incorporate an antibiotic or lack a factor necessary for growth of the untransformed host, such as a nutrient or growth factor. An introduced marker gene may confer antibiotic resistance, or encode an essential growth factor or enzyme, thereby permitting growth on selective media when expressed in the transformed host. Selection of a transformed host can also occur when the expressed marker protein can be detected, either directly or indirectly. Additional selection techniques are described in U.S. Pat. No. 7,238,482 and U.S. Pat. No. 7,259,255.
Preferred host cells for expression of coding regions of interest operably linked to the YAT1 promoter fragments herein are yeast cells, where oleaginous yeast are most preferred where the desired use is for the production of microbial oils, infra. Oleaginous yeast are naturally capable of oil synthesis and accumulation, wherein the total oil content can comprise greater than about 25% of the dry cell weight [“DCW”], more preferably greater than about 30% of the DCW, and most preferably greater than about 40% of the DCW. Genera typically identified as oleaginous yeast include, but are not limited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. More specifically, illustrative oil-synthesizing yeast include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R. graminis and Yarrowia lipolytica (formerly classified as Candida lipolytica). In alternate embodiments, a non-oleaginous organism can be genetically modified to become oleaginous, e.g., yeast such as Saccharomyces cerevisiae. 
Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in a further embodiment, most preferred are the Y. lipolytica strains designated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol., 82(1):43-9 (2002)). The Y. lipolytica strain designated as ATCC #20362 was the particular strain from which the YAT1 promoter was isolated therefrom.
The transformed recombinant host cell is grown under conditions that optimize expression of the chimeric gene(s). In general, media conditions may be optimized for regulated expression of a particular coding region of interest by modifying the type and amount of carbon source, the type and amount of nitrogen source, the carbon-to-nitrogen ratio, the amount of different mineral ions, the oxygen level, growth temperature, pH, length of the biomass production phase, length of the oil accumulation phase and the time and method and the time of cell harvest.
Microorganisms of interest, such as oleaginous yeast, are generally grown in a complex media, such as yeast extract-peptone-dextrose broth [“YPD”] or a defined minimal media that lacks a component necessary for growth and thereby forces selection of the desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.)).
Fermentation media for the methods and host cells described herein must contain a suitable carbon source, such as are taught in U.S. Pat. No. 7,238,482. Although it is contemplated that the source of carbon utilized may encompass a wide variety of carbon-containing sources, preferred carbon sources are sugars, glycerol, and/or fatty acids. Most preferred is glucose and/or fatty acids containing between 10-22 carbons.
Nitrogen may be supplied from an inorganic (e.g., (NH4)2SO4) or organic (e.g., urea, glutamate, or yeast extract) source. Although the YAT1 promoter is active in media containing nitrogen (e.g., up to about 0.5% ammonium sulfate), the activity of the promoter increases when the host cell is grown in nitrogen-limiting conditions (e.g., in medium containing very low levels of ammonium, or lacking ammonium). Thus, a preferred medium would be one that contains less than about 0.1% ammonium sulfate, or other suitable ammonium salts.
In a more preferred embodiment, the YAT1 promoter is induced when the host cell is grown in media with a high carbon to nitrogen (i.e., C:N) ratio, such as a high glucose medium containing about 1-15% glucose, and about 0.1% or less ammonium sulfate. These conditions are also sufficient to induce oleaginy in those yeast that are oleaginous (e.g., Yarrowia lipolytica).
In addition to appropriate carbon and nitrogen sources, the fermentation media must also contain suitable minerals, salts, cofactors, buffers, vitamins, and other components known to those skilled in the art suitable for the growth of the microorganism.
Preferred growth media for the methods and host cells described herein are common commercially prepared media, such as minimal media made with Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.). Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science. A suitable pH range for the fermentation is typically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.5 is preferred as the range for the initial growth conditions. The fermentation may be conducted under aerobic or anaerobic conditions, wherein microaerobic conditions are preferred.
Host cells comprising a suitable coding region of interest operably linked to the promoters of the present disclosure may be cultured using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing regulated expression of the coding region of interest. Furthermore, where commercial production of a product that relies on the instant genetic chimera is desired, a variety of culture methodologies may be applied. For example, large-scale production of a specific gene product over-expressed from a recombinant host may be produced by a batch, fed-batch or continuous fermentation process, as is well known in the art (see, e.g., Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd ed., (1989) Sinauer Associates: Sunderland, Mass.; or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227 (1992), each herein incorporated by reference).
As was discussed above, the instant invention can be practiced in regulatingexpression of any suitable coding region of interest in an oleaginous yeast. In particularcoding regions of the enzymes involved in the production of microbial oils, including ω-6 and ω-3 fatty acids, are of interest. Examples of such enzymes include, but are not limited to, expression of desaturases and elongases.
The term “fatty acids” refers to long chain aliphatic acids (alkanoic acids) of varying chain lengths, from about C12 to C22 (although both longer and shorter chain-length acids are known). The predominant chain lengths are between C16 and C22. The structure of a fatty acid is represented by a simple notation system of “X:Y”, where X is the total number of carbon [“C”] atoms in the particular fatty acid and Y is the number of double bonds. Additional details concerning the differentiation between “saturated fatty acids” versus “unsaturated fatty acids”, “monounsaturated fatty acids” versus “polyunsaturated fatty acids” [“PUFAs”], and “ω-6 fatty acids” [“ω-6” or “n-6”] versus “ω-3 fatty acids” [“ω-3” or “n-3”] are provided in U.S. Pat. No. 7,238,482, which is hereby incorporated herein by reference. Nomenclature used to describe PUFAs herein is shown below in Table 2. In the column titled “Shorthand Notation”, the omega-reference system is used to indicate the number of carbons, the number of double bonds and the position of the double bond closest to the omega carbon, counting from the omega carbon (which is numbered 1 for this purpose). The remainder of the Table summarizes the common names of ω-3 and ω-6 fatty acids and their precursors, the abbreviations that will be used throughout the remainder of the specification, and the chemical name of each compound.







TABLE 2







Nomenclature Of Polyunsaturated Fatty Acids













Shorthand


Common Name
Abbreviation
Chemical Name
Notation





Myristic
—
tetradecanoic
14:0


Palmitic
Palmitate
hexadecanoic
16:0


Palmitoleic
—
9-hexadecenoic
16:1


Stearic
—
octadecanoic
18:0


Oleic
—
cis-9-octadecenoic
18:1


Linoleic
LA
cis-9,12-octadecadienoic
18:2 ω-6


γ-Linolenic
GLA
cis-6,9,12-octadecatrienoic
18:3 ω-6


Eicosadienoic
EDA
cis-11,14-eicosadienoic
20:2 ω-6


Dihomo-γ-
DGLA
cis-8,11,14-eicosatrienoic
20:3 ω-6


linolenic


Sciadonic
SCI
cis-5,11,14-eicosatrienoic
20:3b ω-6


Arachidonic
ARA
cis-5,8,11,14-
20:4 ω-6




eicosatetraenoic


α-Linolenic
ALA
cis-9,12,15-octadecatrienoic
18:3 ω-3


Stearidonic
STA
cis-6,9,12,15-
18:4 ω-3




octadecatetraenoic


Eicosatrienoic
ETrA
cis-11,14,17-eicosatrienoic
20:3 ω-3


Eicosa-tetraenoic
ETA
cis-8,11,14,17-
20:4 ω-3




eicosatetraenoic


Juniperonic
JUP
cis-5,11,14,17-
20:4b ω-3




eicosatetraenoic


Eicosa-
EPA
cis-5,8,11,14,17-
20:5 ω-3


pentaenoic

eicosapentaenoic


Docosa-
DTA
cis-7,10,13,16-
22:4 ω-6


tetraenoic

docosatetraenoic


Docosa-
DPAn-6
cis-4,7,10,13,16-
22:5 ω-6


pentaenoic

docosapentaenoic


Docosa-
DPA
cis-7,10,13,16,19-
22:5 ω-3


pentaenoic

docosapentaenoic


Docosa-
DHA
cis-4,7,10,13,16,19-
22:6 ω-3


hexaenoic

docosahexaenoic









The process of de novo synthesis of palmitate (16:0) in oleaginous microorganisms is described in commonly owned U.S. Pat. No. 7,238,482. This fatty acid is the precursor of longer-chain saturated and unsaturated fatty acid derivates, which are formed through the action of elongases and desaturases. For example, palmitate is converted to its unsaturated derivative [palmitoleic acid (16:1)] by the action of a Δ9 desaturase; similarly, palmitate is elongated to form stearic acid (18:0), which can be converted to its unsaturated derivative by a Δ9 desaturase to thereby yield oleic (18:1) acid.
The metabolic process that converts oleic acid to ω-6 fatty acids such as LA, EDA, GLA, DGLA, ARA, DTA and DPAn-6 and ω-3 fatty acids such as ALA, STA, ETrA, ETA, EPA, DPA and DHA is well described in the literature and is schematically depicted in FIG. 2 (e.g., see U.S. Pat. Appl. Pub. No. 2006-0115881-A1 and U.S. Pat. Appl. Pub. No. 2009-0093543-A1). Simplistically, this process involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds, via a series of special elongation and desaturation enzymes present in the endoplasmic reticulum membrane (and hereinafter referred to as “PUFA biosynthetic pathway enzymes”).
More specifically, “PUFA biosynthetic pathway enzymes” or “ω-3/ω-6 biosynthetic pathway enzymes” will refer to any of the following enzymes (and genes which encode said enzymes) associated with the biosynthesis of a PUFA, including: Δ4 desaturase, Δ5 desaturase, Δ6 desaturase, Δ12 desaturase, ≢15 desaturase, Δ17 desaturase, Δ9 desaturase, Δ8 desaturase, Δ9 elongase, C14/16 elongase, C16/18 elongase, C18/20 elongase and/or C20/22 elongase. For further clarity within the present disclosure, the term “desaturase” refers to a polypeptide that can desaturate one or more fatty acids to produce a mono- or polyunsaturated fatty acid or precursor of interest.
Thus, despite use of the omega-reference system to refer to specific fatty acids, it is more convenient to indicate the activity of a desaturase by counting from the carboxyl end of the source using the delta-system. For example, a Δ17 desaturase will desaturate a fatty acid between the 17th and 18th carbon atom numbered from the carboxyl-terminal end of the molecule and can, for example, catalyze the conversion of ARA to EPA and/or DGLA to ETA. In contrast, the term “elongase” refers to a polypeptide that can elongate a fatty acid carbon chain to produce a mono- or polyunsaturated fatty acid that is 2 carbons longer than the fatty acid source that the elongase acts upon. This process of elongation occurs in a multi-step mechanism in association with fatty acid synthase, whereby CoA is the acyl carrier (Lassner et al., The Plant Cell, 8:281-292 (1996)).
As will be understood by one skilled in the art, the particular functionalities required to be introduced into a host organism for production of a particular PUFA final product will depend on the host cell (and its native PUFA profile and/or desaturase/elongase profile), the availability of substrate and the desired end product(s). As shown in FIG. 2, LA, GLA, EDA, DGLA, ARA, DTA, DPAn-6, ALA, STA, ETrA, ETA, EPA, DPA and DHA may all be produced in oleaginous yeast, by introducing various combinations of the following PUFA enzyme functionalities: Δ4 desaturase, Δ5 desaturase, Δ6 desaturase, Δ12 desaturase, Δ15 desaturase, Δ17 desaturase, Δ9 desaturase, Δ8 desaturase, Δ9 elongase, C14/16 elongase, C16/18 elongase, C18/20 elongase and/or C20/22 elongase. One skilled in the art will be able to identify various candidate genes encoding each of the above enzymes, according to publicly available literature (e.g., GenBank), the patent literature, and experimental analysis of microorganisms having the ability to produce PUFAs. Thus, a variety of desaturases and elongases are suitable as coding regions of interest in the present disclosure. These coding regions of interest could be operably linked to the YAT1 promoters herein, and used as chimeric genes for expression of various ω-6 and ω-3 fatty acids, using techniques well known to those skilled in the art (e.g., see U.S. Pat. No. 7,238,482, U.S. Pat. Appl. Pub. No. 2006-0115881-A1 and U.S. Pat. Appl. Pub. No. 2009-0093543-A1).
Following PUFA biosynthesis, the fatty acids are generally incorporated into the “oil” fraction of oleaginous organisms, which constitutes a major part of the total lipid. More specifically, the term “oil” refers to a lipid substance that is liquid at 25° C. and usually polyunsaturated. “Oil” is composed primarily of triacylglycerols [“TAGs”] but may also contain other neutral lipids, phospholipids and free fatty acids. The fatty acid composition in the oil and the fatty acid composition of the total lipid are generally similar; thus, an increase or decrease in the concentration of PUFAs in the total lipid will correspond with an increase or decrease in the concentration of PUFAs in the oil, and vice versa.
“Neutral lipids” refer to those lipids commonly found in cells in lipid bodies as storage fats and are so called because at cellular pH, the lipids bear no charged groups. Generally, they are completely non-polar with no affinity for water. Neutral lipids generally refer to mono-, di-, and/or triesters of glycerol with fatty acids, also called monoacylglycerol [“MAG”], diacylglycerol [“DAG”] or triacylglycerol [“TAG”], respectively, or collectively, acylglycerols. A hydrolysis reaction must occur to release free fatty acids from acylglycerols.
Finally, for clarity, the term “triacylglycerols” [“TAGs”] refers to neutral lipids composed of three fatty acyl residues esterified to a glycerol molecule. TAGs can contain long chain PUFAs and saturated fatty acids, as well as shorter chain saturated and unsaturated fatty acids.
Thus, following PUFA biosynthesis, the fatty acids are generally stored as an energy resource within the cell in the form of neutral lipid (i.e., MAGs, DAGs, TAGs) and/or in the form of phospholipids. Various acyltransferases, responsible for transferring an acyl group from a donor lipid to an acceptor lipid molecule, enable this process, including: diacylglycerol cholinephosphotransferases, acyl-CoA:lysophospholipid acyltransferases (e.g., lysophosphatidic acid acyltransferases, lysophosphatidylcholine acyltransferases lysophosphatidylethanolamine acyltransferases, lysophosphatidylserine acyltransferases, lysophosphatidylglycerol acyltransferases and lysophosphatidylinositol acyltransferases), diacylglycerol acyltransferases and phospholipid:diacylglycerol acyltransferases, for example.
In preferred embodiments, the nucleic acid sequence of the promoter region is selected from the group consisting of: SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36. The coding region of interest is preferably any desaturase or elongase suitable for regulated expression in the oleaginous yeast for the production of ω-3 or ω-6 fatty acids, wherein the enzyme product is a PUFA selected from the group consisting of LA, EDA, ALA, GLA, STA, ETrA, DGLA, ETA, ARA, DTA, DPAn-6, EPA, DPAn-3 and DHA. Alternately, the coding region of interest is preferably any acyltransferase suitable for production of neutral lipids or phospholipids. And, the transformed oleaginous yeast is preferably cultured under conditions that induce oleaginy (i.e., conditions of nitrogen limitation).
More specifically, for production of the greatest and the most economical yield of PUFAs, the transformed oleaginous yeast host cell is grown under conditions that optimize expression of chimeric genes comprising a promoter region of a yat1 gene and a coding region of interest encoding a PUFA, neutral lipid or phospholipid biosynthetic pathway enzyme. Typically, accumulation of high levels of PUFAs in oleaginous yeast cells requires a two-stage process, since the metabolic state must be “balanced” between growth and synthesis/storage of fats. Thus, most preferably, a two-stage fermentation process is necessary for the production of PUFAs in oleaginous yeast. In this approach, the first stage of the fermentation is dedicated to the generation and accumulation of cell mass and is characterized by rapid cell growth and cell division. In the second stage of the fermentation, it is preferable to establish conditions of nitrogen deprivation in the culture to promote high levels of lipid accumulation.
The effect of this nitrogen deprivation is two-fold. First, the nitrogen deprivation reduces the effective concentration of AMP in the cells, thereby reducing the activity of the NAD-dependent isocitrate dehydrogenase of mitochondria. When this occurs, citric acid will accumulate, thus forming abundant pools of acetyl-CoA in the cytoplasm and priming fatty acid synthesis. Secondly, the nitrogen deprivation induces the YAT1 promoter, thereby promoting expression of any chimeric genes comprising the YAT1 promoter and a coding region of interest encoding an enzyme of the ω-3/ω-6 fatty acid biosynthetic pathway, neutral lipid biosynthetic pathway or phospholipid biosynthetic pathway. Thus, this second phase of the two-stage fermentation is characterized by the cessation of cell division followed by the synthesis of fatty acids and accumulation of oil.
Although cells are typically grown at about 30° C., some studies have shown increased synthesis of unsaturated fatty acids at lower temperatures (Yongmanitchai and Ward, Appl. Environ. Microbiol., 57:419-25 (1991)). Based on process economics, this temperature shift should likely occur after the first phase of the two-stage fermentation, when the bulk of the organisms' growth has occurred.
Additionally, particular attention is given to several metal ions (e.g., Fe+2, Cu+2, Mn+2, Co+2, Zn+2, Mg+2) that promote synthesis of lipids and PUFAs in the fermentation media (Nakahara, T. et al. Ind. Appl. Single Cell Oils, D. J. Kyle and R. Colin, eds. pp 61-97 (1992)).
The PUFAs produced in a host microorganism as described herein may also be found as free fatty acids or sulfolipids or glycolipids.
PUFAs may be extracted from the host cell through a variety of means well-known in the art. One review of extraction techniques, quality analysis and acceptability standards for yeast lipids is that of Z. Jacobs (Critical Reviews in Biotechnology, 12(5/6):463-491 (1992)). A brief review of downstream processing is also available by A. Singh and O. Ward (Adv. Appl. Microbiol., 45:271-312 (1997)).
In general, means for the purification of fatty acids (including PUFAs) may include extraction (e.g., U.S. Pat. No. 6,797,303 and U.S. Pat. No. 5,648,564) with organic solvents, sonication, supercritical fluid extraction (e.g., using carbon dioxide), saponification and physical means such as presses, or combinations thereof. One is referred to the teachings of U.S. Pat. No. 7,238,482 for additional details.
EXAMPLES
The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
General Methods
Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by: 1) Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); 2) T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and 3) Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).
Materials and methods suitable for the maintenance and growth of microbial cultures are well known in the art. Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, Eds), American Society for Microbiology: Washington, D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd ed., Sinauer Associates: Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of microbial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.) or Sigma Chemical Company (St. Louis, Mo.), unless otherwise specified.
General molecular cloning was performed according to standard methods (Sambrook et al., supra). Oligonucleotides were synthesized by Sigma-Genosys (Spring, Tex.). When polymerase chain reaction [“PCR”] was involved in subcloning, the constructs were sequenced to confirm that no errors had been introduced to the sequence. PCR products were cloned into Promega's pGEM-T-easy vector (Madison, Wis.).
Manipulations of genetic sequences were accomplished using the suite of programs available from the Genetics Computer Group Inc. (Wisconsin Package Version 9.0, Genetics Computer Group [“GCG”], Madison, Wis.). The GCG program “Pileup” was used with the gap creation default value of 12, and the gap extension default value of 4. The GCG “Gap” or “Bestfit” programs were used with the default gap creation penalty of 50 and the default gap extension penalty of 3. Unless otherwise stated, in all other cases GCG program default parameters were used.
The meaning of abbreviations is as follows: “sec” means second(s), “min” means minute(s), “h” means hour(s), “d” means day(s), “μL” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” means micromolar, “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” means microgram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means base pair(s) and “kB” means kilobase(s).
Nomenclature For Expression Cassettes
The structure of an expression cassette will be represented by a simple notation system of “X::Y::Z”, wherein X describes the promoter fragment, Y describes the gene fragment, and Z describes the terminator fragment, which are all operably linked to one another.
Transformation And Cultivation Of Yarrowia lipolytica 
Y. lipolytica strain ATCC #20362 was purchased from the American Type Culture Collection (Rockville, Md.). Strains were routinely grown at 28-30° C. on YPD agar (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar) or in YPD liquid medium (2% bacto-yeast extract, 3% bactopeptone, 2% glucose).
Transformation of Y. lipolytica was performed as described in U.S. Pat. Appl. Pub. No. 2009-0093543-A1, hereby incorporated herein by reference.
For selection of transformants, minimal medium (“MM”) was generally used; the composition of MM is as follows: 0.17% yeast nitrogen base (DIFCO Laboratories, Detroit, Mich.) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, pH 6.1 and 20 g/L agar).
“SD” media comprises: 0.67% yeast nitrogen base with ammonium sulfate, without amino acids and 2% glucose. And finally, to promote conditions of oleaginy, High Glucose Media (“HGM”) was prepared as follows: 14 g/L KH2PO4, 4 g/L K2HPO4, 2 g/L MgSO4.7H2O, 80 g/L glucose (pH 6.5).
Fatty Acid Analysis Of Yarrowia lipolytica 
For fatty acid analysis, cells were collected by centrifugation and lipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can. J. Biochem. Physiol., 37:911-917 (1959)). Fatty acid methyl esters [“FAMEs”] were prepared by transesterification of the lipid extract with sodium methoxide (Roughan, G., and Nishida I., Arch Biochem Biophys., 276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard 6890 GC fitted with a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The oven temperature was from 170° C. (25 min hold) to 185° C. at 3.5° C./min.
For direct base transesterification, Yarrowia culture (3 mL) was harvested, washed once in distilled water, and dried under vacuum in a Speed-Vac for 5-10 min. Sodium methoxide (100 μl of 1%) was added to the sample, and then the sample was vortexed and rocked for 20 min. After adding 3 drops of 1 M NaCl and 400 μl hexane, the sample was vortexed and spun. The upper layer was removed and analyzed by GC as described above.
Example 1
Construction And Sequencing Of A Yarrowia lipolytica cDNA Library
The present Example describes the construction of a cDNA library of Y. lipolytica, grown under oleaginous conditions. More specifically, Y. lipolytica cells accumulate considerable amounts of oil when switched to a medium with a high carbon to nitrogen ratio (>80). A cDNA library was constructed from cells grown in an oleaginous medium containing no nitrogen source for 4 hrs, using the BD-Clontech Creator Smart® cDNA library kit (Mississauga, ON, Canada) according to the manufacturer's protocol.
Synthesis of the cDNA library first required growth of Y. lipolytica strain ATCC #20362 in 30 mL YPD medium overnight at 30° C. with shaking. Cells were diluted in two cultures of 100 mL fresh YPD to OD600=0.4, then grown until OD600=1.5 (Perkin-Elmer Lambda 20 UV/VIS Spectrophotometer). Cells from each culture were pelleted twice by centrifugation at 3750 rpm in a Beckman GH3.8 rotor for 5 min and washed with sterile water, then resuspended in 100 mL HGM. Cells were then shaken at 30° C. for 4 hrs. Each 100 mL culture was divided into three equal portions and pelleted by centrifugation at 3750 rpm in a Beckman GH3.8 rotor for 5 min.
Total RNA was extracted from the pellets using the Qiagen Rneasy Midi Kit. Cells were resuspended in 600 μl buffer RLT from the kit, with β-mercaptoethanol added at a concentration of 10 μl/mL. Resuspended cells were transferred to six 2 mL screw cap tubes each containing 0.6 mL of 0.5 mm glass beads. The cells were homogenized at the HOMOGENIZE setting on a Biospec (Bartlesville, Okla.) mini bead beater for 2 min. The tubes were briefly spun to settle the beads. Liquid was transferred to 4 fresh 2 mL microfuge tubes and 600 μl of the RLT/BME mix was added to the beads. The bead tubes were vortexed and all liquid above the beads was transferred to the fresh 2 mL tubes. The fresh tubes were spun for 2 min in a microfuge to pellet cells debris, and the supernatent from each set of three tubes that came from a single culture were pooled in a 10 mL screwcap centrifuge tube. The procedure was then completed following the manufacturer's protocol. The two total RNA samples were then combined and mRNA was isolated from the combined sample using the Qiagen Oligotex Midi kit, following the manufacturer's protocol. Purified poly(A)+FRNA was obtained with a concentration of 35.7 ng/μl.
cDNA was generated, using the LD-PCR method specified by BD-Clontech and 0.1 μl of polyA(+) RNA sample. Specifically, for 1st strand cDNA synthesis, 3 μl of the poly(A)+RNA sample was mixed with 1 μl of SMART IV oligo nucleotide (SEQ ID NO:4) and 1 μl of CDSIII/3′ PCR primer (SEQ ID NO:5). The mixture was heated at 72° C. for 2 min and cooled on ice for 2 min. To the tube was added the following: 2 μl 1st strand buffer, 1 μl 20 mM DTT, 1 μl 10 mM dNTP mix and 1 μl Powerscript reverse transcriptase. The mixture was incubated at 42° C. for 1 hr and cooled on ice.
The 1st strand cDNA synthesis mixture was used as template for the PCR reaction. Specifically, the reaction mixture contained the following: 2 μl of the 1st strand cDNA mixture, 2 μl 5′-PCR primer (SEQ ID NO:6), 2 μl CDSIII/3′-PCR primer (SEQ ID NO:5), 80 μl water, 10 μl 10× Advantage 2 PCR buffer, 2 μl 50× dNTP mix and 2 μl 50× Advantage 2 polymerase mix. The thermocycler conditions were set for 95° C. for 20 sec, followed by 14 cycles of 95° C. for 5 sec and 68° C. for 6 min on a GenAmp 9600 instrument. PCR product was quantitated by agarose gel electrophoresis and ethidium bromide staining.
Seventy-five μl of the above PCR products (cDNA) were mixed with 3 μl of 20 μg/μl proteinase K supplied with the kit. The mixture was incubated at 45° C. for 20 min, then 75 μl of water was added and the mixture was extracted with 150 μl phenol:chloroform:isoamyl alcohol mixture (25:24:1). The aqueous phase was further extracted with 150 μl chloroform:isoamyl alcohol (25:1). The aqueous phase was then mixed with 15 μl of 3 M sodium acetate, 2 μl of 20 μg/μl glycogen and 400 μl of 100% ethanol. The mixture was immediately centrifuged at room temperature for 20 min at 14000 rpm in a microfuge. The pellet was washed once with 150 μl of 80% ethanol, air dried and dissolved in 79 μl of water.
Dissolved cDNA was subsequently digested with Sfil (79 μl of the cDNA was mixed with 10 μl of 10× Sfil buffer, 10 μl of Sfil enzyme and 1 μl of 100× BSA and the mixture was incubated at 50° C. for 2 hrs). Xylene cyanol dye (2 μl of 1%) was added. The mixture was then fractionated on the Chroma Spin-400 column provided with the kit, following the manufacturer's procedure exactly. Fractions collected from the column were analyzed by agarose gel electrophoresis. The first three fractions containing cDNA were pooled and cDNA precipitated with ethanol. The precipitated cDNA was redissolved in 7 μl of water, and ligated into kit-supplied pDNR-LIB.
Library Sequencing
The ligation products were used to transform E. coli XL-1 Blue electroporation competent cells (Stratagene). An estimated total of 5.4×106 colonies was obtained. Sequencing of the cDNA library was carried out by Agencourt Bioscience Corporation (Beverly, Mass.), using a M13 forward primer (SEQ ID NO:7)
Example 2
Identification Of The Yarrowia lipolvtica Gene Encoding YAT1 As A Highly Expressed Gene Under Oleaginous Conditions
This example describes the identification of YAT1 as a highly expressed gene under oleaginous conditions. Specifically, the relative abundance of each species of cDNA was examined (based on the sequencing results from Example 1) to identify those genes that were highly expressed under oleaginous conditions. One of the genes that appeared multiple times (16/9984 or 0.16%) was Yali0E27203 (GenBank Accession No. XM—504457; SEQ ID NO:1 herein), annotated as a homolog of the high affinity, low capacity ammonia transporter MEP2 of Saccharomyces cerevisiae (GenBank Accession No. X83608; see also A. M. Marini et al., EMBO J., 13(15):3456-3463 (1994); A. M. Marini et al., Mol Cell Biol., 17(8):4282-93 (1997)).
BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)) searches were conducted using the corresponding amino acid sequence of Yali0E27203 (SEQ ID NO:2) as a query against the S. cerevisiae MEP1, MEP2 and MEP3 isozymes. The results of these BLAST comparisons are shown below in Table 3 and are reported according to the % identity, % similarity and Expectation value.







TABLE 3







Comparison Of Yarrowia lipolytica Homolog To Saccharomyces


cerevisiae MEP1, MEP2 And MEP3











%
%



Similarity Identified
Identitya
Similarityb
E-valuec





MEP1 [GenBank Accession No.
35
56
1.8e−79


X77608]


MEP2 [GenBank Accession No.
52
66
 1.9e−132


X83608]


MEP3 [GenBank Accession No.
37
57
3.6e−83


P53390]





a% Identity is defined as percentage of amino acids that are identical between the two proteins.


b% Similarity is defined as percentage of amino acids that are identical or conserved between the two proteins.


cExpect value. The Expect value estimates the statistical significance of the match, specifying the number of matches, with a given score, that are expected in a search of a database of this size absolutely by chance.






It was hypothesized that YALI0E27203 encoded a Y. lipolytica ammonia transporter of high affinity; thus, YALI0E27203 was tentatively named yat1.

Example 3
Isolation Of The 5′ Upstream Region Of YAT1 From Yarrowia lipolytica 
To isolate the YAT1 promoter region upstream of the yat1 gene identified in Example 2, primers 27203-F and 27203-R (SEQ ID NOs:8 and 9) were designed, based on the sequence of Y. lipolytica chromosome E between positions 3,222,879 and 3,223,875. These primers were expected to amplify a 778 by fragment (SEQ ID NO:3), including 775 by of the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of the yat1 gene (from −775 to −1 [wherein the ‘A’ position of the ‘ATG’ translation initiation codon was designated as +1]) and the ‘ATG’ translation initiation codon of the yat1 gene (i.e., the +1 to +3 region).
PCR amplification was performed in a 50 μl total volume using a 1:1 dilution of a premixed 2× PCR solution (TaKaRa ExTaq, TaKaRa Bio Inc., Otsu Shiga, 520-2193, Japan). The final composition contained 25 mM TAPS, pH 9.3, 50 mM KCl, 2 mM MgCl2, 1 mM 2-mercaptoethanol, 200 μM each deoxyribonucleotide triphosphate, 10 pmole each primer (supra), 50 ng Y. lipolytica ATCC #20362 genomic DNA, and 1.25 units of TaKaRa ExTaqTM DNA polymerase. The reaction mixture was first heated to 94° C. for 150 sec. Amplification was carried out for 30 cycles at 94° C. for 30 sec, 55° C. for 30 sec and 72° C. for 1 min. This was followed by a final extension for 7 min at 72° C.
The PCR product was analyzed by agarose gel electrophoresis and was shown to contain a single DNA fragment of  ̃780 bp. This fragment was purified using a Qiagen PCR purification kit following the manufacturer's protocol.
Sequence analyses permitted construction of a single contig of DNA (FIG. 1; 2236 by total length, corresponding to SEQ ID NO:24). This contig therefore contained the −775 to +1461 region of the YAT1 gene, wherein the ‘A’ position of the ‘ATG’ translation initiation codon was designated as +1.
Example 4
Synthesis Of Plasmids pY5-30, pYGPAT-GUS and pDMW214
A series of plasmids were created comprising a chimeric gene composed of various native Y. lipolytica promoters and the “GUS” reporter gene, wherein “GUS” corresponds to the E. coli gene encoding β-glucuronidase (Jefferson, R. A., Nature, 342(6251):837-838 (1989)). This was required for comparative studies investigating the promoter activity of TEF, YAT1, FBAIN and GPAT, as described in Example 8.
Synthesis Of Plasmid pY5-30 (TEF::GUS::XPR)
The synthesis of plasmid pY5-30, comprising a TEF::GUS::XPR chimeric gene, is described in U.S. Pat. No. 7,259,255. More specifically, plasmid pY5-30 (FIG. 3A; SEQ ID NO:10) contained: a Yarrowia autonomous replication sequence (ARS18); a CoIE1 plasmid origin of replication; an ampicillin-resistance gene (AmpR) for selection in E. coli; a Yarrowia LEU2 gene for selection in Yarrowia; and the chimeric TEF::GUS::XPR gene.
Synthesis Of Plasmid pDMW214 (FBAIN::GUS::XPR)
The synthesis of plasmid pDMW214, comprising a FBAIN::GUS::XPR chimeric gene, is described in U.S. Pat. No. 7,202,356. Briefly, the FBAIN promoter region (SEQ ID NO:11; which includes both an upstream DNA sequence and a downstream sequence from the putative ‘ATG’ translation initiation codon of the fructose-bisphosphate aldolase (fba1) gene [wherein the downstream region comprises an intron]) was amplified by PCR, digested with NcoI and SalI, and then purified following gel electrophoresis. The NcoI/SallI-digested PCR products were ligated to NcoI/SalI digested pY5-30 vector to produce plasmid “pDMW214” (FIG. 3B).
Synthesis Of pYGPAT-GUS (GPAT::GUS::XPR)
The synthesis of plasmid pYGPAT-GUS (FIG. 3C), comprising a GPAT::GUS::XPR chimeric gene, is described in U.S. Pat. No. 7,264,949. Briefly, synthesis of the plasmid required identification and isolation of the Y. lipolytica glycerol-3-phosphate O-acyltransferase (gpat) gene, isolation of the promoter region upstream of the putative ‘ATG’ translation initiation codon via genome-walking, and then cloning of the GPAT-Pro promoter region (SEQ ID NO:12) into a derivative of pY5-30 (supra).
Example 5
Synthesis of pYAT-GUS
The present Example describes the synthesis of pYAT-GUS (comprising a YAT1::GUS::XPR chimeric gene). Synthesis of this plasmid first required amplification of the putative YAT1 promoter region. Then, the putative promoter region was cloned into pYGPAT-GUS (supra, Example 4).
The purified YAT1 PCR product from Example 3 was digested with HindllI and SalI and a  ̃600 by fragment was isolated by agarose gel electrophoresis followed by purification with Qiagen MinElute Gel purification kit according to the manufacturer's protocol. Furthermore, the YAT1 PCR product was also digested with NcoI and HindllI, and a  ̃200 by fragment was isolated and purified as above. Finally, plasmid pYGPAT-GUS was digested with SalI and NcoI, and a  ̃9.5 kB fragment was isolated and purified. The three DNA fragments were ligated together to create pYAT-GUS (FIG. 3D). In the resulting plasmid, the YAT1 promoter region (SEQ ID NO:25; corresponding to 775 by of the 5′ upstream region of YALI0E27203 (i.e., the −775 to −1 upstream region of the yat1 gene) was fused to the GUS gene, followed by the XPR terminator region.
Example 6
Generation Of ARA-Producinq Yarrowia lipolvtica ATCC #20362 Strain Y2034
The present Example describes the construction of strain Y2034, derived from Yarrowia lipolytica ATCC #20362, capable of producing significant concentrations of ARA relative to the total lipids. Comparison of the TEF, YAT1, GPAT and FBAIN promoters was examined in this ARA-producing strain based on analysis of GUS expression, as described in Example 8 (infra).
The development of strain Y2034, producing 10% ARA, required the construction of strain M4 (producing 8% DGLA). Further details regarding the construction of strains M4 and Y2034 are described in Example 10 of U.S. Pat. No. 7,273,746, hereby incorporated herein by reference. Briefly, however, integration of expression plasmids pKUNF12T6E (SEQ ID NO:13) and pDMW232 (SEQ ID NO:23) into wildtype Yarrowia lipolytica ATCC #20362 resulted in expression of the following chimeric genes in strain Y2034: FBAIN::EL1S:Pex20, TEF::Δ6S::Lip1, FBA::F.Δ12::Lip2, TEF::EL2S::XPR, FBAIN::MAΔ5::Pex20 and TEF::MAΔ5::Lip1 [wherein EL1S is a codon-optimized elongase 1 gene (SEQ ID NO:14), derived from Mortierella alpina (GenBank Accession No. AX464731); Δ6S is a codon-optimized Δ6 desaturase gene (SEQ ID NO:16), derived from Mortierella alpina (GenBank Accession No. AF465281); F.Δ12 is a Fusarium moniliforme Δ12 desaturase gene (SEQ ID NO:19; U.S. Pat. No. 7,504,259); EL2S is a codon-optimized elongase gene (SEQ ID NO:21), derived from Thraustochytrium aureum (U.S. Pat. No. 6,677,145); and, MAΔ5 is a Mortierella alpina Δ5 desaturase gene (Gen Bank Accession No. AF067654)].
Example 7
Transformation Of Y. lipolytica With pY5-30, pYAT-GUS, pYGPAT-GUS And pDMW214
The plasmids pY5-30 (Example 4; comprising a TEF::GUS::XPR chimeric gene), pYAT-GUS (Example 5; comprising a YAT1::GUS::XPR chimeric gene), pYGPAT-GUS (Example 4; comprising a GPAT::GUS::XPR chimeric gene) and pDMW214 (Example 4; comprising a FBAIN::GUS::XPR chimeric gene) were transformed separately into Y. lipolytica ATCC #20362 strain Y2034, according to the General Methods. Selection was performed on SD medium comprising 2.5% agar.
Using this technique, transformants were obtained that contained pY5-30, pYAT-GUS, pYGPAT-GUS and pDMW214, respectively.
Example 8
Comparative Analysis Of The TEF, YAT1, GPAT and FBAIN Promoter Activities In Yarrowia lipolytica, As Determined By Histochemical Assay
The activity of the TEF, YAT1, GPAT and FBAIN promoters was determined in Y. lipolytica containing the pY5-30, pYAT-GUS, pYGPAT-GUS and pDMW214 constructs, each of which possessed a GUS reporter gene and an XPR terminator (from Example 7). GUS activity in each expressed construct was measured by histochemical assays (Jefferson, R. A., Plant Mol. Biol. Reporter, 5:387-405 (1987)).
Specifically, Y. lipolytica strains containing plasmids pY5-30, pYAT-GUS, pYGPAT-GUS and pDMW214, respectively, were grown from single colonies in 5 mL SD media at 30° C. for 24 hrs to an OD600 ̃8.0. Then, 1 mL of cells were collected by centrifugation. The remaining cultures were centrifuged and washed 2× with HGM, resuspended in 5 mL each of HGM and allowed to grow at 30° C. further. After 24 and 120 hrs,  ̃0.25 mL of each culture were centrifuged to collect the cells. Cell samples were resuspended individually in 100 μl of histochemical staining buffer [Staining buffer prepared by dissolving 5 mg of 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) in 50 μl dimethyl formamide, followed by addition of 5 mL 50 mM NaPO4, pH 7.0.]. Zymolase 20T (5 μl of 1 mg/mL; ICN Biomedicals, Costa Mesa, Calif.) was added to each, and the mixture incubated at 30° C.
The results of histochemical staining showed that the YAT1 promoter in construct pYAT-GUS was active. Comparatively, the YAT1 promoter appeared to be stronger than the TEF promoter (FIG. 4A) but significantly weaker than the FBAIN promoter and GPAT promoter, when cells were grown in SD medium for 24 hrs. More interestingly, however, it appeared that the YAT1 promoter was stronger than the GPAT promoter and comparable with the FBAIN promoter in cells grown in HGM for 24 hrs (FIG. 4B). This remained true after 120 hrs in HGM (FIG. 4C). Thus, the YAT1 promoter appeared to be induced in HGM, a medium that promotes oleaginous growth conditions due to nitrogen limitation.
Example 9
Comparative Analysis Of The TEF, YAT1, FBAIN And GPAT Promoter Activities In Yarrowia lipolytica, As Determined by Fluorometric Assay
A variety of methods are available to compare the activity of various promoters, to thereby facilitate determination of each promoter's strength for use in future applications wherein a suite of promoters would be necessary to construct chimeric genes. Thus, although it may be useful to indirectly quantitate promoter activity based on reporter gene expression using histochemical staining (Example 8), quantification of GUS expression using more quantitative means may be desirable. One suitable method to assay GUS activity is by fluorometric determination of the production of 4-methylumbelliferone (4-MU) from the corresponding β-glucuronide (4-MUG; see Jefferson, R. A., Plant Mol. Biol. Reporter, 5:387-405 (1987)).
Yarrowia lipolytica strain Y2034 containing plasmids pY5-30, pYAT-GUS, pYGPAT-GUS and pDMW214 constructs, respectively (from Example 7), were grown from single colonies in 10 mL SD medium at 30° C. for 48 hrs to an OD600 ̃5.0. Two mL of each culture was collected for GUS activity assays, as described below, while 5 mL of each culture was switched into HGM.
Specifically, cells from the 5 mL aliquot were collected by centrifugation, washed once with 5 mL of HGM and resuspended in HGM medium. The cultures in HGM were then grown in a shaking incubator at 30° C. for 24 hrs. Two mL of each HGM culture were collected for GUS activity assay, while the remaining culture was allowed to grow for an additional 96 hrs before collecting an additional 2 mL of each culture for the assay.
Each 2 mL culture sample in SD medium was resuspended in 1 mL of 0.5× cell culture lysis reagent (Promega). Resuspended cells were mixed with 0.6 mL of glass beads (0.5 mm diameter) in a 2.0 mL screw cap tube with a rubber O-ring. The cells were then homogenized in a Biospec mini beadbeater (Bartlesville, Okla.) at the highest setting for 90 sec. The homogenization mixtures were centrifuged for 2 min at 14,000 rpm in an Eppendof centrifuge to remove cell debris and beads. The supernatant was used for GUS assay and protein determination.
For each fluorometric assay, 200 μl of extract was added to 800 μl of GUS assay buffer (2 mM 4-methylumbelliferyl-β-D-glucuronide [“MUG”] in extraction buffer) and placed at 37° C. Aliquots of 100 μl were taken at 0, 30 and 60 min time points and added to 900 μl of stop buffer (1 M Na2CO3). Each time point was read using a Fluorimeter (CytoFluorR Series 4000, Framingham, Mass.) set to an excitation wavelength of 360 nm and an emission wavelength of 455 nm. Total protein concentration of each sample was determined using 20 μl of extract and 980 μl of BioRad Bradford reagent (Bradford, M. M., Anal. Biochem., 72:248-254 (1976)). GUS activity is expressed as nmoles of 4-MU per minute per mg of total protein.
As shown in the Table below, the activity of the YAT1 promoter was highly induced after 24 hrs in HGM.







TABLE 5







Comparison of TEF, FBAIN, YAT1 And GPAT Promoter Activity


Under Various Growth Conditions











Culture
Promoter














Conditions
TEF
FBAIN
YAT1
GPAT

















 48 hr, SD
0.401
43.333
0.536
5.252



 24 hr, HGM
0.942
30.694
19.154
2.969



120 hr HGM
0.466
17.200
13.400
3.050










Based on the data above wherein the activity of the YAT1 promoter was quantitated based on GUS activity of cell extracts, the activity of the YAT1 promoter increased by  ̃37 fold when cells were switched from SD medium into HGM and grown for 24 hrs. After 120 hrs in HGM, the activity was reduced somewhat but was still 25× higher than the activity in SD medium. In contrast, the activity of the FBAIN promoter and the GPAT promoter was reduced by 30% and 40%, respectively, when switched from SD medium into HGM for 24 hrs. The activity of the TEF promoter increased by 2.3 fold after 24 hrs in HGM. Thus, the YAT1 promoter is inducible under oleaginous conditions and will thereby function as an effective promoter for the regulated expression of heterologous genes in Yarrowia lipolytica. 
Example 10
Construction And Expression Of Modified YAT1 Promoters In Yarrowa lipolytica 
The present Example describes the construction and expression of various modified YAT1 promoters derived from the exemplary wildtype 775 by YAT1 promoter set forth as SEQ ID NO:25, which corresponds to the −775 to −1 upstream region of the yat1 gene. These modified promoters range in length from 675 bases to 783 bases and comprise various insertions and substitutions. Each of the modified YAT1 promoters was found to enable successful expression of the coding region to which it was linked, upon expression in Yarrowia lipolytica. Thus, it is demonstrated herein that DNA fragments of diminished length may have identical promoter activity to the promoter region provided by the full length wildtype 775 by YAT1 promoter (SEQ ID NO:25) and constitute promoter regions that differ from SEQ ID NO:25.
Construction Of Modified YAT1 Promoters
Mutations to the wildtype 775 by YAT1 promoter set forth as SEQ ID NO:25 were made using a site-directed mutagenesis kit (Stratagene, La Jolla, Calif.) and recommended protocols, using plasmid pYAT-GUS1 (FIG. 5) as template.
Plasmid pYAT-GUS1 (SEQ ID NO:45) was generated by EcoRI digestion of pYAT-GUS (Example 5), and then the large fragment of the digestion (i.e., 6645 bp) was self-ligated. This self-ligation excised a portion of the vector backbone and left an intact YAT1::GUS::XPR chimeric gene, wherein the sequence of the YAT1 promoter is set forth as SEQ ID NO:25 (i.e., the wildtype YAT1 promoter, corresponding to the −775 to −1 region upstream of the yat1 gene).
Using plasmid pYAT-GUS1 as template, 11 different modified YAT1 promoters were thus created, as described below in Table 6.







TABLE 6







WildType And Modified YAT1 Promoters
















Promoter Region




SEQ ID

Promoter
With Respect to


Promoter
NO
Mutations with Respect to SEQ ID NO: 25
Length
yat1 Gene*





Wildtype YAT1
SEQ ID
NONE
775 bp
Comprises the



promoter
NO: 25


−775 to −1 region


 


Modified
SEQ ID
CC added at +776
777 bp
Comprises the


YAT1-CC promoter
NO: 26


−775 to −1 region


 


Modified
SEQ ID
SalI site (G/TCGAC) added at +1;
783 bp
Comprises the


YAT1-CC-SalI
NO: 27
CC added at +776

−775 to −1 region


promoter


 


Modified
SEQ ID
Internal NcoI site (C/CATGG) mutated to CTATGG
777 bp
Comprises the


YAT1-CC-NcoI*
NO: 28
(C to T mutation at +414);

−775 to −1 region


promoter

CC added at +776


 


Modified
SEQ ID
SalI site (G/TCGAC) added at +1;
783 bp
Comprises the


YAT1-CC-Ncol*-SalI
NO: 29
Internal NcoI site (C/CATGG) mutated to CTATGG

−775 to −1 region


promoter

(C to T) mutation at +414);




CC added at +776


 


Modified
SEQ ID
Deletion of +1 to +26;
751 bp
Comprises the


YAT1-CC-NcoI*-26
NO: 30
Internal NcoI site (C/CATGG) mutated to CTATGG

−749 to −1 region


promoter

(C to T) mutation at +414);




CC added at +776


 


Modified
SEQ ID
Deletion of +1 to +26;
757 bp
Comprises the


YAT1-CC-NcoI*-26-
NO: 31
ClaI site (AT/CGAT) added at +27;

−749 to −1 region


ClaI promoter

Internal NcoI site (C/CATGG) mutated to CTATGG




(C to T) mutation at +414);




CC added at +776


 


Modified
SEQ ID
Deletion of +1 to +26;
759 bp
Comprises the


YAT1-CC-NcoI*-26-
NO:32
SwaI site (ATTT/AAAT) added at +27;

−749 to −1 region


SwaI promoter

Internal NcoI site (C/CATGG) mutated to CTATGG




(C to T mutation at +414);




CC added at +776


 


Modified
SEQ ID
Deletion of +1 to +48;
729 bp
Comprises the


YAT1-CC-NcoI*-48
NO: 33
Internal NcoI site (C/CATGG) mutated to CTATGG

−727 to −1 region


promoter

(C to T mutation at +414);




CC added at +776


 


Modified
SEQ ID
Deletion of +1 to +48;
737 bp
Comprises the


YAT1-CC-NcoI*-48-
NO: 34
PmeI site (GTTT/AAAC) added at +49;

−727 to −1 region


PmeI promoter

Internal NcoI site (C/CATGG) mutated to CTATGG




(C to T mutation at +414);




CC added at +776


 


Modified
SEQ ID
Deletion of +1 to +102;
675 bp
Comprises the


YAT1-CC-NcoI*-102
NO: 35
Internal NcoI site (C/CATGG) mutated to CTATGG

−673 to −1 region


promoter

(C to T mutation at +414);




CC added at +776


 


Modified
SEQ ID
Deletion of +1 to +102;
681 bp
Comprises the


YAT1-CC-NcoI*-102-
NO: 36
EcoRI site (G/AATTC) added at +103 (creating

−673 to −1 region


EcoRI promoter

effectively a deletion of only +1 to +97 of SEQ




ID NO: 25 and a G to T mutation at +100);




Internal NcoI site (C/CATGG) mutated to CTATGG




(C to T mutation at +414);




CC added at +776





*Promoter region with respect to Yarrowia lipolytica yat1 gene is described based on nucleotide numbering such that the ‘A’ position of the ‘ATG’ translation initiation codon is designated as +1.






A multiple sequence alignment of these promoters (i.e., the YAT1-CC promoter [SEQ ID NO:26], the YAT1-CC-SalI promoter [SEQ ID NO:27], the YAT1-CC-NcoI* promoter [SEQ ID NO:28], the YAT1-CC-NcoI*-SalI promoter [SEQ ID NO:29], the YAT1-CC-NcoI*-26 promoter [SEQ ID NO:30], the YAT1-CC-NcoI*-26-ClaI promoter [SEQ ID NO:31], the YAT1-CC-NcoI*-26-SwaI promoter [SEQ ID NO:32], the YAT1-CC-NcoI*-48 promoter [SEQ ID NO:33], the YAT1-CC-NcoI*-48-PmeI promoter [SEQ ID NO:34], the YAT1-CC-NcoI*-102 promoter [SEQ ID NO:35] and the YAT1-CC-NcoI*-102-EcoRI promoter [SEQ ID NO:36]), as well as the wildtype YAT1 promoter (SEQ ID NO:25) which corresponds to the −775 to −1 region upstream of the yat1 gene, is shown in FIG. 6. The alignment was performed using default parameters [gap opening penalty=15, gap extension penalty=6.66, and gap separation penalty range=8] of Vector NTI®'s Advance 9.1.0 AlignX program (Invitrogen Corporation, Carlsbad, Calif.).
Expression Of Modified YAT1 Promoters: YAT1-CC-SalI, YAT1-CC-NcoI*-SalI, YAT1-CC-NcoI*-26-ClaI, YAT1-CC-NcoI*-48-PmeI And YAT1-CC-NcoI*-102-EcoRI
Using standard cloning methodology, the resultant modified YAT1 promoters (i.e., YAT1-CC-SalI, YAT1-CC-NcoI*-SalI, YAT1-CC-NcoI*-26-ClaI, YAT1-CC-NcoI*-48-PmeI and YAT1-CC-NcoI*-102-EcoRI) were operably linked to the coding regions of several different PUFA biosynthetic pathway genes and suitable terminators derived from Yarrowia in various plasmid vectors.
The various plasmid vectors were transformed separately into several different strains of Y. lipolytica derived from Y. lipolytica ATCC #20362 that had been previously engineered to produce the substrate appropriate for the introduced gene (see General Methods). Thus, e.g., a host producing suitable quantities of either LA or ALA was required to enable expression of an introduced Δ9 elongase, since the Δ9 elongase converts LA to EDA and/or ALA to ETrA. Similarly, a host producing suitable quantities of either EDA or ETrA was required to enable expression of an introduced Δ8 desaturase, since the Δ8 desaturase converts EDA to DGLA and/or ETrA to ETA. See, FIG. 2.
Single colonies from each transformation were streaked onto MM selection plates and grown at 30° C. for 24 to 48 hrs. A loop of cells from each MM selection plate was then inoculated into liquid MM at 30° C.; the cells were shaken at 250 rpm/min for 2 days, collected by centrifugation and lipids were extracted. FAMEs were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
The promoter activity of each of the mutant YAT1 promoters (i.e., YAT1-CC-SalI, YAT1-CC-NcoI*-SalI, YAT1-CC-NcoI*-26-ClaI, YAT1-CC-NcoI*-48-PmeI and YAT1-CC-NcoI*-102-EcoRI) was determined based on the substrate conversion efficiency of the particular gene to which the promoter was operably linked. More specifically, the conversion efficiency refers to the efficiency by which a particular enzyme can convert substrate to product and was calculated according to the following formula: ([product]/[substrate+product])*100, where ‘product’ includes the immediate product and all products in the pathway derived from it.
The mutant promoter was deemed active if suitable substrate conversion was observed. Suitable conversion was determined by comparing with the substrate conversion observed in the untransformed, parent strain of Y. lipolytica. 
Based on the above analyses, each of the modified YAT1 promoters (i.e., YAT1-CC-SalI [SEQ ID NO:27], YAT1-CC-NcoI*-SalI [SEQ ID NO:29], YAT1-CC-NcoI*-26-ClaI [SEQ ID NO:31], YAT1-CC-NcoI*-48-PmeI [SEQ ID NO:34] and YAT1-CC-NcoI*-102-EcoRI [SEQ ID NO:36]) was deemed active. Thus, SEQ ID NO:25 could be truncated from its 5′ end in multiple ways, i.e., by having deleted from its sequence any set of consecutive nucleotides starting from nucleotide 1 (at its 5′ end) and including up to nucleotide 97. For example: 1) deleting the region defined as +1 to +97 by of SEQ ID NO:25 results in the active mutant promoter described herein as YAT1-CC-NcoI*-102-EcoRI, which corresponds to bases 98 to 775 of SEQ ID NO:25 (i.e., also corresponding to the −673 to −1 region of the Yarrowia lipolytica yat1 gene); 2) deleting the region defined as +1 to +26 by of SEQ ID NO:25 results in the active mutant promoter described herein YAT1-CC-NcoI*-26-ClaI, which corresponds to bases 27 to 775 of SEQ ID NO:25 (i.e., also corresponding to the −749 to −1 region of the Yarrowia lipolytica yat1 gene); and 3) deleting the region defined as +1 to +48 by of SEQ ID NO:25 results in the active mutant promoter described herein YAT1-CC-NcoI*-48-PmeI, which corresponds to bases 49 to 775 of SEQ ID NO:25 (i.e., also corresponding to the −727 to −1 region of the Yarrowia lipolytica yat1 gene).
In addition to tolerating various truncations from the 5′ end of SEQ ID NO:25, the modified YAT1-CC-SalI (SEQ ID NO:27), YAT1-CC-NcoI*-SalI (SEQ ID NO:29), YAT1-CC-NcoI*-26-ClaI (SEQ ID NO:31), YAT1-CC-NcoI*-48-PmeI (SEQ ID NO:34) and YAT1-CC-NcoI*-102-EcoRI (SEQ ID NO:36) promoters are demonstrated to sustain mutations in the active region (i.e., in the region corresponding to bases 98 to 775 of SEQ ID NO:25) that do not change the active status of the promoter.
Specifically, for all modified YAT1 promoters, a CC insertion at by +776 does not impair their active status. As described in U.S. Pat. No. 7,125,672, the preferred consensus sequence of the codon-optimized translation initiation site for optimal expression of genes in Y. lipolytica is ‘MAMMATGNHS’ (SEQ ID NO:37), wherein the nucleic acid degeneracy code used is as follows: M=A/C; S=C/G; H=A/C/T; and N=A/C/G/T. While the four nucleotides immediately proceeding the ‘ATG’ translation initiation site are ‘ACAA’ in the wildtype YAT1 promoter set forth as SEQ ID NO:25 (therefore not corresponding to the preferred consensus sequence), the CC insertion at by +776 in the modified YAT1 promoters results in a more preferred sequence of ‘AACC’ immediately upstream of the ‘ATG’ translation initiation site.
For YAT1-CC-NcoI*-SalI (SEQ ID NO:29), YAT1-CC-NcoI*-26-ClaI (SEQ ID NO:31), YAT1-CC-NcoI*-48-PmeI (SEQ ID NO:34) and YAT1-CC-NcoI*-102-EcoRI (SEQ ID NO:36) promoters, a substitution at by +414 from C to T (effectively removing the internal NcoI site from the promoter region) does not impair the active status of the mutant promoter. It is hypothesized that a substitution at by +414 from C to G or from C to A would also result in a functional promoter. Furthermore, for the YAT1-CC-NcoI*-102-EcoRI (SEQ ID NO:36) promoter, a substitution at by +100 from G to T (effectively inserting a EcoRI site at the 5′ region of the promoter) does not impair its active status.
Based on the results obtained for YAT1-CC-SalI (SEQ ID NO:27), YAT1-CC-NcoI*-SalI (SEQ ID NO:29), YAT1-CC-NcoI*-26-ClaI (SEQ ID NO:31), YAT1-CC-NcoI*-48-PmeI (SEQ ID NO:34) and YAT1-CC-NcoI*-102-EcoRI (SEQ ID NO:36), it is also hypothesized herein that related promoters (i.e., those that comprise at least the −673 to −1 region upstream of the Y. lipolytica yat1 gene, optionally with or without either a C to A, G or T mutation at +414 [to remove the internal NcoI site] or a CC insertion at +776 [to optimize the consensus sequence around the translation initiation codon]) will also be active.
Thus, for example, it is expected that the YAT1-CC (SEQ ID NO:26) promoter, the YAT1-CC-NcoI* (SEQ ID NO:28) promoter, the YAT1-CC-NcoI*-26 (SEQ ID NO:30) promoter, the YAT1-CC-NcoI*-26-SwaI (SEQ ID NO:32) promoter, the YAT1-CC-NcoI*-48 (SEQ ID NO:33) promoter, and the YAT1-CC-NcoI*-102 (SEQ ID NO:35) promoter will all be active promoters for the purposes of enabling expression of a coding region of interest that is expressible in a yeast cell, when the promoter region is operably linked to the coding region. More specifically, the YAT1-CC promoter set forth as SEQ ID NO:26 is identical to the YAT1-CC-SalI promoter of SEQ ID NO:27, with the exception that a SalI restriction enzyme site was added upstream of the promoter sequence (corresponding to the −775 to −1 region upstream of the Y. lipolytica yat1 gene) in the latter, for cloning convenience. Since SEQ ID NO:27 was proven to be active, one can assume that SEQ ID NO:26 will also be active. Thus, description of a functional modified promoter will not be limited by the particular restriction enzyme that is introduced immediately preceding the promoter sequence. Similarly, since the YAT1-CC-NcoI*-SalI (SEQ ID NO:29) promoter was proven active (also corresponding to the −775 to −1 region upstream of the Y. lipolytica yat1 gene), the related YAT1-CC-NcoI* (SEQ ID NO:28) promoter which lacks only the upstream SalI restriction enzyme site is also expected to be active. Likewise, since the YAT1-CC-NcoI*-26-ClaI (SEQ ID NO:31) promoter was proven active (corresponding to the −749 to −1 region upstream of the Y. lipolytica yat1 gene), the related YAT1-CC-NcoI*-26 (SEQ ID NO:30) promoter which lacks only the upstream ClaI restriction enzyme site is also expected to be active, as is the YAT1-CC-NcoI*-26-SwaI (SEQ ID NO:32) promoter which has an upstream SwaI restriction enzyme site in place of the upstream ClaI restriction enzyme site. Similar conclusions can be drawn concerning the YAT1-CC-NcoI*-48 (SEQ ID NO:33) promoter based on the activity of the YAT1-CC-NcoI*-48-PmeI (SEQ ID NO:34) promoter and concerning the YAT1-CC-NcoI*-102 (SEQ ID NO:35) promoter, based on the activity of the YAT1-CC-NcoI*-102-EcoRI (SEQ ID NO:36) promoter.
Example 11
Use Of Select Modified YAT1 Promoters In Yarrowa lipolytica Strain Y4259, Producing 46.5% Eicosapentaenoic Acid
The present Example describes the construction of strain Y4259, derived from Yarrowia lipolytica ATCC #20362, capable of producing about 46.5% EPA relative to the total lipids via expression of a Δ9 elongase/Δ8 desaturase pathway. The development of strain Y4259 (FIG. 7) required the construction of strains Y2224, Y4001, Y4001 U, Y4036, Y4036U, Y4070, Y4086, Y4086U1, Y4128 [deposited with the American Type Culture Collection on Aug. 23, 2007, bearing the designation ATCC PTA-8614], Y4128U3 (Ura-), Y4217 and Y4217U2 (Ura-).
The final genotype of strain Y4259 with respect to wild type Yarrowia lipolytica ATCC #20362 included eight chimeric genes described as: YAT1::FmD12::OCT, YAT1::ME3S::Pex16, YAT1::EgD9eS::Lip2, YAT1::EgD8M::Aco, YAT1::EgD5S::Aco, YAT1::RD5S::OCT, YAT1::PaD17S::Lip1 and YAT1::YICPT1::ACO. The YAT1 promoter in each of these cassettes corresponds to one of the modified YAT1 promoters of Example 10, as summarized in Table 7 and described in additional detail in the Example below (FIG. 7).








TABLE 7







Use Of Modified YAT1 Promoters In Genetically Engineered



Strains of Yarrowia lipolytica Producing PUFAs













Plasmid

Promoter





SEQ ID

SEQ ID



Plasmid
NO
Promoter
NO
Chimeric Gene





pZKLeuN-29E3
SEQ ID
YAT1-CC-
SEQ ID
YAT1::ME3S::




NO: 38
NcoI*-102-
NO: 36
Pex16




EcoRI


 


pKO2UF8289
SEQ ID
YAT1-CC-
SEQ ID
YAT1::FmD12::



NO: 39
NcoI*-26-SwaI
NO: 32
OCT


 


pZKSL-555R
SEQ ID
YAT1-CC-
SEQ ID
YAT1::RD5S::



NO: 40
NcoI*-26-ClaI
NO: 31
OCT


 


pZP3-Pa777U
SEQ ID
YAT1-CC-
SEQ ID
YAT1::PaD17S::



NO: 41
NcoI*-26-ClaI
NO: 31
Lip1


 


pZP2-2988
SEQ ID
YAT1-CC-
SEQ ID
YAT1::EgD8M::



NO: 42
NcoI*-26-SwaI
NO: 32
ACO


 


pZKL2-5U89GC
SEQ ID
YAT1-CC-
SEQ ID
YAT1::YICPT1::



NO:43
NcoI*-26-SwaI
NO: 32
Aco


 


pZKL2-5U89GC
SEQ ID
YAT1-CC-
SEQ ID
YAT1::EgD5S::



NO: 43
NcoI*-102-
NO: 36
ACO




EcoRI


 


pZKL1-25P98C
SEQ ID
YAT1-CC-
SEQ ID
YAT1::EgD9eS::



NO: 44
NcoI*-48-PmeI
NO: 34
Lip2









Generation Of Strain Y4001 To Produce About 17% EDA Of Total Lipids

The generation of strain Y4001 is described in Example 7 of Intl. App. Pub. No. WO 2008/073367, hereby incorporated herein by reference. Briefly, construct pZKLeuN-29E3 was integrated into the Leu2 loci of strain Y2224 (a FOA resistant mutant from an autonomous mutation of the Ura3 gene of wildtype Yarrowia strain ATCC #20362). Although construct pZKLeuN-29E3 (SEQ ID NO:38) comprised four chimeric genes (i.e., a Δ12 desaturase, a C16/18 elongase and two Δ9 elongases), the chimeric YAT1::ME3S::Pex16 gene is of relevance to the present disclosure. Specifically, the ME3S gene, corresponding to a codon-optimized C16/18 elongase gene derived from Mortierella alpina (U.S. Pat. No. 7,470,532), was operably linked to a YAT1 promoter sequence that corresponds to YAT1-CC-NcoI*-102-EcoRI (SEQ ID NO:36) (Example 10).
Generation Of Strain Y4036 To Produce About 18% DGLA Of Total Lipids
The generation of strain Y4036 is described in Example 7 of Intl. App. Pub. No. WO 2008/073367, hereby incorporated herein by reference. Briefly, following the isolation of strain Y4001 U, having a Leu- and Ura-phenotype, construct pKO2UF8289 was integrated into the Δ12 loci of strain Y4001U1. This resulted in isolation of strain Y4036, producing about 18.2% DGLA of total lipids. Although construct pKO2UF8289 (SEQ ID NO:39) comprised four chimeric genes (i.e., a Δ12 desaturase, one Δ9 elongase and two mutant Δ8 desaturases), the chimeric YAT1::FmD12::OCT gene is of relevance to the present disclosure. Specifically, the FmD12 gene, corresponding to a Fusarium moniliforme Δ12 desaturase gene (U.S. Pat. No. 7,504,259), was operably linked to a YAT1 promoter sequence that corresponds to YAT1-CC-NcoI*-26-SwaI (SEQ ID NO:32) (Example 10).
Generation Of Strain Y4070 To Produce About 12% ARA Of Total Lipids
The generation of strain Y4070 is described in Example 7 of Intl. App. Pub. No. WO 2008/073367, hereby incorporated herein by reference. Briefly, following the isolation of strain Y4036U, having a Leu- and Ura-phenotype, construct pZKSL-555R was integrated into the Lys loci of strain Y4036U. This resulted in isolation of strain Y4070, producing about 12% ARA of total lipids. Although construct pZKSL-555R (SEQ ID NO:40) comprised three chimeric AS desaturase genes, the chimeric YAT1::RD5S::OCT gene is of relevance to the present disclosure. Specifically, the RD5S gene, corresponding to a codon-optimized Δ5 desaturase derived from Peridinium sp. CCMP626 (U.S. Pat. Appl. Pub. No. 2007-0271632-A1), was operably linked to a YAT1 promoter sequence that corresponds to YAT1-CC-NcoI*-26-ClaI (SEQ ID NO:31) (Example 10).
Generation Of Y4086 Strain To Produce About 14% EPA Of Total Lipids
The generation of strain Y4086 is described in Example 1 of U.S. Pat. Appl. Pub. No. 2009-0093543-A1, hereby incorporated herein by reference. Briefly, construct pZP3-Pa777U was integrated into the Pox3 loci (GenBank Accession No. AJ001301) of strain Y4070. This resulted in isolation of strain Y4086, producing about 14% EPA of total lipids. Although construct pZP3-Pa777U (SEQ ID NO:41) comprised three chimeric Δ17 desaturase genes, the chimeric YAT1::PaD17S::Lip1 gene is of relevance to the present disclosure. Specifically, the PaD17S gene, corresponding to a codon-optimized Δ17 desaturase derived from Pythium aphanidermatum (Intl. App. Pub. No. WO 2008/054565), was operably linked to a YAT1 promoter sequence that corresponds to YAT1-CC-NcoI*-26-ClaI (SEQ ID NO:31) (Example 10).
Generation Of Y4128 Strain To Produce About 37% EPA Of Total Lipids
The generation of strain Y4128 is described in Example 2 of U.S. Pat. Appl. Pub. No. 2009-0093543-A1, hereby incorporated herein by reference. Briefly, following the isolation of strain Y4086U1, having a Ura-phenotype, construct pZP2-2988 was integrated into the Pox2 loci (GenBank Accession No. AJ001300) of strain Y4086U1. This resulted in isolation of strain Y4128, producing about 37% EPA of total lipids. Although construct pZP2-2988 (SEQ ID NO:42) comprised four chimeric genes (i.e., a Δ12 desaturase gene, two Δ8 desaturase genes and a Δ9 elongase), the chimeric YAT1::EgD8M::ACO gene is of relevance to the present disclosure. Specifically, the EgD8M gene, corresponding to a synthetic mutant Δ8 desaturase (U.S. Pat. Appl. Pub. No. 2008-0138868 A1) derived from Euglena gracilis (U.S. Pat. No. 7,256,033), was operably linked to a YAT1 promoter sequence that corresponds to YAT1-CC-NcoI*-26-SwaI (SEQ ID NO:32) (Example 10).
Generation Of Y4217 Strain To Produce About 42% EPA Of Total Lipids
The generation of strain Y4217 is described in Example 3 of U.S. Pat. Appl. Pub. No. 2009-0093543-A1, hereby incorporated herein by reference. Briefly, following the isolation of strain Y4128U3, having a Ura-phenotype, construct pZKL2-5U89GC was integrated into the Lip2 loci (GenBank Accession No. AJ012632) of strain Y4128U3. This resulted in isolation of strain Y4217, producing about 42% EPA of total lipids. Although construct pZKL2-5U89GC (SEQ ID NO:43) comprised four chimeric genes (i.e., a Δ9 elongase gene, a Δ8 desaturase gene, a Δ5 desaturase gene and a Yarrowia lipolytica diacylglycerol cholinephosphotransferase gene), the chimeric YAT1::YICPT1::Aco gene and chimeric YAT1::EgD5S::ACO gene are of relevance to the present disclosure. Specifically, the YICPT1gene, corresponding to a Yarrowia lipolytica diacylglycerol cholinephosphotransferase (Intl. App. Pub. No. WO 2006/052870), was operably linked to a YAT1 promoter sequence that corresponds to YAT1-CC-NcoI*-26-SwaI (SEQ ID NO:32) (Example 10). The EgD5S gene, corresponding to a codon-optimized Δ5 desaturase derived from Euglena gracilis (Intl. App. Pub. No. WO 2007/136671), was operably linked to a YAT1 promoter sequence that corresponds to YAT1-CC-NcoI*-102-EcoRI (SEQ ID NO:36) (Example 10).
Generation Of Y4259 Strain To Produce About 46.5% EPA Of Total Lipids
The generation of strain Y4259 is described in Example 3 of U.S. Pat. Appl. Pub. No. 2009-0093543-A1, hereby incorporated herein by reference. Briefly, following the isolation of strain Y4217U2, having a Ura-phenotype, construct pZKL1-2SP98C was integrated into the Lip1 loci (GenBank Accession No. Z50020) of strain Y4217U2. This resulted in isolation of strain Y4259, producing about 46.5% EPA of total lipids. Although construct pZKL1-2SP98C (SEQ ID NO:44) comprised four chimeric genes (i.e., a Δ9 elongase gene, a Δ8 desaturase gene, a Δ12 desaturase gene and a Yarrowia lipolytica diacylglycerol cholinephosphotransferase gene), the chimeric YAT1::EgD9eS::Lip2 gene is of relevance to the present disclosure. Specifically, the EgD9eS gene, corresponding to a codon-optimized Δ9 elongase gene derived from Euglena gracilis (Intl. App. Pub. No. WO 2007/061742), was operably linked to a YAT1 promoter sequence that corresponds to YAT1-CC-NcoI*-48-PmeI (SEQ ID NO:34) (Example 10).
Thus, four different modified mutant YAT promoters derived from the exemplary 775 by YAT1 promoter set forth as SEQ ID NO:25 (corresponding to the −775 to −1 upstream region of the yat1 gene) were utilized in various chimeric genes within strain Y4259, to enable expression of various PUFA biosynthetic pathway genes. These truncated promoters comprise various regions of the upstream yat1 gene, including the −673 to −1 region, the −727 to −1 region and the −749 to −1 region, and comprise various insertions and substitutions. More specifically, each of the modified YAT1 promoters utilized within pZKLeuN-29E3 (SEQ ID NO:38), pKO2UF8289 (SEQ ID NO:39), pZKSL-555R (SEQ ID NO:40), pZP3-Pa777U (SEQ ID NO:41), pZP2-2988 (SEQ ID NO:42), pZKL2-5U89GC (SEQ ID NO:43) and pZKL1-2SP98C (SEQ ID NO:44) was found to enable successful expression of the coding region to which it was linked, upon expression in Yarrowia lipolytica. Thus, it is demonstrated herein that DNA fragments of diminished length may have identical promoter activity as the promoter region provided by the full length of SEQ ID NO:25 and constitute promoter regions that differ from SEQ ID NO:25.

## Claims

1. A recombinant gene expression cassette comprising an isolated DNA sequence comprising a polynucleotide encoding a polypeptide expressible in an oleaginous yeast cell wherein said polynucleotide is operably linked to a promoter region comprising the nucleotide sequence a position 103 to 775 of SEQ ID NO:25 or a mutant of the nucleotide sequence at position 103 to 775 of SEQ ID NO:25 wherein deoxycytidine at position 414 of SEQ ID NO:25 is replaced by deoxythymidine, deoxyadenosine, or deoxyguanosine.

2. The recombinant gene expression cassette of claim 1 wherein a restriction enzyme site is inserted upstream and adjacent to the promoter region.

3. The recombinant gene expression cassette of claim 1 or claim 2 wherein said promoter region comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

4. The recombinant gene expression cassette of claim 1, wherein two deoxycytidines are adjacent to the 3′ end of said nucleotides 103 to 775 of SEQ ID NO:25 or said mutant of the nucleotide sequence at position 103 to 775 of SEQ ID NO:25.

5. The recombinant gene expression cassette of claim 1, wherein said polynucleotide encodes at least one enzyme selected from the group consisting of an enzyme of the ω-3/ω-6 fatty acid biosynthetic pathway, an enzyme of the neutral lipid biosynthetic pathway, and an enzyme of the phospholipid biosynthetic pathway.