# Patent

## Title
High arachidonic acid producing strains of Yarrowia lipolytica

## Abstract

Engineered strains of the oleaginous yeast Yarrowia lipolytica capable of producing greater than 10% arachidonic acid (ARA, an ω-6 polyunsaturated fatty acid) in the total oil fraction are described. These strains comprise various chimeric genes expressing heterologous desaturases, elongases and acyltransferases, and optionally comprise various native desaturase and acyltransferase knockouts to enable synthesis and high accumulation of ARA. Production host cells are claimed, as are methods for producing ARA within said host cells.

## Background
This application claims the benefit of U.S. Provisional Application No. 60/624,812, filed Nov. 4, 2004.


FIELD OF THE INVENTION
This invention is in the field of biotechnology. More specifically, this invention pertains to an engineered strain of the oleaginous yeast Yarrowia lipolytica that is capable of efficiently producing arachidonic acid (an ω-6 polyunsaturated fatty acid) in high concentrations.
BACKGROUND OF THE INVENTION
Arachidonic acid (ARA; cis-5, 8, 11, 14-eicosatetraenoic; ω-6) is an important precursor in the production of eicosanoids (e.g., prostaglandins, thromboxanes, prostacyclin and leukot). Additionally, ARA is recognized as: (1) an essential long-chain polyunsaturated fatty acid (PUFA); (2) the principal ω-6 fatty acid found in the human brain; and, (3) an important component of breast milk and many infant formulas, based on its role in early neurological and visual development. Although adults obtain ARA readily from the diet in foods such as meat, eggs and milk (and can also inefficiently synthesize ARA from dietary linolenic acid (LA)), commercial sources of ARA oil are typically produced from natural vegetarian sources (e.g., microorganisms in the genera Mortierella (filamentous fungus), Entomophthora, Pythium and Porphyridium (red alga)). Most notably, Martek Biosciences Corporation (Columbia, Md.) produces an ARA-containing fungal oil (ARASCO®; U.S. Pat. No. 5,658,767) which is substantially free of EPA and which is derived from either Mortierella alpina or Pythium insidiuosum. One of the primary markets for this oil is infant formula; e.g., formulas containing Martek's ARA oils are now available in more than 60 countries worldwide.
Despite the availability of ARA from natural microbial sources such as those described above, microbial production of ARA using recombinant means is expected to have several advantages over production from natural microbial sources. For example, recombinant microbes having preferred characteristics for oil production can be used, since the naturally occurring microbial fatty acid profile of the host can be altered by the introduction of new biosynthetic pathways in the host and/or by the suppression of undesired pathways, thereby resulting in increased levels of production of desired PUFAs (or conjugated forms thereof) and decreased production of undesired PUFAs. Secondly, recombinant microbes can provide PUFAs in particular forms which may have specific uses. And, finally, microbial oil production can be manipulated by controlling culture conditions, notably by providing particular substrate sources for microbially expressed enzymes, or by addition of compounds/genetic engineering to suppress undesired biochemical pathways. Thus, for example, it is possible to modify the ratio of ω-3 to ω-6 fatty acids so produced, or engineer production of a specific PUFA (e.g., ARA) without significant accumulation of other PUFA downstream or upstream products. The latter possibility is of particular interest in some embodiments of the invention herein, wherein it is desirable to provide a recombinant source of microbial oil containing high concentrations of ARA and that is additionally devoid of gamma-linolenic acid (GLA; γ-linolenic acid; cis-6, 9, 12-octadecatrienoic acid; ω-6).
GLA is an important intermediate in the biosynthesis of biologically active prostaglandin from LA. Although also recognized as an essential ω-6 PUFA having tremendous clinical, physiological and pharmaceutical value, there are some applications in which GLA acts in opposition to ARA. Thus, commercial production of an oil comprising ARA and devoid of GLA would have utility in some applications.
Most microbially produced ARA is synthesized via the Δ6 desaturase/Δ6 elongase pathway (which is predominantly found in, algae, mosses, fungi, nematodes and humans) and wherein: 1.) oleic acid is converted to LA by the action of a Δ12 desaturase; 2.) LA is converted to GLA by the action of a Δ6 desaturase; 3.) GLA is converted to DGLA by the action of a C18/20 elongase; and 3.) DGLA is converted to ARA by the action of a Δ5 desaturase (FIG. 1). However, an alternate Δ9 elongase/Δ8 desaturase pathway for the biosynthesis of ARA operates in some organisms, such as euglenoid species, where it is the dominant pathway for formation of C20 PUFAs (Wallis, J. G., and Browse, J. Arch. Biochem. Biophys. 365:307-316 (1999); WO 00/34439; and Qi, B. et al. FEBS Letters. 510:159-165 (2002)). In this pathway, LA is converted to EDA by a Δ9 elongase, EDA is converted to DGLA by a Δ8 desaturase, and DGLA is converted to ARA by a Δ5 desaturase.
Although genes encoding the Δ6 desaturase/Δ6 elongase and the Δ9 elongase/Δ8 desaturase pathways have now been identified and characterized from a variety of organisms, and some have been heterologously expressed in combination with other PUFA desaturases and elongases, neither of these pathways have been introduced into a microbe, such as a yeast, and manipulated via complex metabolic engineering to enable economical production of commercial quantities of ARA (i.e., greater than 10% with respect to total fatty acids). Additionally, considerable discrepancy exists concerning the most appropriate choice of host organism for such engineering.
Recently, Picataggio et al. (WO 2004/101757 and co-pending U.S. Patent Application No. 60/624,812) have explored the utility of oleaginous yeast, and specifically, Yarrowia lipolytica (formerly classified as Candida lipolytica), as a preferred class of microorganisms for production of PUFAs such as ARA and EPA. Oleaginous yeast are defined as those yeast that are naturally capable of oil synthesis and accumulation, wherein oil accumulation can be up to about 80% of the cellular dry weight. Despite a natural deficiency in the production of ω-6 and ω-3 fatty acids in these organisms (since naturally produced PUFAs are limited to 18:2 fatty acids (and less commonly, 18:3 fatty acids)), Picataggio et al. (supra) have demonstrated production of 1.3% ARA and 1.9% EPA (of total fatty acids) in Y. lipolytica using relatively simple genetic engineering approaches and up to 28% EPA using more complex metabolic engineering. However, similar work has not been performed to enable economic, commercial production of ARA in this particular host organism.
Applicants have solved the stated problem by engineering various strains of Yarrowia lipolytica that are capable of producing greater than 10-14% ARA in the total oil fraction, using either the Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8 desaturase pathway (thereby producing 10-11% ARA-oil with 25-29% GLA or 14% ARA-oil that is devoid of GLA, respectively). Additional metabolic engineering and fermentation methods are provided to further enhance ARA productivity in this oleaginous yeast.
SUMMARY OF THE INVENTION
The invention relates to recombinant production hosts of the genus Yarrowia having enzymatic pathways useful of the production of arachidonic acid.
Accordingly the invention provides a recombinant production host cell for the production of arachidonic acid comprising a background Yarrowia sp. comprising a gene pool comprising the following genes of the ω-3/ω-6 fatty acid biosynthetic pathway:

    
    
        a) at least one gene encoding Δ6 desaturase;
        b) at least one gene encoding C18/20 elongase; and,
        c) at least one gene encoding Δ5 desaturase;

wherein at least one of said ω-3/ω-6 fatty acid biosynthetic pathway genes is over-expressed.

    
    


In another embodiment the invention provides a recombinant production host cell for the production of arachidonic acid comprising a background Yarrowia sp. comprising a gene pool comprising the following genes of the ω-3/ω-6 fatty acid biosynthetic pathway:

    
    
        a) at least one gene encoding Δ9 elongase;
        b) at least one gene encoding Δ8 desaturase; and,
        c) at least one gene encoding Δ5 desaturase;

wherein at least one of said ω-3/ω-6 fatty acid biosynthetic pathway genes is over-expressed.

    
    


In specific embodiments recombinant production hosts of the invention may additionally comprise additional pathway elements including, but not limited to: at least one gene encoding Δ12 desaturase; at least one gene encoding Δ9 desaturase; at least one gene encoding C16/18 elongase; and at least one gene encoding C14/16 elongase and at least one gene encoding an acyltransferase.
In one specific embodiment the invention provides a recombinant production host cell for the production of arachidonic acid comprising a background Yarrowia sp. comprising a gene pool comprising the following genes of the ω-3/ω-6 fatty acid biosynthetic pathway:

    
    
        a) at least one gene encoding Δ6 desaturase; and,
        b) at least one gene encoding C18/20 elongase; and,
        c) at least one gene encoding Δ5 desaturase; and,
        d) at least one gene encoding Δ12 desaturase;
    
    


wherein the background Yarrowia sp. is devoid of any native gene encoding an isopropyl malate dehydrogenase (Leu2-) enzyme; and,
wherein at least one of said ω-3/ω-6 fatty acid biosynthetic pathway genes is over-expressed.
In another specific embodiment the invention provides a recombinant production host cell for the production of arachidonic acid comprising a background Yarrowia sp. comprising a gene pool comprising the following genes of the ω-3/ω-6 fatty acid biosynthetic pathway:

    
    
        a) at least one gene encoding Δ9 elongase; and,
        b) at least one gene encoding Δ8 desaturase; and,
        c) at least one gene encoding Δ5 desaturase; and,
        d) at least one gene encoding Δ12 desaturase; and,
        e) at least one gene encoding C16/18 elongase;
    
    


wherein the background Yarrowia sp. is devoid of any native gene encoding a saccharopine dehydrogenase (Lys5-) enzyme; and,
wherein at least one of said ω-3/ω-6 fatty acid biosynthetic pathway genes is over-expressed.
In another embodiment the invention provides a method for the production of a microbial oil comprising arachidonic acid comprising:

    
    
        a) culturing the production host of any of claims 1, or 2 wherein a microbial oil comprising arachidonic acid is produced; and
        b) optionally recovering the microbial oil of step (a).
    
    


In another embodiment the invention provides a microbial oil produced by the methods of the invention and using the recombinant production hosts of the invention.
In an alternate embodiment the invention provides a food product comprising an effective amount of a microbial oil produced by the method of the invention.
In a specific embodiment the invention provides product selected from the group consisting of a medical food, a dietary supplement; infant formula and a pharmaceutical comprising an effective amount of a microbial oil produced by the method of the invention.
In an alternate embodiment the invention provides an animal feed comprising an effective amount of the microbial oil produced by the method of the invention.
The invention additionally provides methods of making a product a food product, or an animal feed supplemented with arachidonic acid comprising combining a microbial oil produced by the methods of the invention with product, food product or animal feed.
In another embodiment the invention provides a method for providing a human, animal or aquaculture organism diet supplement enriched with arachidonic acid (ARA) comprising providing a microbial oil produced by the method of the invention containing arachidonic acid in a form consumable or usable by humans or animals.
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.
















Accession
Date of



Biological Material
Number
Deposit









Plasmid pY89-5
ATCC PTA-6048
Jun. 4th, 2004



Yarrowia lipolytica Y2047
ATCC PTA-7186
Oct. 26, 2005













BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS
FIG. 1 illustrates the ω-3/ω-6 fatty acid biosynthetic pathway.
FIG. 2 is a schematic illustration of the biochemical mechanism for lipid accumulation in oleaginous yeast.
FIG. 3 is a schematic illustration describing the role of various acyltransferases in lipid accumulation in oleaginous yeast.
FIG. 4 diagrams the development of some Yarrowia lipolytica strains of the invention, producing various fatty acids (including ARA) in the total lipid fraction.
FIG. 5A provides a plasmid map for pY5-30. FIG. 5B illustrates the relative promoter activities of TEF, GPD, GPM, FBA and FBAIN in Yarrowia lipolytica ATCC #76982 strains, as determined by histochemical staining. FIG. 5C illustrates the relative promoter activities of YAT1, TEF, GPAT and FBAIN in Y. lipolytica grown in various media as determined by histochemical staining.
FIG. 6A is a graph comparing the promoter activity of GPD, GPM, FBA and FBAIN in Yarrowia lipolytica ATCC #76982 strains, as determined fluorometrically. FIG. 6B graphically summarizes the results of Real Time PCR relative quanitation, wherein the GUS mRNA in Y. lipolytica ATCC #76982 strains (i.e., expressing GPD::GUS, GPDIN::GUS, FBA::GUS or FBAIN::GUS chimeric genes) was quantified to the mRNA level of the Y. lipolytica strain expressing pY5-30 (i.e., a chimeric TEF::GUS gene).
FIG. 7 provides plasmid maps for the following: (A) pY57.YI.AHAS.w497I; (B) pKUNF12T6E; (C) pDMW232; and (D) pDMW271.
FIG. 8 provides plasmid maps for the following: (A) pKUNT2; (B) pZUF17; (C) pDMW237; (D) pDMW240; and (E) yeast expression vector pY89-5.
FIG. 9 shows a chromatogram of the lipid profile of an Euglena gracilis cell extract.
FIG. 10 shows an alignment of various Euglena gracilis Δ8 desaturase polypeptide sequences. The method of alignment used corresponds to the “Clustal V method of alignment”.
FIG. 11 provides plasmid maps for the following: (A) pKUNFmKF2; (B) pDMW277; (C) pZF5T-PPC; (D) pDMW287F; and (E) pDMW297.
FIG. 12 provides plasmid maps for the following: (A) pZP2C16M899; (B) pDMW314; (C) pDM322; and (D) pZKL5598.
FIG. 13 provides plasmid maps for the following: (A) pZP3L37; (B) pY37/F15; (C) pKO2UF2PE; and (D) pZKUT16.
FIG. 14 provides plasmid maps for the following: (A) pKO2UM25E; (B) pZKUGPI5S; (C) pDMW302T16; and (D) pZKUGPE1S.
FIG. 15 provides plasmid maps for the following: (A) pKO2UM26E; (B) pZUF-Mod-1; (C) pMDAGAT1-17; and (D) pMGPAT-17.
FIG. 16 graphically represents the relationship between SEQ ID NOs:97, 98, 99, 100, 101, 102, 103, 104, 105, 106 and 107, each of which relates to glycerol-3-phosphate o-acyltransferase (GPAT) in Mortierella alpina. 
FIG. 17 graphically represents the relationship between SEQ ID NOs:53, 54, 55, 56, 57, 58, 59 and 60, each of which relates to the C16/18 fatty acid elongase enzyme (ELO3) in Mortierella alpina. 
FIG. 18 provides plasmid maps for the following: (A) pZUF6S; (B) pZUF6S-E3WT; (C) pZKUGPYE1-N; and (D) pZKUGPYE2.
FIG. 19 provides plasmid maps for the following: (A) pZKUGPYE1; (B) pZUF6FYE1; (C) pZP217+Ura; (D) pY20; and (E) pLV13.



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:1-112, 158-168, 209, 252, 255 and 357-364 are ORFs encoding promoters, genes or proteins (or fragments thereof) as identified in Table 1.







TABLE 1







Summary of Gene and Protein SEQ ID Numbers










Nucleic acid
Protein


Description
SEQ ID NO.
SEQ ID NO.





Mortierella alpina Δ6 desaturase
 1 (1374 bp)
 2 (457 AA)


Synthetic Δ6 desaturase, derived from
 3 (1374 bp)
 2 (457 AA)


Mortierella alpina, codon-optimized for


expression in Yarrowia lipolytica


Mortierella alpina Δ6 desaturase “B”
 4 (1521 bp)
 5 (458 AA)


Mortierella alpina Δ5 desaturase
 6 (1341 bp)
 7 (446 AA)


Isochrysis galbana Δ5 desaturase
 8 (1329 bp)
 9 (442 AA)


Synthetic Δ5 desaturase derived from
 10 (1329 bp)
 9 (442 AA)


Isochrysis galbana, codon-optimized for


expression in Yarrowia lipolytica


Homo sapiens Δ5 desaturase
 11 (1335 bp)
 12 (444 AA)


Synthetic Δ5 desaturase derived from
 13 (1335 bp)
 12 (444 AA)


Homo sapiens, codon-optimized for


expression in Yarrowia lipolytica


Saprolegnia diclina Δ17 desaturase
 14 (1077 bp)
 15 (358 AA)


Synthetic Δ17 desaturase gene derived
 16 (1077 bp)
 15 (358 AA)


from Saprolegnia diclina, codon-


optimized for expression in Yarrowia


lipolytica


Mortierella alpina C18/20 elongase
 17 (957 bp)
 18 (318 AA)


Synthetic C18/20 elongase gene derived
 19 (957 bp)
 18 (318 AA)


from Mortierella alpina, codon-


optimized for expression in Yarrowia


lipolytica


Thraustochytrium aureum C18/20
 20 (819 bp)
 21 (272 AA)


elongase


Synthetic C18/20 elongase gene derived
 22 (819 bp)
 21 (272 AA)


from Thraustochytrium aureum, codon-


optimized for expression in Yarrowia


lipolytica


Yarrowia lipolytica Δ12 desaturase
 23 (1936 bp)
 24 (419 AA)


Mortieralla isabellina Δ12 desaturase
 25 (1203 bp)
 26 (400 AA)


Fusarium moniliforme Δ12 desaturase
 27 (1434 bp)
 28 (477 AA)


Aspergillus nidulans Δ12 desaturase
 29 (1416 bp)
 30 (471 AA)


Aspergillus flavus Δ12 desaturase
—
 31 (466 AA)


Aspergillus fumigatus Δ12 desaturase
—
 32 (424 AA)


Magnaporthe grisea Δ12 desaturase
 33 (1656 bp)
 34 (551 AA)


Neurospora crassa Δ12 desaturase
 35 (1446 bp)
 36 (481 AA)


Fusarium graminearium Δ12 desaturase
 37 (1371 bp)
 38 (456 AA)


Mortierella alpina Δ12 desaturase
357 (1403 bp)
358 (400 AA)


Saccharomyces kluyveri Δ12 desaturase
—
359 (416 AA)


Kluyveromyces lactis Δ12 desaturase
360 (1948 bp)
361 (415 AA)


Candida albicans Δ12 desaturase
—
362 (436 AA)


Debaryomyces hansenii CBS767 Δ12
—
363 (416 AA)


desaturase


Isochrysis galbana Δ9 elongase
 39 (792 bp)
 40 (263 AA)


Synthetic Δ9 elongase gene, codon-
 41 (792 bp)
 40 (263 AA)


optimized for expression in Yarrowia


lipolytica


Euglena gracillis Δ8 desaturase gene
 42 (1275 bp)
 43 (419 AA)


(non-functional; GenBank Accession


No. AAD45877)


Euglena gracillis Δ8 desaturase gene
—
252 (422 AA)


(non-functional; Wallis et al. [Archives


of Biochem. Biophys., 365: 307-316


(1999)]; WO 00/34439)


Synthetic Δ8 desaturase gene, codon-
209 (1270 bp)
—


optimized for expression in Yarrowia


lipolytica (D8S-1)


Synthetic Δ8 desaturase gene, codon-
255 (1269 bp)
—


optimized for expression in Yarrowia


lipolytica (D8S-3)


Euglena gracillis Δ8 desaturase gene
 44 (1271 bp)
 45 (421 AA)


(Eg5)


Euglena gracillis Δ8 desaturase gene
 46 (1271 bp)
 47 (421 AA)


(Eg12)


Synthetic Δ8 desaturase gene, codon-
 48 (1272 bp)
 49 (422 AA)


optimized for expression in Yarrowia


lipolytica (D8SF)


Rattus norvegicus C16/18 elongase
 50 (2628 bp)
 51 (267 AA)


Synthetic C16/18 elongase gene derived
 52 (804 bp)
 51 (267 AA)


from Rattus norvegicus, codon-


optimized for expression in Yarrowia


lipolytica


Mortierella alpina C16/18 elongase
 53 (828 bp)
 54 (275 AA)


(ELO3)


Mortierella alpina ELO3-partial cDNA
 55 (607 bp)
—


sequence


Mortierella alpina ELO3-3′ sequence
 56 (1042 bp)
—


obtained by genome walking


Mortierella alpina ELO3-5′ sequence
 57 (2223 bp)
—


obtained by genome walking


Mortierella alpina ELO3-cDNA contig
 58 (3557 bp)
—


Mortierella alpina ELO3-intron
 59 (542 bp)
—


Mortierella alpina ELO3-genomic
 60 (4099 bp)
—


contig


Yarrowia lipolytica C16/18 elongase gene
 61 (915 bp)
 62 (304 AA)


Candida albicans probable fatty acid
—
 63 (353 AA)


elongase (GenBank Accession No.


EAL04510)


Yarrowia lipolytica C14/16 elongase gene
 64 (978 bp)
 65 (325 AA)


Neurospora crassa FEN1 gene
—
 66 (337 AA)


(GenBank Accession No. CAD70918)


Mortierella alpina lysophosphatidic
 67 (945 bp)
 68 (314 AA)


acid acyltransferase (LPAAT1)


Mortierella alpina lysophosphatidic
 69 (927 bp)
 70 (308 AA)


acid acyltransferase (LPAAT2)


Yarrowia lipolytica lysophosphatidic
 71 (1549 bp)
 72 (282 AA)


acid acyltransferase (LPAAT1)


Yarrowia lipolytica lysophosphatidic
 73 (1495 bp)
—


acid acyltransferase (LPAAT2)-genomic


fragment comprising gene


Yarrowia lipolytica lysophosphatidic
 74 (672 bp)
 75 (223 AA)


acid acyltransferase (LPAAT2)


Yarrowia lipolytica
 76 (2326 bp)
 77 (648 AA)


phospholipid:diacylglycerol


acyltransferase (PDAT)


Yarrowia lipolytica acyl-CoA:sterol-
 78 (1632 bp)
 79 (543 AA)


acyltransferase (ARE2)


Caenorhabditis elegans acyl-CoA:1-
—
 80 (282 AA)


acyl lysophosphatidylcholine


acyltransferase (LPCAT)


Yarrowia lipolytica diacylglycerol
 81 (1578 bp)
 82 (526 AA)


acyltransferase (DGAT1)


Mortierella alpina diacylglycerol
 83 (1578 bp)
 84 (525 AA)


acyltransferase (DGAT1)


Neurospora crassa diacylglycerol
—
 85 (533 AA)


acyltransferase (DGAT1)


Gibberella zeae PH-1 diacylglycerol
—
 86 (499 AA)


acyltransferase (DGAT1)


Magnaporthe grisea diacylglycerol
—
 87 (503 AA)


acyltransferase (DGAT1)


Aspergillus nidulans diacylglycerol
—
 88 (458 AA)


acyltransferase (DGAT1)


Yarrowia lipolytica diacylglycerol
 89 (2119 bp)
 90 (514 AA)


acyltransferase (DGAT2)
 91 (1380 bp)
 92 (459 AA)



 93 (1068 bp)
 94 (355 AA)


Mortierella alpina diacylglycerol
 95 (996 bp)
 96 (331 AA)


acyltransferase (DGAT2)


Mortierella alpina glycerol-3-phosphate
 97 (2151 bp)
 98 (716 AA)


acyltransferase (GPAT)


M. alpina GPAT-partial cDNA
 99 (1212 bp)
—


sequence


M. alpina GPAT-genomic fragment
100 (3935 bp)
—


comprising - 1050 bp to + 2886 bp


region


M. alpina GPAT-3′ cDNA sequence
101 (965 bp)
—


obtained by genome walking


M. alpina GPAT-5′ sequence obtained
102 (1908 bp)
—


by genome walking


M. alpina GPAT-internal sequence
103 (966 bp)
—


obtained by genome walking


M. alpina GPAT-intron #1
104 (275 bp)
—


M. alpina GPAT-intron #2
105 (255 bp)
—


M. alpina GPAT-intron #3
106 (83 bp)
—


M. alpina GPAT-intron #4
107 (99 bp)
—


Yarrowia lipolytica diacylglycerol
108 (2133 bp)
—


cholinephosphotransferase (CPT1)-


genomic fragment comprising gene


Yarrowia lipolytica diacylglycerol
109 (1185 bp)
110 (394 AA)


cholinephosphotransferase (CPT1)


Saccharomyces cerevisiae inositol
111 (1434 bp)
112 (477 AA)


phosphosphingolipid-specific


phospholipase C (ISC1)


Yarrowia lipolytica glyceraldehyde-3-
158 (971 bp)
—


phosphate dehydrogenase promoter


(GPD)


Yarrowia lipolytica glyceraldehyde-3-
159 (1174 bp)
—


phosphate dehydrogenase + intron


promoter (GPDIN)


Yarrowia lipolytica phosphoglycerate
160 (878 bp)
—


mutase promoter (GPM)


Yarrowia lipolytica fructose-
161 (1001 bp)
—


bisphosphate aldolase promoter (FBA)


Yarrowia lipolytica fructose-
162 (973 bp)
—


bisphosphate aldolase + intron promoter


(FBAIN)


Yarrowia lipolytica fructose-
163 (924 bp)
—


bisphosphate aldolase + modified intron


promoter (FBAINm)


Yarrowia lipolytica glycerol-3-
164 (1130 bp)
—


phosphate acyltransferase promoter


(GPAT)


Yarrowia lipolytica ammonium
165 (778 bp)
—


transporter promoter (YAT1)


Yarrowia lipolytica translation
166 (436 bp)
—


elongation factor EF1-α promoter


(TEF)


Yarrowia lipolytica chimeric
167 (1020 bp)
—


GPM::FBA intron promoter


(GPM::FBAIN)


Yarrowia lipolytica chimeric
168 (1052 bp)
—


GPM::GPD intron promoter


(GPM::GPDIN)


Yarrowia lipolytica export protein
364 (1000 bp)
—


promoter (EXP1)









SEQ ID NOs:113-157 are plasmids as identified in Table 2.







TABLE 2







Summary of Plasmid SEQ ID Numbers











Plasmid
Corresponding FIG.
SEQ ID NO







pY5-30
 5A
113 (8,953 bp)



pKUNF12T6E
 7B
114 (12,649 bp)



pDMW232
 7C
115 (10,945 bp)



pDMW271
 7D
116 (13,034 bp)



pKUNT2
 8A
117 (6,457 bp)



pZUF17
 8B
118 (8,165 bp)



pDMW237
 8C
119 (7,879 bp)



pY54PC
—
120 (8,502 bp)



pKUNFmkF2
11A
121 (7,145 bp)



pZF5T-PPC
11C
122 (5,553 bp)



pDMW297
11E
123 (10,448 bp)



pZP2C16M899
12A
124 (15,543 bp)



pDMW314
12B
125 (13,295 bp)



pDMW322
12C
126 (11,435 bp)



pZKSL5598
12D
127 (16,325 bp)



pZP3L37
13A
128 (12,690 bp)



pY37/F15
13B
129 (8,194 bp)



pKO2UF2PE
13C
130 (10,838 bp)



pZKUT16
13D
131 (5,833 bp)



pKO2UM25E
14A
132 (12,663 bp)



pZKUGPI5S
14B
133 (6,912 bp)



pDMW302T16
14C
134 (14,864 bp)



pZKUGPE1S
14D
135 (6,540 bp)



pKO2UM26E
15A
136 (13,321 bp)



pZKUM
—
137 (4,313 bp)



pMLPAT-17
—
138 (8,015 bp)



pMLPAT-Int
—
139 (8,411 bp)



pZUF-MOD-1
15B
140 (7,323 bp)



pMDGAT1-17
15C
141 (8,666 bp)



pMDGAT2-17
—
142 (8,084 bp)



pMGPAT-17
15D
143 (9,239 bp)



pZF5T-PPC-E3
—
144 (5,031 bp)



pZUF6S
18A
145 (8,462 bp)



pZUF6S-E3WT
18B
146 (11,046 bp)



pZKUGPYE1-N
18C
147 (6,561 bp)



pZKUGPYE2
18D
148 (6,498 bp)



pZUF6TYE2
—
149 (10,195 bp)



pZKUGPYE1
19A
150 (6,561 bp)



pZUF6FYE1
19B
151 (10,809 bp)



pYCPT1-17
—
152 (8,273 bp)



pZP2I7 + Ura
19C
153 (7,822 bp)



pYCPT1-ZP2I7
—
154 (7,930 bp)



pTEF::ISC1
—
155 (8,179 bp)



pY20
19D
156 (8,196 bp)



pLV13
19E
157 (5,105 bp)










SEQ ID NO:356 corresponds to the codon-optimized translation initiation site for genes optimally expressed in Yarrowia sp.
SEQ ID NOs:169-182 correspond to primers YL211, YL212, YL376, YL377, YL203, YL204, GPAT-5-1, GPAT-5-2, ODMW314, YL341, ODMW320, ODMW341, 27203-F and 27203-R, respectively, used to amplify Yarrowia lipolytica promoter regions.
SEQ ID NOs:183-186 are the oligonucleotides YL-URA-16F, YL-URA-78R, GUS-767F and GUS-891R, respectively, used for Real Time analysis.
SEQ ID NOs:187-202 correspond to 8 pairs of oligonucleotides which together comprise the entire codon-optimized coding region of the /. galbana Δ9 elongase (i.e., IL3-1A, IL3-1B, IL3-2A, IL3-2B, IL3-3A, IL3-3B, IL3-4A, IL3-4B, IL3-5A, IL3-5B, IL3-6A, IL3-6B, IL3-7A, IL3-7B, IL3-8A and IL3-8B, respectively).
SEQ ID NOs:203-206 correspond to primers IL3-1F, IL3-4R, IL3-5F and IL3-8R, respectively, used for PCR amplification during synthesis of the codon-optimized Δ9 elongase gene.
SEQ ID NO:207 is the 417 bp NcoI/PstI fragment described in pT9(1-4); and SEQ ID NO:208 is the 377 bp PstI/Not1 fragment described in pT9(5-8).
SEQ ID NOs:210-235 correspond to 13 pairs of oligonucleotides which together comprise the entire codon-optimized coding region of the E. gracilis Δ8 desaturase (i.e., D8-1A, D8-1B, D8-2A, D8-2B, D8-3A, D8-3B, D8-4A, D8-4B, D8-5A, D8-5B, D8-6A, D8-6B, D8-7A, D8-7B, D8-8A, D8-8B, D8-9A, D8-9B, D8-10A, D8-10B, D8-11A, D8-11B, D8-12A, D8-12B, D8-13A and D8-13B, respectively).
SEQ ID NOs:236-243 correspond to primers D8-1F, D8-3R, D8-4F, D8-6R, D8-7F, D8-9R, D8-10F and D8-13R, respectively, used for PCR amplification during synthesis of the codon-optimized Δ8 desaturase gene.
SEQ ID NO:244 is the 309 bp Nco/BglII fragment described in pT8(1-3); SEQ ID NO:245 is the 321 bp BglII/XhoI fragment described in pT8(4-6); SEQ ID NO:246 is the 264 bp XhoI/SacI fragment described in pT8(7-9); and SEQ ID NO:247 is the 369 bp Sac1/Not1 fragment described in pT8(10-13).
SEQ ID NOs:248 and 249 correspond to primers ODMW390 and ODMW391, respectively, used during synthesis of D8S-2 in pDMW255.
SEQ ID NOs:250 and 251 are the chimeric D8S-1::XPR and D8S-2::XPR genes described in Example 7.
SEQ ID NOs:253 and 254 correspond to primers ODMW392 and ODMW393, used during synthesis of D8S-3.
SEQ ID NOs:256 and 257 correspond to primers Eg5-1 and Eg3-3, respectively, used for amplification of the Δ8 desaturase from Euglena gracilis. 
SEQ ID NOs:258-261 correspond to primers T7, M13-28Rev, Eg3-2 and Eg5-2, respectively, used for sequencing a Δ8 desaturase clone.
SEQ ID NO:262 corresponds to primer ODMW404, used for amplification of D8S-3.
SEQ ID NO:263 is a 1272 bp chimeric gene comprising D8S-3.
SEQ ID NOs:264 and 265 correspond to primers YL521 and YL522, respectively, used to create new restriction enzyme sites in a cloned D8S-3 gene.
SEQ ID NOs:266-279 correspond to primers YL525, YL526, YL527, YL528, YL529, YL530, YL531, YL532, YL533, YL534, YL535, YL536, YL537 and YL538, respectively, used in site directed mutagenesis reactions to produce D8SF.
SEQ ID NO:280 is a mutant AHAS gene comprising a W497L mutation.
SEQ ID NOs:281-283 correspond to BD-Clontech Creator Smart®cDNA library kit primers SMART IV oligonucleotide, CDSIII/3′ PCR primer and 5′-PCR primer, respectively.
SEQ ID NO:284 corresponds to the M13 forward primer used for M. alpina cDNA library sequencing.
SEQ ID NOs:285-288 and 290-291 correspond to primers MLPAT-F, MLPAT-R, LPAT-Re-5-1, LPAT-Re-5-2, LPAT-Re-3-1 and LPAT-Re-3-2, respectively, used for cloning of the M. alpina LPAAT2 ORF.
SEQ ID NOs:289 and 292 correspond to a 5′ (1129 bp) and 3′ (938 bp) region of the Y. lipolytica LPMT1 ORF, respectively.
SEQ ID NOs:293 and 294 correspond to primers pzuf-mod1 and pzuf-mod2, respectively, used for creating “control” plasmid pZUF-MOD-1.
SEQ ID NOs:295 and 296 correspond to primers MACAT-F1 and MACAT-R, respectively, used for cloning of the M. alpina DGAT1 ORF.
SEQ ID NOs:297 and 298 correspond to primers MDGAT-F and MDGAT-R1, respectively, used for cloning of the M. alpina DGAT2 ORF.
SEQ ID NOs:299 and 300 correspond to primers MGPAT-N1 and MGPAT-NR5, respectively, used for degenerate PCR to amplify the M. alpina GPAT.
SEQ ID NOs:301-303 correspond to primers MGPAT-5N1, MGPAT-5N2 and MGPAT-5N3, respectively, used for amplification of the 3′-end of the M. alpina GPAT.
SEQ ID NOs:304 and 305 correspond to the Genome Walker adaptor from ClonTech's Universal GenomeWalkerTM Kit, used for genome-walking.
SEQ ID NOs:306-309 correspond to the PCR primers used in genome-walking: MGPAT-5-1A, Adaptor-1 (AP1), MGPAT-3N1 and Nested Adaptor Primer 2 (AP2), respectively.
SEQ ID NOs:310 and 311 correspond to primers mgpat-cdna-5 and mgpat-cdna-R, respectively, used for amplifying the M. alpina GPAT.
SEQ ID NOs:312 and 313 correspond to primers MA Elong 3′1 and MA elong 3′2, respectively, used for genome-walking to isolate the 3′-end region of the M. alpina ELO3.
SEQ ID NOs:314 and 315 correspond to primers MA Elong 5′1 and MA Elong 5′2, respectively, used for genome-walking to isolate the 5′-end region of the M. alpina ELO3.
SEQ ID NOs:316 and 317 correspond to primers MA ELONG 5′ NcoI 3 and MA ELONG 3′ NotI 1, respectively, used for amplifying the complete ELO3 from M. alpina cDNA.
SEQ ID NOs:318 and 319 correspond to primers YL597 and YL598, respectively, used for amplifying the coding region of Y. lipolytica YE2.
SEQ ID NOs:320-323 correspond to primers YL567, YL568, YL569 and YL570, respectively, used for amplifying the coding region of Y. lipolytica YE1.
SEQ ID NOs:324 and 325 correspond to primers YL571 and YL572, respectively, used for site-directed mutagenesis during cloning of Y. lipolytica YE1.
SEQ ID NOs:326 and 327 correspond to primers CPT1-5′-NcoI and CPT1-3′-NotI, respectively, used for cloning of the Y. lipolytica CPT1 ORF.
SEQ ID NOs: 328 and 329 correspond to primers Isc1F and Isc1R, respectively, used for cloning of the S. cerevisiae ISC1 ORF.
SEQ ID NOs:330 and 331 correspond to primers Pcl1F and Pcl1R, respectively, used for cloning of the S. cerevisiae PCL1 ORF.
SEQ ID NOs:332-335 correspond to primers P95, P96, P97 and P98, respectively, used for targeted disruption of the Y. lipolytica DGAT2 gene.
SEQ ID NOs:336-338 correspond to primers P115, P116 and P112, respectively, used to screen for targeted integration of the disrupted Y. lipolytica DGAT2 gene.
SEQ ID NOs:339-342 correspond to primers P39, P41, P40 and P42, respectively, used for targeted disruption of the Y. lipolytica PDAT gene.
SEQ ID NOs:343-346 correspond to primers P51, P52, P37 and P38, respectively, used to screen for targeted integration of the disrupted Y. lipolytica PDAT gene.
SEQ ID NOs:347 and 348 are the degenerate primers identified as P201 and P203, respectively, used for the isolation of the Y. lipolytica DGAT1.
SEQ ID NOs:349-353 correspond to primers P214, P215, P216, P217 and P219, respectively, used for the creation of a targeting cassette for targeted disruption of the putative DGAT1 gene in Y. lipolytica. 
SEQ ID NOs:354 and 355 correspond to primers P226 and P227, respectively, used to screen for targeted integration of the disrupted Y. lipolytica DGAT1 gene.
SEQ ID NOs:365-370 correspond to primers 410, 411, 412, 413, 414 and 415, respectively, used for synthesis of a mutant Yarrowia lipolytica AHAS gene, comprising a W497L mutation.
SEQ ID NOs:371 and 372 correspond to primers YL325 and YL326, respectively, used to amplify a NotI/PacI fragment containing the Aco 3′ terminator.
SEQ ID NO:373 corresponds to a His Box 1 motif found in fungal Δ15 and Δ12 desaturases.
SEQ ID NO:374 corresponds to a motif that is indicative of a fungal protein having Δ15 desaturase activity, while SEQ ID NO:375 corresponds to a motif that is indicative of a fungal protein having Δ12 desaturase activity.
DETAILED DESCRIPTION OF THE INVENTION
All patents, patent applications, and publications cited herein are incorporated by reference in their entirety. This specifically includes, the following Applicants' Assignee's copending applications:
U.S. patent application Ser. No. 10/840,478 (filed May 6, 2004),
U.S. patent application Ser. No. 10/840,579 (filed May 6, 2004),
U.S. patent application Ser. No. 10/840,325 (filed May 6, 2004)
U.S. patent application Ser. No. 10/869,630 (filed Jun. 16, 2004),
U.S. patent application Ser. No. 10/882,760 (filed Jul. 1, 2004),
U.S. patent application Ser. No. 10/985,109 (filed Nov. 10, 2004),
U.S. patent application Ser. No. 10/987,548 (filed Nov. 12, 2004)
U.S. patent application Ser. No. 60/624,812 (filed Nov. 4, 2004), U.S. patent applications Ser. No. 11/024,545 and No. 11/024,544 (filed Dec. 29, 2004),
U.S. patent application Ser. No. 60/689,031 (filed Jun. 9, 2005),
U.S. patent application Ser. No. 11/183,664 (filed Jul. 18, 2005),
U.S. patent application Ser. No. 11/185,301 (filed Jul. 20, 2005),
U.S. patent application Ser. No. 11/190,750 (filed Jul. 27, 2005),
U.S. patent application Ser. No. 11/225,354 (filed Sep. 13, 2005).
In accordance with the subject invention, Applicants provide production host strains of Yarrowia lipolytica that are capable of producing greater than 10% arachidonic acid (ARA, 20:4, ω-6). Accumulation of this particular polyunsaturated fatty acid (PUFA) is accomplished by introduction of either of two different functional ω-3/ω-6 fatty acid biosynthetic pathways. The first pathway comprises proteins with Δ6 desaturase, C18/20 elongase and Δ5 desaturase activities into the oleaginous yeast host for high-level recombinant expression, wherein the ARA oil also comprises GLA; the latter pathway comprises proteins with Δ9 elongase, Δ8 desaturase and Δ5 desaturase activities and thereby enables production of an ARA oil that is devoid of any GLA. Thus, this disclosure demonstrates that Y. lipolytica can be engineered to enable commercial production of ARA and derivatives thereof. Methods of production are also claimed.
The subject invention finds many applications. PUFAs, or derivatives thereof, made by the methodology disclosed herein can be used as dietary substitutes, or supplements, particularly infant formulas, for patients undergoing intravenous feeding or for preventing or treating malnutrition. Alternatively, the purified PUFAs (or derivatives thereof) may be incorporated into cooking oils, fats or margarines formulated so that in normal use the recipient would receive the desired amount for dietary supplementation. The PUFAs may also be incorporated into infant formulas, nutritional supplements or other food products and may find use as anti-inflammatory or cholesterol lowering agents. Optionally, the compositions may be used for pharmaceutical use (human or veterinary). In this case, the PUFAs are generally administered orally but can be administered by any route by which they may be successfully absorbed, e.g., parenterally (e.g., subcutaneously, intramuscularly or intravenously), rectally, vaginally or topically (e.g., as a skin ointment or lotion).
Supplementation of humans or animals with PUFAs produced by recombinant means can result in increased levels of the added PUFAs, as well as their metabolic progeny. For example, treatment with ARA can result not only in increased levels of ARA, but also downstream products of ARA such as eicosanoids. Complex regulatory mechanisms can make it desirable to combine various PUFAs, or add different conjugates of PUFAs, in order to prevent, control or overcome such mechanisms to achieve the desired levels of specific PUFAs in an individual.
In alternate embodiments, PUFAs, or derivatives thereof, made by the methodology disclosed herein can be utilized in the synthesis of aquaculture feeds (i.e., dry feeds, semi-moist and wet feeds) since these formulations generally require at least 1-2% of the nutrient composition to be ω-3 and/or ω-6 PUFAs.
Definitions
In this disclosure, a number of terms and abbreviations are used. The following definitions are provided.
“Open reading frame” is abbreviated ORF.
“Polymerase chain reaction” is abbreviated PCR.
“American Type Culture Collection” is abbreviated ATCC.
“Polyunsaturated fatty acid(s)” is abbreviated PUFA(s).
“Diacylglycerol acyltransferase” is abbreviated DAG AT or DGAT.
“Phospholipid:diacylglycerol acyltransferase” is abbreviated PDAT.
“Glycerol-3-phosphate acyltransferase” is abbreviated GPAT.
“Lysophosphatidic acid acyltransferase” is abbreviated LPAAT.
“Acyl-CoA:1-acyl lysophosphatidylcholine acyltransferase” is abbreviated “LPCAT”.
“Acyl-CoA:sterol-acyltransferase” is abbreviated ARE2.
“Diacylglycerol” is abbreviated DAG.
“Triacylglycerols” are abbreviated TAGs.
“Co-enzyme A” is abbreviated CoA.
“Phosphatidyl-choline” is abbreviated PC.
The term “Fusarium moniliforme” is synonymous with “Fusarium verticillioides”.
The term “food product” refers to any food generally suitable for human consumption. Typical food products include but are not limited to meat products, cereal products, baked foods, snack foods, dairy products and the like.
The term “functional food” refers to those foods that encompass potentially healthful products including any modified food or ingredient that may provide a health benefit beyond the traditional nutrients it contains. Functional foods can include foods like cereals, breads and beverages which are fortified with vitamins, herbs and nutraceuticals. Functional foods contain a substance that provides health benefits beyond its nutritional value, wherein the substance either is naturally present in the food or is deliberately added.
As used herein the term “medical food” refers to a food which is formulated to be consumed or administered enterally under the supervision of a physician and which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation [see section 5(b) of the Orphan Drug Act (21 U.S.C. 360ee(b)(3))]. A food is a “medical food” only if: (i) It is a specially formulated and processed product (as opposed to a naturally occurring foodstuff used in its natural state) for the partial or exclusive feeding of a patient by means of oral intake or enteral feeding by tube; (ii) It is intended for the dietary management of a patient who, because of therapeutic or chronic medical needs, has limited or impaired capacity to ingest, digest, absorb, or metabolize ordinary foodstuffs or certain nutrients, or who has other special medically determined nutrient requirements, the dietary management of which cannot be achieved by the modification of the normal diet alone; (iii) It provides nutritional support specifically modified for the management of the unique nutrient needs that result from the specific disease or condition, as determined by medical evaluation; (iv) It is intended to be used under medical supervision; and (v) It is intended only for a patient receiving active and ongoing medical supervision wherein the patient requires medical care on a recurring basis for, among other things, instructions on the use of the medical food. Thus, unlike dietary supplements or conventional foods, a medical food that is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements have been established, may bear scientifically valid claims relating to providing distinctive nutritional support for a specific disease or condition. Medical foods are distinguished from the broader category of foods for special dietary use (e.g., hypoallergenic foods) and from foods that make health claims (e.g., dietary supplements) by the requirement that medical foods be used under medical supervision.
The term “medical nutritional” is a medical food as defined herein typically refers to a fortified beverage that is specifically designed for special dietary needs. The medical nutritional generally comprises a dietary composition focused at a specific medical or dietary condition. Examples of commercial medical nuturitionals include, but are not limited to Ensure® and Boost®.
The term “pharmaceutical” as used herein means a compound or substance which if sold in the United States would be controlled by Section 505 or 505 of the Federal Food, Drug and Cosmetic Act.
The term “infant formula” means a food which is designed exclusively for consumption by the human infant by reason of its simulation of human breast milk. Typical commercial examples of infant formula include bur are not limited to Similac®, and Isomil®.
The term “dietary supplement” refers to a product that: (i) is intended to supplement the diet and thus is not represented for use as a conventional food or as a sole item of a meal or the diet; (ii) contains one or more dietary ingredients (including, e.g., vitamins, minerals, herbs or other botanicals, amino acids, enzymes and glandulars) or their constituents; (iii) is intended to be taken by mouth as a pill, capsule, tablet, or liquid; and (iv) is labeled as being a dietary supplement.
A “food analog” is a food-like product manufactured to resemble its food counterpart, whether meat, cheese, milk or the like, and is intended to have the appearance, taste, and texture of its counterpart. Thus, the term “food” as used herein also encompasses food analogs.
The terms “aquaculture feed” and “aquafeed” refer to manufactured or artificial diets (formulated feeds) to supplement or to replace natural feeds in the aquaculture industry. Thus, an aquafeed refers to artificially compounded feeds that are useful for farmed finfish and crustaceans (i.e., both lower-value staple food fish species [e.g., freshwater finfish such as carp, tilapia and catfish] and higher-value cash crop species for luxury or niche markets [e.g., mainly marine and diadromous species such as shrimp, salmon, trout, yellowtail, seabass, seabream and grouper]). These formulate feeds are composed of several ingredients in various proportions complementing each other to form a nutritionally complete diet for the aquacultured species.
The term “animal feed” refers to feeds intended exclusively for consumption by animals, including domestic animals (pets, farm animals etc.) or for animals raised for the production of food e.g. fish farming.
The term “feed nutrient” means nutrients such as proteins, lipids, carbohydrates, vitamins, minerals, and nucleic acids that may be derived from the yeast biomass comprising the recombinant production hosts of the invention.
As used herein the term “biomass” refers specifically to spent or used yeast cellular material from the fermentation of a recombinant production host production EPA in commercially significant amounts.
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” (or “PUFAs”), and “omega-6 fatty acids” (ω-6 or n-6) versus “omega-3 fatty acids” (ω-3 or n-3) are provided in WO2004/101757.
Nomenclature used to describe PUFAs in the present disclosure is shown below in Table 3. 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 specification and each compounds' chemical name.







TABLE 3







Nomenclature of Polyunsaturated Fatty Acids And Precursors













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-Linoleic
GLA
cis-6,9,12-
18:3 ω-6




octadecatrienoic


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


Dihomo-γ-
DGLA
cis-8,11,14-
20:3 ω-6


Linoleic

eicosatrienoic


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




eicosatetraenoic


α-Linolenic
ALA
cis-9,12,15-
18:3 ω-3




octadecatrienoic


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




octadecatetraenoic


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




eicosatrienoic


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




eicosatetraenoic


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




eicosapentaenoic


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




docosapentaenoic


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




docosahexaenoic









The term “high-level ARA production” refers to production of at least about 5% ARA in the total lipids of the microbial host, preferably at least about 10% ARA in the total lipids, more preferably at least about 15% ARA in the total lipids, more preferably at least about 20% ARA in the total lipids and most preferably at least about 25-30% ARA in the total lipids. The structural form of the ARA is not limiting; thus, for example, the ARA may exist in the total lipids as free fatty acids or in esterified forms such as acylglycerols, phospholipids, sulfolipids or glycolipids.
The term “devoid of any GLA” refers to lack of any detectable GLA in the total lipids of the microbial host, when measured by GC analysis using equipment having a detectable level down to about 0.1%.
The term “essential fatty acid” refers to a particular PUFA that an organism must ingest in order to survive, being unable to synthesize the particular essential fatty acid de novo. For example, mammals can not synthesize the essential fatty acids LA (18:2, ω-6) and ALA (18:3, ω-3). Other essential fatty acids include GLA (ω-6), DGLA (ω-6), ARA (ω-6), EPA (ω-3) and DHA (ω-3).
“Microbial oils” or “single cell oils” are those oils naturally produced by microorganisms (e.g., algae, oleaginous yeasts and filamentous fungi) during their lifespan. The term “oil” refers to a lipid substance that is liquid at 25° C. and usually polyunsaturated. In contrast, the term “fat” refers to a lipid substance that is solid at 25° C. and usually saturated.
“Lipid bodies” refer to lipid droplets that usually are bounded by specific proteins and a monolayer of phospholipid. These organelles are sites where most organisms transport/store neutral lipids. Lipid bodies are thought to arise from microdomains of the endoplasmic reticulum that contain TAG-biosynthesis enzymes; and, their synthesis and size appear to be controlled by specific protein components.
“Neutral lipids” refer to those lipids commonly found in cells in lipid bodies as storage fats and oils 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, diacylglycerol or TAG, respectively (or collectively, acylglycerols). A hydolysis reaction must occur to release free fatty acids from acylglycerols.
The terms “triacylglycerol”, “oil” and “TAGs” refer to neutral lipids composed of three fatty acyl residues esterified to a glycerol molecule (and such terms will be used interchangeably throughout the present disclosure herein). Such oils can contain long chain PUFAs, as well as shorter saturated and unsaturated fatty acids and longer chain saturated fatty acids. Thus, “oil biosynthesis” generically refers to the synthesis of TAGs in the cell.
The term “acyltransferase” refers to an enzyme responsible for transferring a group other than an amino-acyl group (EC 2.3.1.-).
The term “DAG AT” refers to a diacylglycerol acyltransferase (also known as an acyl-CoA-diacylglycerol acyltransferase or a diacylglycerol O-acyltransferase) (EC 2.3.1.20). This enzyme is responsible for the conversion of acyl-CoA and 1,2-diacylglycerol to TAG and CoA (thereby involved in the terminal step of TAG biosynthesis). Two families of DAG AT enzymes exist: DGAT1 and DGAT2. The former family shares homology with the acyl-CoA:cholesterol acyltransferase (ACAT) gene family, while the latter family is unrelated (Lardizabal et al., J. Biol. Chem. 276(42):38862-38869 (2001)).
The term “PDAT” refers to a phospholipid:diacylglycerol acyltransferase enzyme (EC 2.3.1.158). This enzyme is responsible for the transfer of an acyl group from the sn-2 position of a phospholipid to the sn-3 position of 1,2-diacylglycerol, thus resulting in lysophospholipid and TAG (thereby involved in the terminal step of TAG biosynthesis). This enzyme differs from DGAT (EC 2.3.1.20) by synthesizing TAG via an acyl-CoA-independent mechanism.
The term “ARE2” refers to an acyl-CoA:sterol-acyltransferase enzyme (EC 2.3.1.26; also known as a sterol-ester synthase 2 enzyme), catalyzing the following reaction: acyl-CoA+sterol=CoA+sterol ester.
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 “LPAAT” refers to a lysophosphatidic acid-acyltransferase enzyme (EC 2.3.1.51). This enzyme is responsible for the transfer of an acyl-CoA group onto 1-acyl-sn-glycerol 3-phosphate (i.e., lysophosphatidic acid) to produce CoA and 1,2-diacyl-sn-glycerol 3-phosphate (phosphatidic acid). The literature also refers to LPAAT as acyl-CoA:1-acyl-sn-glycerol-3-phosphate 2-O-acyltransferase, 1-acyl-sn-glycerol-3-phosphate acyltransferase and/or 1-acylglycerolphosphate acyltransferase (abbreviated as AGAT).
The term “LPCAT” refers to an acyl-CoA:1-acyl lysophosphatidyl-choline acyltransferase. This enzyme is responsible for the exchange of acyl groups between CoA and phosphatidyl choline (PC). Herein it also refers to enzymes involved the acyl exchange between CoA and other phospholipids, including lysophosphatidic acid (LPA).
“Percent (%) PUFAs in the total lipid and oil fractions” refers to the percent of PUFAs relative to the total fatty acids in those fractions. The term “total lipid fraction” or “lipid fraction” both refer to the sum of all lipids (i.e., neutral and polar) within an oleaginous organism, thus including those lipids that are located in the phosphatidylcholine (PC) fraction, phosphatidyletanolamine (PE) fraction and triacylglycerol (TAG or oil) fraction. However, the terms “lipid” and “oil” will be used interchangeably throughout the specification.
The term “phosphatidylcholine” or “PC” refers to a phospholipid that is a major constituent of cell membranes. The chemical structure of PC can generally be described as comprising the following: a choline molecule, a phosphate group and glycerol, wherein fatty acyl chains are attached as R groups on the sn-1 and sn-2 positions of the glycerol molecule.
The term “PUFA biosynthetic pathway enzyme” refers to any of the following enzymes (and genes which encode said enzymes) associated with the biosynthesis of a PUFA, including: a Δ4 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ12 desaturase, a Δ15 desaturase, a Δ17 desaturase, a Δ9 desaturase, a Δ8 desaturase, a Δ9 elongase, a C14/16 elongase, a C16/18 elongase, a C18/20 elongase and/or a C20/22 elongase.
The term “ω-3/ω-6 fatty acid biosynthetic pathway” refers to a set of genes which, when expressed under the appropriate conditions encode enzymes that catalyze the production of either or both ω-3 and (0-6 fatty acids. Typically the genes involved in the ω-3/ω-6 fatty acid biosynthetic pathway encode some or all of the following enzymes: Δ12 desaturase, Δ6 desaturase, C18/20 elongase, C20/22 elongase, Δ9 elongase, Δ5 desaturase, Δ17 desaturase, Δ15 desaturase, Δ9 desaturase, Δ8 desaturase, and Δ4 desaturase. A representative pathway is illustrated in FIG. 1, providing for the conversion of oleic acid through various intermediates to DHA, which demonstrates how both ω-3 and ω-6 fatty acids may be produced from a common source. The pathway is naturally divided into two portions where one portion will generate ω-3 fatty acids and the other portion, only ω-6 fatty acids. That portion that only generates ω-3 fatty acids will be referred to herein as the ω-3 fatty acid biosynthetic pathway, whereas that portion that generates only ω-6 fatty acids will be referred to herein as the ω-6 fatty acid biosynthetic pathway.
The term “functional” as used herein in context with the ω-3/ω-6 fatty acid biosynthetic pathway means that some (or all of) the genes in the pathway express active enzymes, resulting in in vivo catalysis or substrate conversion. It should be understood that “ω-3/ω-6 fatty acid biosynthetic pathway” or “functional ω-3/ω-6 fatty acid biosynthetic pathway” does not imply that all the genes listed in the above paragraph are required, as a number of fatty acid products will only require the expression of a subset of the genes of this pathway.
The term “Δ6 desaturase/Δ6 elongase pathway” will refer to an ARA fatty acid biosynthetic pathway that minimally includes the following genes: Δ6 desaturase, C18/20 elongase and Δ5 desaturase. In a related manner, the term “Δ9 elongase/Δ8 desaturase pathway” will refer to an ARA fatty acid biosynthetic pathway that minimally includes the following genes: Δ9 elongase, Δ8 desaturase and Δ5 desaturase.
The term “desaturase” refers to a polypeptide that can desaturate, i.e., introduce a double bond, in one or more fatty acids to produce a fatty acid or precursor of interest. Despite use of the omega-reference system throughout the specification 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 substrate using the delta-system. Of particular interest herein are: 1.) Δ8 desaturases that desaturate a fatty acid between the 8th and 9th carbon atom numbered from the carboxyl-terminal end of the molecule and which, for example, catalyze the conversion of EDA to DGLA and/or ETrA to ETA; 2.) Δ6 desaturases that catalyze the conversion of LA to GLA and/or ALA to STA; 3.) Δ5 desaturases that catalyze the conversion of DGLA to ARA and/or ETA to EPA; 4.) Δ4 desaturases that catalyze the conversion of DPA to DHA; 5.) Δ12 desaturases that catalyze the conversion of oleic acid to LA; 6.) Δ15 desaturases that catalyze the conversion of LA to ALA and/or GLA to STA; 7.) Δ17 desaturases that catalyze the conversion of ARA to EPA and/or DGLA to ETA; and 8.) Δ9 desaturases that catalyze the conversion of palmitate to palmitoleic acid (16:1) and/or stearate to oleic acid (18:1).
The term “bifunctional” as it refers to Δ15 desaturases of the invention means that the polypeptide has the ability to use both oleic acid and linoleic acid as an enzymatic substrate. By “enzymatic substrate” it is meant that the polypeptide binds the substrate at an active site and acts upon it in a reactive manner.
The term “elongase system” refers to a suite of four enzymes that are responsible for elongation of a fatty acid carbon chain to produce a fatty acid that is 2 carbons longer than the fatty acid substrate that the elongase system acts upon. More specifically, the process of elongation occurs in association with fatty acid synthase, whereby CoA is the acyl carrier (Lassner et al., The Plant Cell 8:281-292 (1996)). In the first step, which has been found to be both substrate-specific and also rate-limiting, malonyl-CoA is condensed with a long-chain acyl-CoA to yield CO2 and a β-ketoacyl-CoA (where the acyl moiety has been elongated by two carbon atoms). Subsequent reactions include reduction to β-hydroxyacyl-CoA, dehydration to an enoyl-CoA and a second reduction to yield the elongated acyl-CoA. Examples of reactions catalyzed by elongase systems are the conversion of GLA to DGLA, STA to ETA and EPA to DPA.
For the purposes herein, an enzyme catalyzing the first condensation reaction (i.e., conversion of malonyl-CoA to β-ketoacyl-CoA) will be referred to generically as an “elongase”. In general, the substrate selectivity of elongases is somewhat broad but segregated by both chain length and the degree and type of unsaturation. Accordingly, elongases can have different specificities. For example, a C14/16 elongase will utilize a C14 substrate (e.g., myristic acid), a C16/18 elongase will utilize a C16 substrate (e.g., palmitate), a C18/20 elongase will utilize a C18 substrate (e.g., GLA, STA) and a C20/22 elongase will utilize a C20 substrate (e.g., EPA). In like manner, a Δ9 elongase is able to catalyze the conversion of LA and ALA to EDA and ETrA, respectively. It is important to note that some elongases have broad specificity and thus a single enzyme may be capable of catalyzing several elongase reactions (e.g., thereby acting as both a C16/18 elongase and a C18/20 elongase). In preferred embodiments, it is most desirable to empirically determine the specificity of a fatty acid elongase by transforming a suitable host with the gene for the fatty acid elongase and determining its effect on the fatty acid profile of the host.
The term “high affinity elongase” or “EL1 S” or “ELO1” refers to a C18/20 elongase whose substrate specificity is preferably for GLA (with DGLA as a product of the elongase reaction [i.e., a Δ6 elongase]). One such elongase is described in WO 00/12720 and is provided herein as SEQ ID NOs:17 and 18. However, the Applicants have shown that this enzyme also has some activity on 18:2 (LA) and 18:3 (ALA); thus, SEQ ID NO:18 shows Δ9 elongase activity (in addition to its Δ6 elongase activity). It is therefore concluded that the C18/20 elongase provided herein as SEQ ID NO:18 can function both within the Δ6 desaturase/Δ6 elongase pathway as described in the invention herein and within the Δ9 elongase/Δ8 desaturase pathway, as a substitute for e.g., the Isochrysis galbana Δ9 elongase (SEQ ID NO:40).
The term “EL2S” or “ELO2” refers to a C18/20 elongase whose substrate specificity is preferably for GLA (with DGLA as a product of the elongase reaction) and/or STA (with STA as a product of the elongase reaction). One such elongase is described in U.S. Pat. No. 6,677,145 and is provided herein as SEQ ID NOs:20 and 21.
The term “ELO3” refers to a Mortierella alpina C16/18 fatty acid elongase enzyme (provided herein as SEQ ID NO:54), encoded by the elo3 gene (SEQ ID NO:53). The term “YE2” refers to a Yarrowia lipolytica C16/18 fatty acid elongase enzyme (provided herein as SEQ ID NO:62), encoded by the gene provided herein as SEQ ID NO:61. Based on data reported herein, both ELO3 amd YE2 preferentially catalyze the conversion of palmitate (16:0) to stearic acid (18:0).
The term “YE1” refers to a Yarrowia lipolytica C14/16 fatty acid elongase enzyme (provided herein as SEQ ID NO:65), encoded by the gene provided herein as SEQ ID NO:64. Based on data reported herein, YE2 preferentially catalyzes the conversion of myristic acid (14:0) to palmitate (16:0).
The terms “conversion efficiency” and “percent substrate conversion” refer to the efficiency by which a particular enzyme (e.g., a desaturase or elongase) can convert substrate to product. The conversion efficiency is measured 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 term “oleaginous” refers to those organisms that tend to store their energy source in the form of lipid (Weete, In: Fungal Lipid Biochemistry, 2nd Ed., Plenum, 1980). Generally, the cellular oil content of these 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)).
The term “oleaginous yeast” refers to those microorganisms classified as yeasts that can make oil. Generally, the cellular oil or triacylglycerol 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. 
The term “fermentable carbon source” means a carbon source that a microorganism will metabolize to derive energy. Typical carbon sources of the invention include, but are not limited to: monosaccharides, oligosaccharides, polysaccharides, alkanes, fatty acids, esters of fatty acids, monoglycerides, diglycerides, triglycerides, carbon dioxide, methanol, formaldehyde, formate and carbon-containing amines.
As used herein, an “isolated nucleic acid fragment” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
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 (e.g., 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 fragment 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 fragment comprising the sequence.
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.
“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.
“Chemically synthesized”, as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well-established procedures; or, automated chemical synthesis can be performed using one of a number of commercially available machines. “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.
“Gene” refers to a nucleic acid fragment that expresses a specific protein, and that 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. “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, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.
“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 “GPAT promoter” or “GPAT promoter region” refers to the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of a glycerol-3-phosphate O-acyltransferase enzyme (E.C. 2.3.1.15) encoded by the gpat gene and that is necessary for expression. Examples of suitable Yarrowia lipolytica GPAT promoter regions are described in U.S. patent application Ser. No. 11/225,354.
The term “GPD promoter” or “GPD promoter region” refers to the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of a glyceraldehyde-3-phosphate dehydrogenase enzyme (E.C. 1.2.1.12) encoded by the gpd gene and that is necessary for expression. Examples of suitable Yarrowia lipolytica GPD promoter regions are described in WO 2005/003310.
The term “GPM promoter” or “GPM promoter region” refers to the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of a phosphoglycerate mutase enzyme (EC 5.4.2.1) encoded by the gpm gene and that is necessary for expression. Examples of suitable Yarrowia lipolytica GPM promoter regions are described in WO 2005/003310.
The term “FBA promoter” or “FBA promoter region” refers to the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of a fructose-bisphosphate aldolase enzyme (E.C. 4.1.2.13) encoded by the fba1 gene and that is necessary for expression. Examples of suitable Yarrowia lipolytica FBA promoter regions are described in WO 2005/049805.
The term “FBAIN promoter” or “FBAIN promoter region” refers to the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of the fba1 gene and that is necessary for expression, plus a portion of 5′ coding region that has an intron of the fba1 gene. Examples of suitable Yarrowia lipolytica FBAIN promoter regions are described in WO 2005/049805.
The term “GPDIN promoter” or “GPDIN promoter region” refers to the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of the gpd gene and that is necessary for expression, plus a portion of 5′ coding region that has an intron of the gpd gene. Examples of suitable Yarrowia lipolytica GPDIN promoter regions are described in U.S. patent application Ser. No. 11/183,664.
The term “YAT1 promoter” or “YAT1 promoter region” refers to the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of an ammonium transporter enzyme (TC 2.A.49; GenBank Accession No. XM—504457) encoded by the yat1 gene and that is necessary for expression. Examples of suitable Yarrowia lipolytica YAT1 promoter regions are described in U.S. patent application Ser. No. 11/185,301.
The term “EXP1 promoter” or “EXP1 promoter region” refers to the 5′ upstream untranslated region in front of the ‘ATG’ translation initiation codon of a protein encoded by the Yarrowia lipolytica “YALI0C12034g” gene (GenBank Accession No. XM—501745) and that is necessary for expression. Based on significant homology of “YALI0C12034g” to the sp|Q12207 S. cerevisiae non-classical export protein 2 (whose function is involved in a novel pathway of export of proteins that lack a cleavable signal sequence), this gene is herein designated as the exp1 gene, encoding a protein designated as EXP1. An example of a suitable Yarrowia lipolytica EXP1 promoter region is described as SEQ ID NO:364, but this is not intended to be limiting in nature. One skilled in the art will recognize that since the exact boundaries of the EXP1 promoter sequence have not been completely defined, DNA fragments of increased or diminished length may have identical promoter activity.
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.
“Introns” are sequences of non-coding DNA found in gene sequences (either in the coding region, 5′ non-coding region, or 3′ non-coding region) in most eukaryotes. Their full function is not known; however, some enhancers are located in introns (Giacopelli F. et al., Gene Expr. 11: 95-104 (2003)). These intron sequences are transcribed, but removed from within the pre-mRNA transcript before the mRNA is translated into a protein. This process of intron removal occurs by self-splicing of the sequences (exons) on either side of the intron.
The term “enhancer” refers to a cis-regulatory sequence that can elevate levels of transcription from an adjacent eukaryotic promoter, thereby increasing transcription of the gene. Enhancers can act on promoters over many tens of kilobases of DNA and can be 5′ or 3′ to the promoter they regulate. Enhancers can also be located within introns.
The terms “3′non-coding sequences” and “transcription terminator” refer 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; WO 99/28508). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or the coding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that is not translated and yet has an effect on cellular processes.
The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment 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 (i.e., 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 the nucleic acid fragments of the invention. Expression may also refer to translation of mRNA into a polypeptide.
“Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. “Precursor” protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be (but are not limited to) intracellular localization signals.
The term “recombinase” refers to an enzyme(s) that carries out site-specific recombination to alter the DNA structure and includes transposases, lambda integration/excision enzymes, as well as site-specific recombinases.
“Recombinase site” or “site-specific recombinase sequence” means a DNA sequence that a recombinase will recognize and bind to. It will be appreciated that this may be a wild type or mutant recombinase site, as long as functionality is maintained and the recombinase enzyme may still recognize the site, bind to the DNA sequence, and catalyze the recombination between two adjacent recombinase sites.
“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 fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.
The terms “plasmid”, “vector” and “cassette” 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 a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
The term “homologous recombination” refers to the exchange of DNA fragments between two DNA molecules (during cross over). The fragments that are exchanged are flanked by sites of identical nucleotide sequences between the two DNA molecules (i.e., “regions of homology”). The term “regions of homology” refer to stretches of nucleotide sequence on nucleic acid fragments that participate in homologous recombination that have homology to each other. Effective homologous recombination will generally take place where these regions of homology are at least about 10 bp in length where at least about 50 bp in length is preferred. Typically fragments that are intended for recombination contain at least two regions of homology where targeted gene disruption or replacement is desired.
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. Editor(s): Suhai, Sandor. 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.
The term “conserved domain” or “motif” means a set of amino acids conserved at specific positions along an aligned sequence of evolutionarily related proteins. While amino acids at other positions can vary between homologous proteins, amino acids that are highly conserved at specific positions indicate amino acids that are essential in the structure, the stability, or the activity of a protein. Because they are identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers, or “signatures”, to determine if a protein with a newly determined sequence belongs to a previously identified protein family. A motif that is indicative of a fungal protein having Δ15 desaturase activity is provided as SEQ ID NO:374, while a motif that is indicative of a fungal protein having Δ12 desaturase activity is provided as SEQ ID NO:375.
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 (1987).
A Preferred Microbial Host for ARA Production: Yarrowia lipolytica 
Prior to work by the Applicants (see, Picataggio et al., WO2004/101757), oleaginous yeast have not been examined previously as a class of microorganisms suitable for use as a production platform for PUFAs. 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 revkaufl, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R. graminis and Yarrowia lipolytica (formerly classified as Candida lipolytica).
Oleaginous yeast were considered to have several qualities that would faciliate their use as a host organism for economical, commercial production of ARA. First, the organisms are defined as those that are naturally capable of oil synthesis and accumulation, wherein the oil can comprise greater than about 25% of the cellular dry weight, more preferably greater than about 30% of the cellular dry weight and most preferably greater than about 40% of the cellular dry weight. Secondly, 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 (DD259637); lipases (SU1454852, WO2001083773, DD279267); polyhydroxyalkanoates (WO2001088144); citric acid (RU2096461, RU2090611, DD285372, DD285370, DD275480, DD227448, PL160027); erythritol (EP770683); 2-oxoglutaric acid (DD267999); γ-decalactone (U.S. Pat. No. 6,451,565, FR2734843); γ-dodecalactone (EP578388); and pyruvic acid (JP09252790).
Of those organisms classified as oleaginous yeast, Yarrowia lipolytica was selected as the preferred microbial host for the purposes herein. This selection was based on the knowledge that oleaginous strains were available that were capable of incorporating ω-6 and ω-3 fatty acids into the TAG fraction, the organism was amenable to genetic manipulation, and previous use of the species as a Generally Recognized As Safe (“GRAS”, according to the U.S. Food and Drug Administration) source of food-grade citric acid. 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)), due to preliminary studies targeted toward identification of wildtype strains having high lipid content (measured as a percent dry weight) and high volumetric productivity (measured as g/L h−1).
As described in WO 2004/101757, Yarrowia lipolytica was previously genetically engineered to produce 1.3% ARA and 1.9% EPA, respectively, by introduction and expression of genes encoding the ω-3/ω-6 biosynthetic pathway. More specifically, two different DNA expression constructs (comprising either a Δ6 desaturase, Δ5 desaturase and high-affinity PUFA C18/20 elongase for ARA synthesis or a Δ6 desaturase, Δ5 desaturase, high-affinity PUFA C18/20 elongase and codon-optimized Δ17 desaturase for EPA synthesis) were separately transformed and integrated into the Y. lipolytica chromosomal URA3 gene encoding the enzyme orotidine-5′-phosphate decarboxylase (EC 4.1.1.23). GC analysis of the host cells fed with appropriate substrates detected production of ARA and EPA. Although suitable to demonstrate proof-of-concept for the ability of oleaginous hosts to be genetically engineered for production of ω-6 and ω-3 fatty acids, this work failed to perform the complex metabolic engineering required to enable synthesis of greater than 5% ARA in the total oil fraction, or more preferably greater than 10% ARA in the total oil fraction, or even more preferably greater than 15-20% ARA in the total oil fraction, or most preferably greater than 25-30% ARA in the total oil fraction.
In co-pending U.S. Patent Application No. 60/624,812, complex metabolic engineering within Yarrowia lipolytica was performed to: (1) identify preferred desaturases and elongases that allow for the synthesis and high accumulation of EPA; (2) manipulate the activity of acyltransferases that allow for the transfer of omega fatty acids into storage lipid pools; (3) over-express desaturases, elongases and acyltransferases by use of strong promoters, expression in multicopy, and/or codon-optimization; (4) down-regulate the expression of specific genes within the PUFA biosynthetic pathway that diminish overall accumulation of EPA; and, (5) manipulate pathways and global regulators that affect EPA production. This resulted in the production of up to 28% EPA in one particular recombinant strain of Yarrowia lipolytica. 
In the present Application, analogous complex metabolic engineering is performed to result in the production of 10-14% ARA in the total oil fraction in recombinant strains of Yarrowia lipolytica. More specifically, strains Y2034 and Y2047 were genetically engineered to utilize the Δ6 desaturase/Δ6 elongase pathway and produced oil comprising 10% ARA and 11% ARA, respectively; strain Y2214 was genetically engineered to utilize the Δ9 elongase/Δ8 desaturase pathway and produced oil comprising 14% ARA that was devoid of GLA. Aspects of the metabolic engineering utilized will be discussed below, as will additional engineering and fermentation methods that could be performed to further enhance ARA productivity in this oleaginous yeast.
An Overview: Microbial Biosynthesis of Fatty Acids and Triacylglycerols
In general, lipid accumulation in oleaginous microorganisms is triggered in response to the overall carbon to nitrogen ratio present in the growth medium. This process, leading to the de novo synthesis of free palmitate (16:0) in oleaginous microorganisms, is described in detail in WO 2004/101757. Palmitate 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 by a C16/18 fatty acid elongase 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.
TAGs (the primary storage unit for fatty acids) are formed by a series of reactions that involve: 1.) the esterification of one molecule of acyl-CoA to glycerol-3-phosphate via an acyltransferase to produce lysophosphatidic acid; 2.) the esterification of a second molecule of acyl-CoA via an acyltransferase to yield 1,2-diacylglycerol phosphate (commonly identified as phosphatidic acid); 3.) removal of a phosphate by phosphatidic acid phosphatase to yield 1,2-diacylglycerol (DAG); and 4.) the addition of a third fatty acid by the action of an acyltransferase to form TAG (FIG. 2).
A wide spectrum of fatty acids can be incorporated into TAGs, including saturated and unsaturated fatty acids and short-chain and long-chain fatty acids. Some non-limiting examples of fatty acids that can be incorporated into TAGs by acyltransferases include: capric (10:0), lauric (12:0), myristic (14:0), palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1), vaccenic (18:1), LA, eleostearic (18:3), ALA, GLA, arachidic (20:0); EDA, ETrA, DGLA, ETA, ARA, EPA, behenic (22:0), DPA, DHA, lignoceric (24:0), nervonic (24:1), cerotic (26:0), and montanic (28:0) fatty acids. In preferred embodiments of the present invention, incorporation of ARA into TAG is most desirable.
Biosynthesis of ARA, An ω-6 Fatty Acid
The metabolic process wherein oleic acid is converted to ARA involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds. This requires a series of special desaturation and elongation enzymes present in the endoplasmic reticulim membrane. However, as seen in FIG. 1 and as described below, two alternate pathways exist for ARA production.
Specifically, both pathways require the initial conversion of oleic acid to LA (18:2), the first of the ω-6 fatty acids, by the action of a Δ12 desaturase. Then, using the “Δ6 desaturase/Δ6 elongase pathway” for ARA biosynthesis, PUFAs are formed as follows: (1) LA is converted to GLA by the activity of a Δ6 desaturase; (2) GLA is converted to DGLA by the action of a C18/20 elongase; and (3) DGLA is converted to ARA by the action of a Δ5 desaturase.
Alternatively, via the “Δ9 elongase/Δ8 desaturase pathway”, LA is converted to EDA by the action of a Δ9 elongase; then, a Δ8 desaturase converts EDA to DGLA. Subsequent desaturation of DGLA by the action of a Δ5 desaturase yields ARA, as described above.
For the sake of clarity, each of these pathways will be summarized in the Table below, as well as their distinguishing characteristics:







TABLE 4







Alternate Biosynthetic Pathways For ARA Biosynthesis










Minimum




Required Genes


Name
For ARA*
Pathway





Δ6 desaturase/Δ6 elongase
Δ6D, C18/20
—


pathway
ELO, Δ5D


Δ9 elongase/Δ8 desaturase
Δ9 ELO,
produces oil that is


pathway
Δ8D, Δ5D
devoid of GLA


Combination Δ6 desaturase/Δ6
Δ6D, C18/20
—


elongase and Δ9 elongase/Δ8
ELO, Δ9 ELO,


desaturase pathway
Δ8D, Δ5D





*Abbreviations: “D” = desaturase; “ELO” = elongase.






If desirable, several other PUFAs can be produced using ARA as substrate. For example, ARA can be further desaturated to EPA by a Δ17 desaturase and subsequently converted to DHA by the action of a C20/22 elongase and a Δ4 desaturase.
Selection of Microbial Genes for ARA Synthesis
The particular functionalities required to be introduced into Yarrowia lipolytica for production of ARA 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). With respect to the native host cell, it is known that Y. lipolytica can naturally produce 18:2 fatty acids and thus possesses a native Δ12 desaturase (SEQ ID NOs:23 and 24; see WO 2004/104167). With respect to the desired end products, the consequences of Δ6 desaturase/Δ6 elongase pathway expression as opposed to Δ9 elongase/Δ8 desaturase pathway expression have been described above, in terms of the final fatty acid profile of oil so produced (i.e., % GLA in the final composition of high ARA oil).
In some embodiments, it will therefore be desirable to produce ARA via the Δ6 desaturase/Δ6 elongase pathway. Thus, at a minimum, the following genes must be introduced into the host organism and expressed for ARA biosythesis: a Δ6 desaturase, a C18/20 elongase and a Δ5 desaturase. In a further preferred embodiment, the host strain additionally includes at least one of the following: a Δ9 desaturase, a Δ12 desaturase, a C14/16 elongase and a C16/18 elongase.
In alternate embodiments, it is desirable to produce ARA without co-synthesis of GLA (thus requiring expression of the Δ9 elongase/Δ8 desaturase pathway). This strategy thereby minimally requires the following genes to be introduced into the host organism and expressed for ARA biosythesis: a Δ9 elongase, a Δ8 desaturase and a Δ5 desaturase. In a further preferred embodiment, the host strain additionally includes at least one of the following: a Δ9 desaturase, a Δ12 desaturase, a C14/16 elongase and a C16/18 elongase.
One skilled in the art will be able to identify various candidate genes encoding each of the enzymes desired for ARA biosynthesis. Useful desaturase and elongase sequences may be derived from any source, e.g., isolated from a natural source (from bacteria, algae, fungi, plants, animals, etc.), produced via a semi-synthetic route or synthesized de novo. Although the particular source of the desaturase and elongase genes introduced into the host is not critical to the invention, considerations for choosing a specific polypeptide having desaturase or elongase activity include: 1.) the substrate specificity of the polypeptide; 2.) whether the polypeptide or a component thereof is a rate-limiting enzyme; 3.) whether the desaturase or elongase is essential for synthesis of a desired PUFA; and/or 4.) co-factors required by the polypeptide. The expressed polypeptide preferably has parameters compatible with the biochemical environment of its location in the host cell. For example, the polypeptide may have to compete for substrate with other enzymes in the host cell. Analyses of the KM and specific activity of the polypeptide therefore may be considered in determining the suitability of a given polypeptide for modifying PUFA production in a given host cell. The polypeptide used in a particular host cell is one that can function under the biochemical conditions present in the intended host cell but otherwise can be any polypeptide having desaturase or elongase activity capable of modifying the desired PUFA.
In additional embodiments, it will also be useful to consider the conversion efficiency of each particular desaturase and/or elongase. More specifically, since each enzyme rarely functions with 100% efficiency to convert substrate to product, the final lipid profile of un-purified oils produced in a host cell will typically be a mixture of various PUFAs consisting of the desired ARA, as well as various upstream intermediary PUFAs (e.g., as opposed to 100% ARA oil). Thus, consideration of each enzyme's conversion efficiency is also an important variable when optimizing biosynthesis of ARA, that must be considered in light of the final desired lipid profile of the product.
With each of the considerations above in mind, candidate genes having the appropriate desaturase and elongase activities can be identified according to publicly available literature (e.g., GenBank), the patent literature, and experimental analysis of microorganisms having the ability to produce PUFAs. For instance, the following GenBank Accession Numbers refer to examples of publicly available genes useful in ARA biosynthesis: AY131238, Y055118, AY055117, AF296076, AF007561, L11421, NM—031344, AF465283, AF465281, AF110510, AF465282, AF419296, AB052086, AJ250735, AF126799, AF126798 (Δ6 desaturases); AF390174 (Δ9 elongase); AF139720 (Δ8 desaturase); AF199596, AF226273, AF320509, AB072976, AF489588, AJ510244, AF419297, AF07879, AF067654, AB022097 (Δ5 desaturases); MG36933, AF110509, AB020033, ML13300, AF417244, AF161219, AY332747, MG36933, AF110509, AB020033, AAL13300, AF417244, AF161219, X86736, AF240777, AB007640, AB075526, AP002063 (Δ12 desaturases); AF338466, AF438199, E11368, E11367, D83185, U90417, AF085500, AY504633, NM—069854, AF230693 (Δ9 desaturases); and NP—012339, NP—009963, NP—013476, NP—599209, BAB69888, AF244356, AAF70417, AAF71789, AF390174, AF428243, NP—955826, AF206662, AF268031, AY591335, AY591336, AY591337, AY591338, AY605098, AY605100, AY630573 (C14/16, C16/18 and C18/20, elongases). Similarly, the patent literature provides many additional DNA sequences of genes (and/or details concerning several of the genes above and their methods of isolation) involved in PUFA production [e.g., WO 02/077213 (Δ9 elongases); WO 00/34439 and WO 04/057001 (Δ8 desaturases); U.S. Pat. No. 5,968,809 (Δ6 desaturases); U.S. Pat. No. 5,972,664 and U.S. Pat. No. 6,075,183 (Δ5 desaturases); WO 94/11516, U.S. Pat. No. 5,443,974, WO 03/099216 and WO 05/047485 (Δ12 desaturases); WO 91/13972 and U.S. Pat. No. 5,057,419 (Δ9 desaturases); and, WO 00/12720, U.S. Pat. No. 6,403,349, U.S. Pat. No. 6,677,145, U.S. 2002/0139974A1, U.S. 2004/0111763 (C14/16, C16/18 and C18/20 elongases)]. Each of these patents and applications are herein incorporated by reference in their entirety.
It is contempalted that the examples above are not intended to be limiting and numerous other genes encoding: (1) Δ6 desaturases, C18/20 elongases and Δ5 desaturases (and optionally other genes encoding Δ9 desaturases, Δ12 desaturases, C14/16 elongases and/or C16/18 elongases); or (2) Δ9 elongases, Δ8 desaturases and Δ5 desaturases (and optionally other genes encoding Δ9 desaturases, Δ12 desaturases, C14/16 elongases and/or C16/18 elongases) derived from different sources would be suitable for introduction into Yarrowia lipolytica. 
Preferred Genes for ARA Synthesis
Despite the wide selection of desaturases and elongases that could be suitable for expression in Yarrowia lipolytica, however, in preferred embodiments of the present invention the desaturases and elongases are selected from the following (or derivatives thereof):







TABLE 5







Preferred Desaturases And Elongases For ARA Biosynthesis In


Yarrowia lipolytica













SEQ





ID


ORF
Organism
Reference
NOs





Δ6
Mortierella
GenBank Accession No.
1, 2


desaturase
alpina
AF465281; U.S. Pat. No. 5,968,809


Δ6
Mortierella
GenBank Accession No.
4, 5


desaturase
alpina
AB070555


C18/20
Mortierella
GenBank Accession No.
17,


elongase
alpina
AX464731; WO 00/12720
18


(“ELO1”)


C18/20
Thrausto-
U.S. Pat. No. 6,677,145
20,


elongase
chytrium

21


(“ELO2”)
aureum


Δ9
Isochrysis
GenBank Accession No.
39,


elongase
galbana
AF390174
40


Δ8
Euglena
Co-pending U.S. patent
44,


desaturase
gracillis
application No. 11/166,993
45


Δ5
Mortierella
GenBank Accession No.
6, 7


desaturase
alpina
AF067654; U.S. Pat. No. 6,075,183


Δ5
Isochrysis
WO 02/081668 A2
8, 9


desaturase
galbana


Δ5
Homo sapiens
GenBank Accession No.
11,


desaturase

NP_037534
12


C16/18
Yarrowia
—
61,


elongase
lipolytica

62


(“YE2”)


C16/18
Mortierella
—
53,


elongase
alpina

54


(“ELO3”)


C16/18
Rattus
GenBank Accession No.
50,


elongase
norvegicus
AB071986
51


(rELO2)


C14/16
Yarrowia
—
64,


elongase
lipolytica

65


(“YE1”)


Δ12
Yarrowia
WO 2004/104167
23,


desaturase
lipolytica

24


Δ12
Mortieralla
GenBank Accession No.
25,


desaturase
isabellina
AF417245
26


Δ12
Fusarium
WO 2005/047485
27,


desaturase
moniliforme

28


(Fm d12)


Δ12
Aspergillus
Contig 1.15 (scaffold 1) in the A.
29,


desaturase
nidulans
nidulans genome project;
30


(An d12)

AAG36933; WO 2005/047485


Δ12
Aspergillus
GenBank Accession No.
31


desaturase
flavus
AY280867 (VERSION




AY280867.1; gi: 30721844);




WO 2005/047485


Δ12
Aspergillus
AFA.133c 344248: 345586
32


desaturase
fumigatus
reverse (AfA5C5.001c) in the


(Afd12p)

Aspergillus fumigatus genome




project; WO 2005/047485


Δ12
Magnaporthe
Locus MG01985.1 in contig
33,


desaturase
grisea
2.375 in the M. grisea genome
34


(Mg d12)

project; WO 2005/047485


Δ12
Neurospora
GenBank Accession No.
35,


desaturase
crassa
AABX01000374;
36


(Nc d12)

WO 2005/047485


Δ12
Fusarium
Contig 1.233 in the F. graminearium
37,


desaturase
graminearium
genome project;
38


(Fg d12)

WO 2005/047485


Δ12
Mortierella
GenBank Accession No.
357,


desaturase
alpina
AB020033
358


(Mad12)


Δ12
Saccharomyces
GenBank Accession No.
359


desaturase
kluyveri
BAD08375


(Skd12)


Δ12
Kluyveromyces
gnl|GLV|KLLA0B00473g ORF
360,


desaturase
lactis
from Klla0B: 35614 . . . 36861
361


(Kld12p)

antisense (m) of K. lactis




database of the “Yeast project




Genolevures” (Center for




Bioinformatics, LaBRI, Talence




Cedex, France)


Δ12
Candida
GenBank Accession No.
362


desaturase
albicans
EAK94955


(Cad12p)


Δ12
Debaryomyces
GenBank Accession No.
363


desaturase
hansenii
CAG90237


(Dhd12p)
CBS767





*Note:


The Aspergillus fumigatus genome project is sponsored by Sanger Institute, collaborators at the University of Manchester and The Institute of Genome Research (TIGR);


the A. nidulans genome project is sponsored by the Center for Genome Research (CGR), Cambridge, MA;


the M. grisea genome project is sponsored by the CGR and International Rice Blast Genome Consortium;


the F. graminearium genome project is sponsored by the CGR and the International Gibberella zeae Genomics Consortium (IGGR).






Applicants have analyzed of various elongases, to either determine or confirm each enzyme's substrate specificity and/or substrate selectivity when expressed in Yarrowia lipolytica. For example, although the coding sequences of the two Y. lipolytica elongases were publically available and each protein was annotated as a putative long-chain fatty-acyl elongase or shared significant homology to other fatty acid elongases, the substrate specificity of these enzymes had never been determined. Based on the analyses performed herein, YE1 was positively determined to be a fatty acid elongase that preferentially used C14 fatty acids as substrates to produce C16 fatty acids (i.e., a C14/16 elongase) and YE2 was determined to be a fatty acid elongase that preferentially used C16 fatty acids as substrates to produce C18 fatty acids (i.e., a C16/18 elongase). Relatedly, upon identification of the novel M. alpina ELO3 gene, the sequence was characterized as homologous to other fatty acid elongases; however, lipid profile analyses were required to confirm the specificity of ELO3 as a C16/18 elongase.
With respect to Δ12 desaturase, Applicants have made the surprising discovery that the Fusarium moniliforme Δ12 desaturase (encoded by SEQ ID NO:27) functions with greater efficiency than the native Yarrowia lipolytica Δ12 desaturase in producing 18:2 in Y. lipolytica (see WO 2005/047485). Specifically, expression of the F. moniliforme Δ12 desaturase under the control of the TEF promoter in Y. lipolytica was determined to produce higher levels of 18:2 (68% product accumulation of LA) than were previously attainable by expression of a chimeric gene encoding the Y. lipolytica Δ12 desaturase under the control of the TEF promoter (59% product accumulation of LA). This corresponds to a difference in percent substrate conversion (calculated as ([18:2+18:3]/[18:1+18:2+18:3])*100) of 85% versus 74%, respectively. On the basis of these results, expression of the present fungal F. moniliforme Δ12 desaturase is preferred relative to other known Δ12 desaturases as a means to engineer a high ARA-producing strain of Y. lipolytica (however, one skilled in the art would expect that the activity of the F. moniliforme Δ12 desaturase could be enhanced in Y. lipolytica, following e.g., codon-optimization).
Despite the current identification of the F. moniliforme Δ12 enzyme as the preferred Δ12 desaturase, five new Δ12 desaturases have recently been identified that could possibly function with improved efficiency in Yarrowia lipolytica. Specifically, the Saccharomyces kluyveri Δ12 desaturase (GenBank Accession No. BAD08375) was described in Watanabe et al. (Biosci. Biotech. Biocheml. 68(3):721-727 (2004)), while that from Mortierella alpina (GenBank Accession No. AB182163) was described by Sakuradani et al. (Eur. J. Biochem. 261(3):812-820 (1999)). Since both sequences were subsequently utilized to identify S. kluyveri and M. alpina Δ15 desaturases (GenBank Accession No. BAD11952 and No. AB182163, respectively), these two pairs of proteins provided additional examples of closely related fungal Δ12 and Δ15 desaturases similar to those of Fusarium moniliforme, Aspergillus nidulans, Magnaporthe grisea, Neurospora crassa and Fusarium graminearium (see WO 2005/047480 and WO 2005/047485). This finding offered additional support to the Applicants' previous hypothesis that “pairs” of fungal Δ12 desaturase-like sequences likely comprise one protein having Δ15 desaturase activity and one protein having Δ12 desaturase activity. Similar “pairs” of Δ12 desaturase-like proteins were thus identified herein in Kluyveromyces lactis, Candida albicans and Debaryomyces hansenii CBS767; and, as predicted, one member of each pair aligned more closely to the previously identified S. kluyveri Δ12 desaturase (i.e., K. lactis gnl|GLV|KLLA0B00473 g ORF, C. albicans GenBank Accession No. EAK94955 and D. hansenii CBS767 GenBank Accession No. CAG90237) while the other aligned more closely to the S. kluyveri Δ15 desaturase (i.e., K. lactis GenBank Accession No. XM—451551, D. hansenii CBS767 GenBank Accession No. CAG88182, C. albicans GenBank Accession No. EAL03493). Thus, based on this analysis, the Applicants have identified the desaturases identified herein as SEQ ID NOs:358, 359, 361, 362 and 363 as putative fungal Δ12 desaturases whose overexpression in Y. lipolytica could be useful to increase production of ω-6 fatty acids.
In additional embodiments, the Applicants have identified a means to readily distinguish fungal sequences having Δ12 desaturase activity as opposed to Δ15 desaturase activity. Specifically, when an amino acid sequence alignment was analysed that comprised Δ12 desaturases (i.e., Mad12, Skd12, Nc d12, Fm d12, Mg d12, An d12, Fg d12, Dhd12p, Kld12p, Cad12p and Afd12p (see Table above)), as well as Δ15 desaturases (i.e., from Fusarium moniliforme, Aspergillus nidulans, Magnaporthe grisea, Neurospora crassa, F. graminearium, Mortierella alpina, K. lactis, C. albicans, Saccharomyces kluyveri, D. hansenii CBS767 and Aspergillus fumigatus), it became apparent that all of the fungal Δ15 or Δ12 desaturases contained either an Ile or Val amino acid residue, respectively, at the position that corresponds to position 102 of the Fusarium moniliforme Δ15 desaturase (SEQ ID NO:2 in WO 2005/047479) and that is only three amino acid residues away from the highly conserved His Box 1 (“HECGH”; SEQ ID NO:373) (Table 6). 



The Applicants conclude that Ile and Val at this position is a determinant of Δ15 and Δ12 desaturase specificity, respectively, in fungal desaturases. More specifically, the Applicants propose that any fungal Δ12 desaturase-like protein with Ile at the corresponding residue(s) (i.e., or the motif IXXHECGH [SEQ ID NO:374]) will be a Δ15 desaturase and any fungal Δ12 desaturase-like protein with Val at the corresponding residue(s) (i.e., or the motif VXXHECGH [SEQ ID NO:375]) will be a Δ12 desaturase. Thus, this single leucine/valine amino acid will be an important residue to consider as future fungal desaturases are identified and annotated. Futhermore, the Applicants also hypothesize that mutation(s) that result in a Ile-to-Val change at this position will alter enzyme specificity, such as towards Δ12 desaturation, in genes encoding fungal Δ12 desaturase-like proteins (e.g., the Fusarium monoliforme desaturase described as SEQ ID NO:2 in WO 2005/047479); and, conversely, those mutations that result in a Val-to-Ile change at this position will alter enzyme specificity, such as towards Δ15 desaturation.
Of course, in alternate embodiments of the present invention, other DNAs which are substantially identical to the desaturases and elongases encoded by SEQ ID NOs:2, 5, 7, 9, 12, 18, 21, 24, 26, 28, 30-32, 34, 36, 38, 40, 45, 51, 54, 62, 65, 358, 359 and 361-363 also can be used for production of ARA in Yarrowia lipolytica. By “substantially identical” is intended an amino acid sequence or nucleic acid sequence exhibiting in order of increasing preference at least 80%, 90% or 95% homology to the selected polypeptides, or nucleic acid sequences encoding the amino acid sequence. For polypeptides, the length of comparison sequences generally is at least 16 amino acids, preferably at least 20 amino acids or most preferably 35 amino acids. For nucleic acids, the length of comparison sequences generally is at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
Homology typically is measured using sequence analysis software, wherein 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.); and 4.) 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, 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. In general, such computer software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.
In more preferred embodiments, codon-optimized genes encoding desaturases and elongases that are substantially identical to those described in SEQ ID NOs:2, 9, 12, 18, 21, 40, 45 and 51 are utilized. Specifically, as is well known to one of skill in the art, the expression of heterologous genes can be increased by increasing the translational efficiency of encoded mRNAs by replacement of codons in the native gene with those for optimal gene expression in the selected host microorganism. Thus, it is frequently useful to modify a portion of the codons encoding a particular polypeptide that is to be expressed in a foreign host, such that the modified polypeptide uses codons that are preferred by the alternate host; and, use of host preferred codons can substantially enhance the expression of the foreign gene encoding the polypeptide.
In general, host preferred codons can be determined within a particular host species of interest by examining codon usage in proteins (preferably those expressed in the largest amount) and determining which are used with highest frequency. Then, the coding sequence for a polypeptide of interest (e.g., a desaturase, elongase, acyltransferase) can be synthesized in whole or in part using the codons preferred in the host species. All (or portions) of the DNA also can be synthesized to remove any destabilizing sequences or regions of secondary structure that would be present in the transcribed mRNA. And, all (or portions) of the DNA also can be synthesized to alter the base composition to one more preferable in the desired host cell.
Additionally, the 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 to obtain optimal gene expression. For expression in yeast, this can be done by site-directed mutagenesis of an inefficiently expressed gene by fusing it in-frame to an endogenous yeast gene, preferably a highly expressed gene. Alternatively, as demonstrated herein for Yarrowia lipolytica, one can determine the consensus translation initiation sequence in the host and engineer this sequence into heterologous genes for their optimal expression in the host of interest.
In the present invention, several desaturase and elongase genes from Table 5 were codon-optimized for expression in Yarrowia lipolytica, based on the host preferences described above. This was possible by first determining the Y. lipolytica codon usage profile (see WO 04/101757) and identifying those codons that were preferred. Then, for further optimization of gene expression in Y. lipolytica, the consensus sequence around the ‘ATG’ initiation codon was determined (i.e., ‘MAMMATGNHS’ (SEQ ID NO:356), 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). Table 7, below, compares the activity of native and codon-optimized genes when expressed in Y. lipolytica and provides details about each codon-optimized gene. % Sub. Conv. is the abbeviation for “percent substrate conversion” and Codon-Opt. is an abbreviation for “codon-optimized”.







TABLE 7







Most Preferred Codon-Optimized Desaturases And Elongases For ARA


Biosynthesis In Yarrowia lipolytica















Codon-





Native
Total Bases
Opt.

Codon-



Gene %
Modified In
Gene

Opt.



Sub.
Codon-Opt.
% Sub.

SEQ


Native Gene
Conv.
Gene
Conv.
Reference
ID NO















M. alpina Δ6
30%
152 of 1374 bp
42%
WO 04/101753
3


desaturase

(corresponding


(GenBank

to 144 codons)


Accession No.


AF465281)


M. alpina high
30%
94 of 957 bp
47%
WO 04/101753
19


affinity C18/20

(corresponding


elongase (GenBank

to 85 codons)


Accession No.


AX464731)


T. aureum C18/20
33%
114 of 817 bp
46%
—
22


elongase (“ELO2”)

(corresponding




to 108 codons)


S. diclina Δ17
23%
127 of 1077 bp
45%
Co-Pending
16


desaturase (US

(corresponding

U.S. patent


2003/0196217 A1)

to 117 codons)

application No.






10/840,478


Isochrysis galbana
—
126 of 789 bp
30%
—
41


Δ9 elongase

(corresponding




to 123 codons)


Euglena gracillis Δ8
—
207 of 1263 bp
75%
Co-pending
48


desaturase

(corresponding

U.S. patent




to 192 codons)

application No.






11/166,993


Isochrysis galbana
 7%
203 of 1323 bp
32%
—
10


Δ5 desaturase

(corresponding




to 193 codons)


Homo sapiens Δ5
—
227 of 1335 bp
30%
—
13


desaturase

(corresponding


(GenBank

to 207 codons)


Accession No.


NP_037534)


Rattus norvegicus
—
127 of 792 bp
43%
—
52


C16/18 elongase

(corresponding


(GenBank

to 125 codons)


Accession No.


AB071986)









In additional alternate embodiments of the invention, other DNAs which, although not substantially identical to the preferred desaturases and elongases presented as SEQ ID NOs:3, 10, 13, 16, 19, 22, 41, 48 and 52 also can be used for the purposes herein. For example, DNA sequences encoding Δ6 desaturase polypeptides that would be useful for introduction into Yarrowia lipolytica according to the teachings of the present invention may be obtained from microorganisms having an ability to produce GLA or STA. Such microorganisms include, for example, those belonging to the genera Mortierella, Conidiobolus, Pythium, Phytophathora, Penicillium, Porphyridium, Coidosporium, Mucor, Fusarium, Aspergillus, Rhodotorula and Entomophthora. Within the genus Porphyridium, of particular interest is P. cruentum. Within the genus Mortierella, of particular interest are M. elongata, M. exigua, M. hygrophila, M. ramanniana var. angulispora and M. alpina. Within the genus Mucor, of particular interest are M. circinelloides and M. javanicus. 
Alternatively, a related desaturase that is not substantially identical to the M. alpina Δ6 desaturase, for example, but which can desaturate a fatty acid molecule at carbon 6 from the carboxyl end of the molecule would also be useful in the present invention as a Δ6 desaturase, assuming the desaturase can still effectively convert LA to GLA and/or ALA to STA. As such, related desaturases and elongases can be identified (or created) by their ability to function substantially the same as the desaturases and elongases disclosed herein.
As suggested above, in another embodiment one skilled in the art could create a fusion protein having e.g., both Δ12 desaturase and Δ6 desaturase activities suitable for the purposes herein. This would be possible by fusing together a Δ12 desaturase and Δ6 desaturase with an adjoining linker. Either the Δ12 desaturase or the Δ6 desaturase could be at the N-terminal portion of the fusion protein. Means to design and synthesize an appropriate linker molecule are readily known by one of skill in the art; for example, the linker can be a stretch of alanine or lysine amino acids and will not affect the fusion enzyme's activity.
Finally, it is well known in the art that methods for synthesizing sequences and bringing sequences together are well established in the literature. Thus, in vitro mutagenesis and selection, site-directed mutagenesis, chemical mutagenesis, “gene shuffling” methods or other means can be employed to obtain mutations of naturally occurring desaturase and/or elongase genes. This would permit production of a polypeptide having desaturase or elongase activity, respectively, in vivo with more desirable physical and kinetic parameters for functioning in the host cell (e.g., a longer half-life or a higher rate of production of a desired PUFA).
In summary, although sequences of preferred desaturase and elongase genes are presented that encode PUFA biosynthetic pathway enzymes suitable for ARA production in Yarrowia lipolytica, these genes are not intended to be limiting to the invention herein. Numerous other genes encoding PUFA biosynthetic pathway enzymes that would be suitable for the purposes herein could be isolated from a variety of sources (e.g., a wildtype, codon-optimized, synthetic and/or mutant enzyme having appropriate desaturase or elongase activity). These alternate desaturases would be characterized by the ability to: 1.) desaturate a fatty acid between the 8th and 9th carbon atom numbered from the carboxyl-terminal end of the molecule and catalyze the conversion of EDA to DGLA (Δ8 desaturases); 2.) catalyze the conversion of LA to GLA (Δ6 desaturases); 3.) catalyze the conversion of DGLA to ARA (Δ5 desaturases); 4.) catalyze the conversion of oleic acid to LA (Δ12 desaturases); and/or 5.) catalyze the conversion of palmitate to palmitoleic acid and/or stearate to oleic acid (Δ9 desaturases). In like manner, suitable elongases for the purposes herein are not limited to those from a specific source; instead, the enzymes having use for the purposes herein are characterized by their ability to elongate a fatty acid carbon chain by 2 carbons relative to the substrate the elongase acts upon, to thereby produce a mono- or polyunsaturated fatty acid. More specifically, these elongases would be characterized by the ability to: 1.) elongate LA to EDA (Δ9 elongases); 2.) elongate a C18 substrate to produce a C20 product (C18/20 elongases); 3.) elongate a C14 substrate to produce a C16 product (C14/16 elongases); and/or 4.) elongate a C16 substrate to produce a C18 product (C16/18 elongases). Again, it is important to note that some elongases may be capable of catalyzing several elongase reactions, as a result of broad substrate specificity.
Acyltransferases and their Role in the Terminal Step of Tag Biosynthesis
Acyltransferases are intimately involved in the biosynthesis of TAGs. Two comprehensive mini-reviews on TAG biosynthesis in yeast, including details concerning the genes involved and the metabolic intermediates that lead to TAG synthesis are: D. Sorger and G. Daum, Appl. Microbiol. Biotechnol. 61:289-299 (2003); and H. Müllner and G. Daum, Acta Biochimica Polonica, 51(2):323-347 (2004). Although the authors of these reviews clearly summarize the different classes of eukaryotic acyltransferase gene families (infra), they also acknowledge that regulatory aspects of TAG synthesis and formation of neutral lipids in lipid particles remain far from clear.
Four eukaryotic acyltransferase gene families have been identified which are involved in acyl-CoA-dependent or independent esterification reactions leading to neutral lipid synthesis:

    (1) The acyl-CoA:cholesterol acyltransferase (ACAT) family, EC 2.3.1.26 (commonly known as sterol acyltransferases). This family of genes includes enzymes responsible for the conversion of acyl-CoA and sterol to CoA and sterol esters. This family also includes DGAT1, involved in the terminal step of TAG biosynthesis.
    (2) The lecithin:cholesterol acyltransferase (LCAT) family, EC 2.3.1.43. This family of genes is responsible for the conversion of phosphatidylcholine and a sterol to a sterol ester and 1-acylglycerophosphocholine. This family also includes the phospholipid:diacylglycerol acyltransferase (PDAT) enzyme involved in the transfer of an acyl group from the sn-2 position of a phospholipid to the sn-3 position of 1,2-diacylglycerol resulting in TAG biosynthesis.
    (3) The diacylglycerol acyltransferase (DAG AT) family, EC 2.3.1.20. This family of genes (which includes DGAT2) is involved in the terminal step of TAG biosynthesis.
    (4) The glycerol-3-phosphate acyltransferase and acyl-CoA lysophosphatidic acid acyltransferase (GPAT/LPMT) family. GPAT (E.C. 2.3.1.15) proteins are responsible for the first step of TAG biosynthesis, while LPMT (E.C. 2.3.1.51) enzymes are involved in the second step of TAG biosynthesis. This family also includes lysophosphatidylcholine acyltransferase (LPCAT) that catalyzes the acyl exchange between phospholipid and CoA.

Together, these 4 acyltransferase gene families represent overlapping biosynthetic systems for neutral lipid formation and appear to be the result of differential regulation, alternate localization, and different substrate specifities (H. Müllner and G. Daum, supra). Each of these four gene families will be discussed herein based on their importance with respect to metabolic engineering in Yarrowia lipolytica, to enable synthesis of greater than 10-30% ARA.

The Functionality of Various Acyltransferases



The interplay between many of these acyltransferases in Yarrowia lipolytica is schematically diagrammed in FIG. 3. Focusing initially on the direct mechanism of TAG biosynthesis, the first step in this process is the esterification of one molecule of acyl-CoA to sn-glycerol-3-phosphate via GPAT to produce lysophosphatidic acid (LPA) (and CoA as a by-product). Then, lysophosphatidic acid is converted to phosphatidic acid (PA) (and CoA as a by-product) by the esterification of a second molecule of acyl-CoA, a reaction that is catalyzed by LPMT. Phosphatidic acid phosphatase is then responsible for the removal of a phosphate group from phosphatidic acid to yield 1,2-diacylglycerol (DAG). And, finally a third fatty acid is added to the sn-3 position of DAG by a DAG AT (e.g., DGAT1, DGAT2 or PDAT) to form TAG.
Historically, DGAT1 was thought to be the only enzyme specifically involved in TAG synthesis, catalyzing the reaction responsible for the conversion of acyl-CoA and DAG to TAG and CoA, wherein an acyl-CoA group is transferred to DAG to form TAG. DGAT1 was known to be homologous to ACATs; however, recent studies have identified a new family of DAG AT enzymes that are unrelated to the ACAT gene family. Thus, nomenclature now distinguishes between the DAG AT enzymes that are related to the ACAT gene family (DGAT1 family) versus those that are unrelated (DGAT2 family) (Lardizabal et al., J. Biol. Chem. 276(42):38862-38869 (2001)). Members of the DGAT2 family have been identified in all major phyla of eukaryotes (fungi, plants, animals and basal eukaryotes).
Even more recently, Dahlqvist et al. (Proc. Nat. Acad. Sci. (USA) 97:6487-6492 (2000)) and Oelkers et al. (J. Biol. Chem. 275:15609-15612 (2000)) discovered that TAG synthesis can also occur in the absence of acyl-CoA, via an acyl-CoA-independent mechanism. Specifically, PDAT removes an acyl group from the sn-2 position of a phosphotidylcholine substrate for transfer to DAG to produce TAG. This enzyme is structurally related to the LCAT family; and although the function of PDAT is not as well characterized as DGAT2, PDAT has been postulated to play a major role in removing “unusual” fatty acids from phospholipids in some oilseed plants (Banas, A. et al., Biochem. Soc. Trans. 28(6):703-705 (2000)).
With respect to TAG synthesis in Saccharomyces cerevisiae, three pathways have been described (Sandager, L. et al., J. Biol. Chem. 277(8):6478-6482 (2002)). First, TAGs are mainly synthesized from DAG and acyl-CoAs by the activity of DGAT2 (encoded by the DGA1 gene). More recently, however, a PDAT (encoded by the LRO1 gene) has also been identified. Finally, two acyl-CoA:sterol-acyltransferases (encoded by the ARE1 and ARE2 genes) are known that utilize acyl-CoAs and sterols to produce sterol esters (and TAGs in low quantities; see Sandager et al., Biochem. Soc. Trans. 28(6):700-702 (2000)). Together, PDAT and DGAT2 are responsible for approximately 95% of oil biosynthesis in S. cerevisiae. 
Based on several publicly available sequences encoding DGAT1s, DGAT2s, PDATs and ARE2s (infra), the Applicants isolated and characterized the genes encoding DGAT1 (SEQ ID NO:81), DGAT2 (SEQ ID NOs:89, 91 and 93 [wherein SEQ ID NO:89 contains at least two additional nested ORFs as provided in SEQ ID NOs:91 and 93; the ORF encoded by SEQ ID NO:93 has a high degree of similarity to other known DGAT enzymes and disruption in SEQ ID NO:93 eliminated DGAT function of the native gene, thereby confirming that the polypeptide of SEQ ID NO:94 has DGAT functionality]), PDAT (SEQ ID NO:76) and ARE2 (SEQ ID NO:78) in Yarrowia lipolytica. In contrast to the model developed in S. cerevisiae, wherein PDAT and DGAT2 are responsible for approximately 95% of oil biosynthesis, however, it was discovered that the PDAT, DGAT2 and DGAT1 of Yarrowia lipolytica are responsible for up to 95% of oil biosynthesis (while ARE2 may additionally be a minor contributor to oil biosynthesis).
The final acyltransferase enzyme whose function could be important in the accumulation of ARA in the TAG fraction of Yarrowia lipolytica is LPCAT. As shown in FIG. 3, this enzyme (EC 2.3.1.23) is hypothesized to be responsible for two-way acyl exchange at the sn-2 position of sn-phosphatidylcholine to enhance ω-6 and ω-3 PUFA biosynthesis. This hypothesis is based on the following studies: (1) Stymne S. and A. K. Stobart (Biochem J. 223(2):305-14(1984)), who hypothesized that LPCAT affected exchange between the acyl-CoA pool and phosphatidylcholine (PC) pool; (2) Domergue, F. et al. (J. Bio. Chem 278:35115 (2003)), who suggested that accumulation of GLA at the sn-2 position of PC and the inability to efficiently synthesize ARA in yeast was a result of the elongation step involved in PUFA biosynthesis occurring within the acyl-CoA pool, while Δ5 and Δ6 desaturation steps occurred predominantly at the sn-2 position of PC; (3) Abbadi, A. et al. (The Plant Cell, 16:2734-2748 (2004)), who suggested that LPCAT plays a criticial role in the successful reconstitution of a Δ6 desaturase/Δ6 elongase pathway, based on analysis on the constraints of PUFA accumulation in transgenic oilseed plants; and, (4) WO 2004/076617 A2 (Renz, A. et al.), who provided a gene encoding LPCAT from Caenorhabditis elegans (T06E8.1) that substantially improved the efficiency of elongation in a genetically introduced Δ6 desaturase/Δ6 elongase pathway in S. cerevisiae. The inventors concluded that LPCAT allowed efficient and continuous exchange of the newly synthesized fatty acids between phospholipids and the acyl-CoA pool, since desaturases catalyze the introduction of double bonds in lipid-coupled fatty acids (sn-2 acyl PC) while elongases exclusively catalyze the elongation of CoA esterified fatty acids (acyl-CoAs).
Selection of Heterologous Acyltransferase Genes for ARA Synthesis
Since naturally produced PUFAs in Yarrowia lipolytica are limited to 18:2 fatty acids (and less commonly, 18:3 fatty acids), it would be likely that the host organism's native genes encoding GPAT, LPAAT (i.e., LPAAT1 or LPMT2), DGAT1, DGAT2, PDAT and LPCAT could have difficulty efficiently synthesizing TAGs comprising fatty acids that were 18:3 and greater in length (e.g., ARA). Thus, in some cases, a heterologous (or “foreign”) acyltransferase could be preferred over a native enzyme.
Numerous acyltransferase genes have been identified in various organisms and disclosed in the public and patent literature. For instance, the following GenBank Accession Numbers refer to examples of publicly available acyltransferase genes useful in lipid biosynthesis: CQ891256, AY441057, AY360170, AY318749, AY093169, AJ422054, AJ311354, AF251795, Y00771, M77003 (GPATs); Q93841, Q22267, Q99943, O15120, Q9NRZ7, Q9NRZ5, Q9NUQ2, O35083, Q9D1E8, Q924S1, Q59188, Q42670, P26647, P44848, Q9ZJN8, O25903 Q42868, Q42870, P26974, P33333, Q9XFW4, CQ891252, CQ891250, CQ891260, CQ891258, CQ891248, CQ891245, CQ891241, CQ891238, CQ891254, CQ891235 (LPAATs); AY445635, BC003717, NM—010046, NM—053437, NM—174693, AY116586, AY327327, AY327326, AF298815 and AF164434 (DGAT1s); and NC—001147 [locus NP—014888], NM—012079, NM—127503, AF051849, AJ238008, NM—026384, NM—010046, AB057816, AY093657, AB062762, AF221132, AF391089, AF391090, AF129003, AF251794 and AF164434 (DGAT2s); P40345, O094680, NP—596330, NP—190069 and AB006704 [gi:2351069] (PDATs). Similarly, the patent literature provides many additional DNA sequences of genes (and/or details concerning several of the genes above and their methods of isolation) involved in TAG production [e.g., U.S. Pat. No. 5,210,189, WO 2003/025165 (GPATs); EP1144649A2, EP1131438, U.S. Pat. No. 5,968,791, U.S. Pat. No. 6,093,568, WO 2000/049156 and WO 2004/087902 (LPMTs); U.S. Pat. No. 6,100,077, U.S. Pat. No. 6,552,250, U.S. Pat. No. 6,344,548, US 2004/0088759A1 and U.S. 20040078836A1 (DGAT1s); US 2003/124126, WO 2001/034814, US 2003/115632, US 2003/0028923 and US 2004/0107459 (DGAT2s); WO 2000/060095 (PDATs); and WO 2004/076617 A2 (LPCATs).
It is contemplated that the examples above are not intended to be limiting and numerous other genes encoding DGAT1, DGAT2, PDAT, GPAT, LPCAT and LPAAT derived from different sources would be suitable for introduction into Yarrowia lipolytica. For example, the Applicants have identified novel DGAT1s from Mortierella alpina (SEQ ID NOs:83 and 84), Neurospora crassa (SEQ ID NO:85), Gibberella zeae PH-1 (SEQ ID NO:86), Magnaporthe grisea (SEQ ID NO:87) and Aspergillus nidulans (SEQ ID NO:88); and, a novel DGAT2 (SEQ ID NOs:95 and 96), GPAT (SEQ ID NOs:97 and 98), LPMT1 (SEQ ID NOs:67 and 68) and LPMT2 (SEQ ID NOs:69 and 70) from Mortierella alpina.
Preferred Acyltransferase Genes for ARA Synthesis
Despite the wide selection of acyltransferases that could be suitable for expression in Yarrowia lipolytica, however, in preferred embodiments of the present invention the DGAT1, DGAT2, PDAT, GPAT, LPAAT and LPCAT are selected from organisms producing significant amounts of longer chain ω-6 (e.g., ARA) and/or ω-3 (e.g., EPA, DHA) PUFAs. Thus, the following enzymes are especially preferred (or derivatives thereof):







TABLE 8







Preferred Heterologous Acyltransferases For Expression In A High


ARA-Producing Strain Of Yarrowia lipolytica













SEQ





ID


ORF
Organism
Reference
NOs





DGAT1
Mortierella
Co-pending U.S. patent
83, 84



alpina
application No. 11/024,544


DGAT2
Mortierella
Co-pending U.S. patent
95, 96



alpina
application No. 11/024,545


GPAT
Mortierella
—
97, 98



alpina


LPAAT1
Mortierella
—
67, 68



alpina


LPAAT2
Mortierella
Co-pending U.S. patent
69, 70



alpina
application No.-60/689,031


LPCAT
Caenorhabditis
Clone T06E8.1;
80



elegans
WO 2004/076617 A2









Although not intended to be limiting in the invention herein, M. alpina was selected as a preferred source of heterologous acyltransferases since the native organism is capable of synthesizing ARA at concentrations greater than 50% of the total fatty acids (TFAs). In similar manner, C. elegans can produce up to 20-30% of its TFAs as EPA.
Of course, in alternate embodiments of the present invention, other DNAs which are substantially identical to the acyltransferases encoded by SEQ ID NOs:67, 68, 69, 70, 80, 83, 84, 95, 96, 97 and 98 also can be used for heterologous expression in Yarrowia lipolytica to facilitate the production and accumulation of ARA in the TAG fraction. In more preferred embodiments, codon-optimized genes encoding acyltransferases that are substantially identical to those described in SEQ ID NOs: 67-70, 80, 83, 84 and 95-98 are utilized.
General Expression Systems, Cassettes, Vectors and Transformation for Expression of Foreign Genes
Microbial expression systems and expression vectors containing regulatory sequences that direct high-level expression of foreign proteins such as those leading to the high-level production of ARA are well known to those skilled in the art. Any of these could be used to construct chimeric genes encoding the preferred desaturases, elongases and acyltransferases. These chimeric genes could then be introduced into Yarrowia lipolytica using standard methods of transformation to provide high-level expression of the encoded enzymes.
Vectors or DNA cassettes useful for the transformation of host cells are well known in the art. The specific choice of sequences present in the construct is dependent upon the desired expression products, the nature of the host cell, and the proposed means of separating transformed cells versus non-transformed cells. 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 (e.g., a promoter) 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 it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.
Where two or more genes are expressed from separate replicating vectors, it is desirable that each vector has a different means of selection and should lack homology to the other constructs 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 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 the gene(s) 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)]), protoplast fusion, bolistic impact, electroporation, microinjection, or any other method that introduces the gene(s) of interest into the host cell. More specific teachings applicable for Yarrowia lipolytica include U.S. Pat. No. 4,880,741 and 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 (e.g., an expression cassette) will be referred to as “transformed” or “recombinant” herein. The transformed host will have at least one copy of the expression construct 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 various selection techniques, as described in WO 2004/101757 and WO 2005/003310.
Preferred selection methods for use herein are resistance to kanamycin, hygromycin and the amino glycoside G418, as well as ability to grow on media lacking uracil, leucine, lysine, tryptophan or histidine. In alternate embodiments, 5-fluoroorotic acid (5-fluorouracil-6-carboxylic acid monohydrate; “5-FOA”) is used for selection of yeast Ura− mutants. The compound is toxic to yeast cells that possess a functioning URA3 gene encoding orotidine 5′-monophosphate decarboxylase (OMP decarboxylase); thus, based on this toxicity, 5-FOA is especially useful for the selection and identification of Ura− mutant yeast strains (Bartel, P. L. and Fields, S., Yeast 2-Hybrid System, Oxford University: New York, v. 7, pp 109-147,1997).
An alternate preferred selection method utilized herein relies on a dominant, non antibiotic marker for Yarrowia lipolytica based on sulfonylurea resistance. The technique is also generally applicable to other industrial yeast strains that may be haploid, diploid, aneuploid or heterozygous. It is expected to overcome two main limitations to the development of genetic transformation systems for industrial yeast strains, wherein: (1) there are almost no naturally auxotrophic strains, and the isolation of spontaneous or induced auxotrophic mutants is hindered by the ploidy of the strains; and, (2) the use of antibiotic resistance markers may limit the commercial application of strains due to restrictions on the release of genetically modified organisms carrying antibiotic resistance genes. Although Puig et al. (J. Agric. Food Chem. 46:1689-1693 (1998)) developed a method to overcome these limitations based on the genetic engineering of a target strain in order to make it auxotrophic for uridine and the subsequent use of the URA3 marker in order to introduce traits of interest, this strategy was deemed too laborious for routine work.
The new sulfonylurea resistance selection marker disclosed herein for transforming Yarrowia lipolytica does not rely on a foreign gene but on a mutant native gene. Thus, it neither requires auxotrophy nor results in auxotrophy and allows transformation of wild type strains. More specifically, the marker gene (SEQ ID NO:280) is a native acetohydroxyacid synthase (AHAS or acetolactate synthase; E.C. 4.1.3.18) that has a single amino acid change (W497L) that confers sulfonyl urea herbicide resistance. AHAS is the first common enzyme in the pathway for the biosynthesis of branched-chain amino acids and it is the target of the sulfonylurea and imidazolinone herbicides. W497L mutation, based on work in Saccharomyces cerevisiae (Falco, S. C., et al., Dev. Ind. Microbiol. 30:187-194 (1989); Duggleby, R. G., et. al. Eur. J. Biochem. 270:2895 (2003)) is known. Initial testing determined that Yarrowia cells were not naturally resistant to the herbicide as a result of: 1.) poor or no uptake of the herbicide; 2.) the presence of a native herbicide-resistant form of AHAS; and/or 3.) use of a herbicide-inactivating mechanism. This enabled synthesis and use of the mutant AHAS gene (SEQ ID NO:280) as a means for selection of transformants.
An additional method for recyling a selection marker relies on site-specific recombinase systems. Briefly, the site-specific recombination system consists of two elements: (1) a recombination site having a characteristic DNA sequence [e.g., LoxP]; and (2) a recombinase enzyme that binds to the DNA sequence specifically and catalyzes recombination (i.e., excision) between DNA sequences when two or more of the recombination sites are oriented in the same direction at a given interval on the same DNA molecule [e.g., Cre]. This methodology has utility as a means of selection, since it is possible to “recycle” a pair of preferred selection markers for their use in multiple sequential transformations.
More specifically, an integration construct is created comprising a target gene that is desirable to insert into the host genome (e.g., a desaturase, elongase, acyltransferase), as well as a first selection marker (e.g., Ura3, hygromycin phosphotransferase [HPT]) that is flanked by recombination sites. Following transformation and selection of the transformants, the first selection marker is excised from the chromosome by the introduction of a replicating plasmid carrying a second selection marker (e.g., sulfonylurea resistance [AHAS]) and a recombinase suitable to recognize the site-specific recombination sites introduced into the genome. Upon selection of those transformants carrying the second marker and confirmation of excision of the first selection marker from the host genome, the replicating plasmid is then cured from the host in the absence of selection. This produces a transformant that possesses neither the first nor second selection marker, and thus the cured strain is available for another round of transformation. One skilled in the art will recognize that the methodology is not limited to the particular selection markers or site-specific recombination system described above.
Overexpression of Foreign Genes in Yarrowia lipolytica 
As is well known to one of skill in the art, merely inserting a gene (e.g., a desaturase) into a cloning vector does not ensure that it will be successfully expressed at the level needed. It may be desirable to manipulate a number of different genetic elements that control aspects of transcription, translation, protein stability, oxygen limitation and secretion from the host cell. More specifically, gene expression may be controlled by altering the following: the nature of the relevant transcriptional promoter and terminator sequences; the number of copies of the cloned gene; whether the gene is plasmid-borne or integrated into the genome of the host cell; the final cellular location of the synthesized foreign protein; the efficiency of translation in the host organism; the intrinsic stability of the cloned gene protein within the host cell; and the codon usage within the cloned gene, such that its frequency approaches the frequency of preferred codon usage of the host cell. Several of these methods of overexpression will be discussed below, and are useful in the present invention as a means to overexpress e.g., desaturases, elongases and acyltransferases in Yarrowia lipolytica. 
Expression of the desired gene(s) can be increased at the transcriptional level through the use of a stronger promoter (either regulated or constitutive) to cause increased expression, 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).
Initiation control regions or promoters which are useful to drive expression of desaturase, elongase and acyltransferase genes in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of directing expression of these genes in Yarrowia lipolytica is suitable for the present invention. Expression in the host cell can be accomplished in a transient or stable fashion. Transient expression can be accomplished by inducing the activity of a regulatable promoter operably linked to the gene of interest; alternatively, stable expression can be achieved by the use of a constitutive promoter operably linked to the gene of interest. As an example, when the host cell is yeast, transcriptional and translational regions functional in yeast cells are provided, particularly from the host species. The transcriptional initiation regulatory regions can be obtained, for example, from: 1.) genes in the glycolytic pathway, such as alcohol dehydrogenase, glyceraldehyde-3-phosphate-dehydrogenase, phosphoglycerate mutase, fructose-bisphosphate aldolase, phosphoglucose-isomerase, phosphoglycerate kinase, glycerol-3-phosphate O-acyltransferase, etc.; or 2.) regulatable genes, such as acid phosphatase, lactase, metallothionein, glucoamylase, the translation elongation factor EF1-α (TEF) protein (U.S. Pat. No. 6,265,185), ribosomal protein S7 (U.S. Pat. No. 6,265,185), ammonium transporter proteins, export proteins, etc. Any one of a number of regulatory sequences can be used, depending upon whether constitutive or induced transcription is desired, the efficiency of the promoter in expressing the ORF of interest, the ease of construction and the like. The examples provided above are not intended to be limiting in the invention herein.
As one of skill in the art is aware, a variety of methods are available to compare the activity of various promoters. This type of comparison is useful to facilitate a determination of each promoter's strength for use in future applications wherein a suite of promoters would be necessary to construct chimeric genes useful for the production of ω-6 and ω-3 fatty acids. Thus, it may be useful to indirectly quantitate promoter activity based on reporter gene expression (i.e., the E. coli gene encoding β-glucuronidase (GUS)). In alternate embodiments, it may sometimes be useful to quantify promoter activity using more quantitative means. One suitable method is the use of real-time PCR (for a general review of real-time PCR applications, see Ginzinger, D. J., Experimental Hematology, 30:503-512 (2002)). Real-time PCR is based on the detection and quantitation of a fluorescent reporter. This signal increases in direct proportion to the amount of PCR product in a reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template. There are two general methods for the quantitative detection of the amplicon: (1) use of fluorescent probes; or (2) use of DNA-binding agents (e.g., SYBR-green I, ethidium bromide). For relative gene expression comparisons, it is necessary to use an endogenous control as an internal reference (e.g., a chromosomally encoded 16S rRNA gene), thereby allowing one to normalize for differences in the amount of total DNA added to each real-time PCR reaction. Specific methods for real-time PCR are well documented in the art. See, for example, the Real Time PCR Special Issue (Methods, 25(4):383481 (2001)).
Following a real-time PCR reaction, the recorded fluorescence intensity is used to quantitate the amount of template by use of: 1.) an absolute standard method (wherein a known amount of standard such as in vitro translated RNA (cRNA) is used); 2.) a relative standard method (wherein known amounts of the target nucleic acid are included in the assay design in each run); or 3.) a comparative CT method (ΔΔCT) for relative quantitation of gene expression (wherein the relative amount of the target sequence is compared to any of the reference values chosen and the result is given as relative to the reference value). The comparative CT method requires one to first determine the difference (ΔCT) between the CT values of the target and the normalizer, wherein: ΔCT=CT(target)−CT (normalizer). This value is calculated for each sample to be quantitated and one sample must be selected as the reference against which each comparison is made. The comparative ΔΔCT calculation involves finding the difference between each sample's ΔCT and the baseline's ΔCT, and then transforming these values into absolute values according to the formula 2−ΔΔCT.
Despite the wide selection of promoters that could be suitable for expression in Yarrowia lipolytica, however, in preferred embodiments of the present invention the promoters are selected from those shown below in Table 9 (or derivatives thereof).







TABLE 9







Native Promoters Preferred For Overexpression In Yarrowia lipolytica

















SEQ


Promoter


Activity

ID


Name
Location*
Native Gene
“Rank”
Reference
NO





TEF
—
translation
1
U.S. Pat. No.
166




elongation factor

6,265,185 (Muller et




EF1-α

al.); GenBank






Accession No.






AF054508


GPD
−968 bp to +3 bp
glyceraldehyde-3-
2
WO 2005/003310
158




phosphate-




dehydrogenase


GPM
−875 bp to +3 bp
phospho-
1
WO 2005/003310
160




glycerate mutase


FBA
−1001 bp to
fructose-
4
WO 2005/049805
161



−1 bp
bisphosphate




aldolase


FBAIN
−804 bp to
fructose-
7
WO 2005/049805
162



+169 bp
bisphosphate



(including a
aldolase



102 bp intron



[+64 to



+165])


FBAINm
−804 bp to
fructose-
5
WO 2005/049805
163



+169 bp with
bisphosphate



modification***
aldolase


GPDIN
−973 bp to
glyceraldehyde-3-
3
Co-pending U.S.
159



+201 bp
phosphate-

patent application



(including a
dehydrogenase

No. 11/183,664



146 bp intron



[+49 to



+194])


GPAT
−1130 to +3 bp
glycerol-3-
5
Co-pending U.S.
164




phosphate O-

patent application




acyltransferase

No. 11/225,354


YAT1
−778 to −1 bp
ammonium
6
Co-pending U.S.
165




transporter

patent application




enzyme

No. 11/185,301


EXP1
−1000 to −1 bp
export protein
6
—
364





*Location is with respect to the native gene, wherein the ‘A’ position of the ‘ATG’ translation initiation codon is designated as +1.


***The FBAINm promoter is a modified version of the FBAIN promoter, wherein FBAINm has a 52 bp deletion between the ATG translation initiation codon and the intron of the FBAIN promoter (thereby including only 22 amino acids of the N-terminus) and a new translation consensus motif after the intron. Furthermore, while the FBAIN promoter generates a fusion protein when fused with the coding region of a gene to be expressed, the FBAINm promoter does not generate such a fusion protein.






The activity of GPM is about the same as TEF, while the activity of GPD, FBA, FBAIN, FBAINm, GPDIN, GPAT, YAT1 and EXP1 are all greater than TEF (activity is quantified in a relative manner in the column titled “Activity Rank”, wherein a ‘1’ corresponds to the promoter with lowest activity, while a ‘7’ corresponds to the promoter with highest activity). This quantitation is based on comparative studies wherein each promoter was used for creation of a chimeric gene possessing the E. coli gene encoding β-glucuronidase (GUS) as a reporter (Jefferson, R. A. Nature. 14;342:837-838 (1989)) and a  ̃100 bp of the 3′ region of the Yarrowia Xpr gene. GUS activity in each expressed construct was measured by histochemical and/or fluorometric assays (Jefferson, R. A. Plant Mol. Biol. Reporter 5:387-405 (1987)) and/or by use of Real Time PCR.
The YAT1 promoter is unique in that it is characterized by the Applicants as the first promoter identified within Yarrowia that is inducible under oleaginous conditions (i.e., nitrogen limitation). Specifically, 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 (HGM) containing about 8-12% 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). Based on GUS activity of cell extracts, the activity of the YAT1 promoter increased by  ̃37 fold when cells were switched from a minimal 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 minimal medium comprising nitrogen (Example 1).
Of course, in alternate embodiments of the present invention, other promoters which are derived from any of the promoter regions described above in Table 9 also can be used for heterologous expression in Yarrowia lipolytica to facilitate the production and accumulation of ARA in the TAG fraction. In particular, modification of the lengths of any of the promoters described above can result in a mutant promoter having identical activity, since the exact boundaries of these regulatory sequences have not been completely defined. In alternate embodiments, the enhancers located within the introns of the FBAIN and GPDIN promoters can be used to create a chimeric promoter having increased activity relative to the native Yarrowia promoter (e.g., chimeric GPM::FBAIN and GPM::GPDIN promoters (SEQ ID NOs:167 and 168) had increased activity relative to the GPM promoter alone, when driving expression of the GUS reporter gene in conjunction with a  ̃100 bp of the 3′ region of the Yarrowia Xpr gene)).
The termination region can be derived 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 where they were derived). The termination region usually is selected more as a matter of convenience rather than because of any particular property. Preferably, the termination region is 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. Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included. Although not intended to be limiting, termination regions useful in the disclosure herein include:  ̃100 bp of the 3′ region of the Yarrowia lipolytica extracellular protease (XPR; GenBank Accession No. Ml 7741); the acyl-coA oxidase (Aco3: GenBank Accession No. AJ001301 and No. CM04661; Pox3: GenBank Accession No. XP—503244) terminators; the Pex20 (GenBank Accession No. AF054613) terminator; the Pex16 (GenBank Accession No. U75433) terminator; the Lip1 (GenBank Accession No. Z50020) terminator; the Lip2 (GenBank Accession No. AJ012632) terminator; and the 3-oxoacyl-coA thiolase (OCT; GenBank Accession No. X69988) terminator.
Additional copies (i.e., more than one copy) of the desaturase, elongase and/or acyltransferase genes described above may be introduced into Yarrowia lipolytica to thereby increase ARA production and accumulation. Specifically, additional copies of genes may be cloned within a single expression construct; and/or, additional copies of the cloned gene(s) may be introduced into the host cell by increasing the plasmid copy number or by multiple integration of the cloned gene into the genome (infra). For example, in one embodiment, a strain of Yarrowia lipolytica (i.e., strain Y2214) was engineered to produce greater than 14% ARA by the introduction and integration into the Yarrowia genome of chimeric genes comprising: 5 copies of a Δ9 elongase, 3 copies of a Δ8 desaturase, 4 copies of a Δ5 desaturase, 1 copy of a Δ12 desaturase and 1 copy of a C16/18 elongase. Similarly, in an alternate embodiment, strain Y2047 of Y. lipolytica was engineered to produce greater than 11% ARA by the introduction and integration into the Yarrowia genome of chimeric genes comprising: 1 copy of a Δ6 desaturase, 2 copies of a C18/20 elongase, 3 copies of a Δ5 desaturase and 1 copy of a Δ12 desaturase.
In general, once the DNA that is suitable for expression in an oleaginous yeast has been obtained (e.g., a chimeric gene comprising a promoter, ORF and terminator), it is placed in a plasmid vector capable of autonomous replication in a host cell; or, it 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. Although not relied on in the present invention, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus where constructs are targeted to an endogenous locus.
In the present invention, the preferred method of expressing genes in Yarrowia lipolytica is by integration of linear DNA into the genome of the host; and, integration into multiple locations within the genome can be particularly useful when high level expression of genes are desired. Toward this end, it is desirable to identify a sequence within the genome that is present in multiple copies.
Schmid-Berger et al. (J. Bact., 176(9):2477-2482 (1994)) discovered the first retrotransposon-like element Ylt1 in Yarrowia lipolytica. This retrotransposon is characterized by the presence of long terminal repeats (LTRs; each approximately 700 bp in length) called zeta regions. Ylt1 and solo zeta elements were present in a dispersed manner within the genome in at least 35 copies/genome and 50-60 copies/genome, respectively; both elements were determined to function as sites of homologous recombination. Further, work by Juretzek et al. (Yeast 18:97-113 (2001)) demonstrated that gene expression could be dramatically increased by targeting plasmids into the repetitive regions of the yeast genome (using linear DNA with LTR zeta regions at both ends), as compared to the expression obtained using low-copy plasmid transformants. Thus, zeta-directed integration can be ideal as a means to ensure multiple integration of plasmid DNA into Y. lipolytica, thereby permitting high-level gene expression. Unfortunately, however, not all strains of Y. lipolytica possess zeta regions (e.g., the strain identified as ATCC #20362). When the strain lacks such regions, it is also possible to integrate plasmid DNA comprising expression cassettes into alternate loci to reach the desired copy number for the expression cassette. For example, preferred alternate loci include: the Lys5 gene locus (GenBank Accession No. M34929), the Ura3 locus (GenBank Accession No. AJ306421), the Leu2 gene locus (GenBank Accession No. AF260230), the Aco2 gene locus (GenBank Accession No. AJ001300), the Pox3 gene locus (Pox3: GenBank Accession No. XP—503244; or, Aco3: GenBank Accession No. AJ001301), the Δ12 desaturase gene locus (SEQ ID NO:23), the Lip1 gene locus (GenBank Accession No. Z50020) and/or the Lip2 gene locus (GenBank Accession No. AJ012632).
Advantageously, the Ura3 gene can be used repeatedly in combination with 5-FOA selection (supra). More specifically, one can first knockout the native Ura3 gene to produce a strain having a Ura− phenotype, wherein selection occurs based on 5-FOA resistance. Then, a cluster of multiple chimeric genes and a new Ura3 gene could be integrated into a different locus of the Yarrowia genome to thereby produce a new strain having a Ura+ phenotype. Subsequent integration would produce a new Ura3− strain (again identified using 5-FOA selection), when the introduced Ura3 gene is knocked out. Thus, the Ura3 gene (in combination with 5-FOA selection) can be used as a selection marker in multiple rounds of transformation and thereby readily permit genetic modifications to be integrated into the Yarrowia genome in a facile manner.
For some applications, it will be useful to direct the instant proteins to different cellular compartments (e.g., the acyl-CoA pool versus the phosphatidylcholine pool). For the purposes described herein, ARA may be found as free fatty acids or in esterified forms such as acylglycerols, phospholipids, sulfolipids or glycolipids. It is envisioned that the chimeric genes described above encoding polypeptides that permit ARA biosynthesis may be further engineered to include appropriate intracellular targeting sequences.
Juretzek et al. (Yeast, 18:97-113 (2001)) note that the stability of integrated plasmid copy number in Yarrowia lipolytica is dependent on the individual transformants, the recipient strain and the targeting platform used. Thus, the skilled artisan will recognize that multiple transformants must be screened in order to obtain a strain displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA blots (Southern, J. Mol. Biol. 98:503 (1975)), Northern analysis of mRNA expression (Kroczek, J. Chromatogr. Biomed. Appl., 618(1-2):133-145 (1993)), Western analysis of protein expression, phenotypic analysis or GC analysis of the PUFA products.
In summary, each of the means described above is useful to increase the expression of a particular gene product (e.g., a desaturase, elongase, acyltransferase) in Yarrowia lipolytica; and, one skilled in the art of biotechnology will readily be capable of selecting the most appropriate combinations of methods to enable high production of ARA.
Pathway Engineering for Increased ARA Production
Although the methodology described above is useful to up-regulate the expression of individual heterologous genes, the challenge of increasing ARA production in Yarrowia lipolytica is much more complex and may require coordinated manipulation of various metabolic pathways. Manipulations in the PUFA biosynthetic pathway will be addressed first, followed by desirable manipulations in the TAG biosynthetic pathway and the TAG degradation pathway.
As previously described, the construction of a Yarrowia lipolytica strain producing greater than 5% ARA in the total oil fraction, or more preferably greater than 10% ARA in the total oil fraction, or even more preferably greater than 15-20% ARA in the total oil fraction, or most preferably greater than 25-30% ARA in the total oil fraction requires at least the following genes for expression of the Δ6 desaturase/Δ6 elongase pathway: a Δ6 desaturase, a C18/20 elongase and a Δ5 desaturase; or, at least the following genes for expression of the Δ9 elongase/Δ8 desaturase pathway: a Δ9 elongase, a Δ8 desaturase and a Δ5 desaturase. In either embodiment, however, it may be desirable to additionally include a Δ9 desaturase, a Δ12 desaturase, a C14/16 elongase and/or a C16/18 elongase in the host strain.
In some cases, it may prove advantageous to replace the native Yarrowia lipolytica Δ12 desaturase with the Fusarium moniliforme Δ12 desaturase, since the latter shows increased percent substrate conversion (WO 2005/047485). More specifically, although both Δ12 desaturases catalyze the conversion of oleic acid to LA, the two enzymes differ in their overall specificity (which thereby affects each enzyme's percent substrate conversion). The Applicants have determined that the F. moniliforme Δ12 desaturase has a higher loading capacity of LA onto the sn-2 position of a phosphotidylcholine substrate (thereby facilitating the subsequent reaction by Δ6 desaturase) than the Y. lipolytica Δ12 desaturase. On this basis, overexpression of the F. moniliforme Δ12 desaturase in conjunction with a knockout of the Y. lipolytica Δ12 desaturase may result in increased product for subsequent conversion to ARA.
In some embodiments, it may be useful to regulate the activity of a host organism's native DAG ATs to thereby enable manipulation of the percent of PUFAs within the lipids and oils of the Y. lipolytica host. Specifically, since oil biosynthesis is expected to compete with polyunsaturation during oleaginy, it is possible to reduce or inactivate the activity of an organism's one or more acyltransferases (e.g., PDAT and/or DGAT1 and/or DGAT2), to thereby reduce the overall rate of oil biosynthesis while concomitantly increasing the percent of PUFAs (relative to the total fatty acids) that are incorporated into the lipid and oil fractions. This results since polyunsaturation is permitted to occur more efficiently; or, in other words, by down-regulating the activity of specific DAG ATs, the substrate competition between oil biosynthesis and polyunsaturation is reduced in favor of polyunsaturation during oleaginy.
One skilled in the art will have the skills necessary to elucidate the optimum level of down-regulation and the means required to achieve such inhibition. For example, in some preferred embodiments, it may be desirable to manipulate the activity of a single DAG AT (e.g., create a DGAT1 knockout, while the activity of PDAT and DGAT2 are not altered). In alternate embodiments, the oleaginous organism comprises at total of “n” native DAG ATs and the activity of a total of “n-1” acyltransferases are modified to result in a reduced rate of oil biosynthesis, while the remaining acyltransferase retains its wildtype activity. And, in some situations, it may be desirable to manipulate the activity of all of the native DAG ATs in some preferred oleaginous organisms, to achieve the optimum rate of oil biosynthesis with respect to the rate of polyunsaturation.
In a similar manner, the Applicants hypothesize that expression of heterologous acyltransferases in conjunction with knockouts of the corresponding native Yarrowia lipolytica acyltransferase can significantly increase the overall ARA that is produced in the host cells. Specifically, as suggested previously, heterologous GPAT, LPMT, DGAT1, DGAT2, PDAT and LPCAT acyltransferases that have specificity for those fatty acids that are C20 and greater could be preferred over the native enzymes, since naturally produced PUFAs in Y. lipolytica are limited to 18:2 fatty acids and the native enzymes may not efficiently catalyze reactions with longer-chain fatty acids. Based on this conclusion, the Applicants identified the genes encoding GPAT, LPAAT, DGAT1 and DGAT2 in M. alpina and expressed these genes in engineered Yarrowia hosts producing EPA, resulting in increased PUFA biosynthesis (Examples 14-17 herein). Subsequently, the activity of several of the native acyltransferases (e.g., DGAT1 and DGAT2) in Y. lipolytica were diminished or knocked-out, as a means to reduce substrate competition between the native and heterologous acyltransferase. Similar results would be expected in an engineered Yarrowia host producing ARA.
One must also consider manipulation of pathways and global regulators that affect ARA production. For example, it is useful to increase the flow of carbon into the PUFA biosynthetic pathway by increasing the availability of the precursors of longer chain saturated and unsaturated fatty acids, such as palmitate (16:0) and stearic acid (18:0). The synthesis of the former is dependent on the activity of a C14/16 elongase, while the synthesis of the latter is dependent on the activity of a C16/18 elongase. Thus, over-expression of the native Yarrowia lipolytica C14/16 elongase (SEQ ID NOs:64 and 65) substantially increased the production of 16:0 and 16:1 fatty acids (22% increase relative to control strains); similarly, over-expression of the native Y. lipolytica C16/18 elongase (SEQ ID NOs:61 and 62) substantially increased the production of 18:0, 18:1, 18:2 and 18:3 fatty acids (18% increase relative to control strains) and reduced the accumulation of C16 fatty acids (22% decrease relative to control strains). Of course, as demonstrated herein and as suggested by the work of Inagaki, K. et al. (Biosci. Biotech. Biochem. 66(3):613-621 (2002)), in some embodiments of the present invention it may be useful to co-express a heterologous C16/18 elongase (e.g., from Rattus norvegicus [GenBank Accession No. AB071986; SEQ ID NOs:50 and 51 herein] and/or from M. alpina [SEQ ID NO:53 and 54. Thus, although a Y. lipolytica host strain must minimally be manipulated to express either a Δ6 desaturase, a C18/20 elongase and a Δ5 desaturase or a Δ9 elongase, a Δ8 desaturase and a Δ5 desaturase for ARA biosynthesis, in further preferred embodiments the host strain additionally includes at least one of the following: a Δ9 desaturase, a Δ12 desaturase, a C14/16 elongase and/or a C16/18 elongase.
In another preferred embodiment, those pathways that affect fatty acid degradation and TAG degradation can be modified in the Yarrowia lipolytica of the present invention, to mimimize the degradation of ARA that accumulates in the cells in either the acyl-CoA pool or in the TAG fraction. These pathways are represented by the acyl-CoA oxidase and lipase genes, respectively. More specifically, the acyl-CoA oxidases (EC 1.3.3.6) catalyze a peroxisomal β-oxidation reaction wherein each cycle of degradation yields an acetyl-CoA molecule and a fatty acid that is two carbon atoms shorter than the fatty acid substrate. Five acyl-CoA oxidase isozymes are present in Yarrowia lipolytica, encoded by the POX1, POX2, POX3, POX4 and POX5 genes (also known as the Aco1, Aco2, Aco3, Aco4 and Aco5 genes), corresponding to GenBank Accession Nos. AJ001299-AJ001303, respectively (see also corresponding GenBank Accession Nos. XP—504703, XP—505264, XP—503244, XP—504475 and XP—502199). Each of the isozymes has a different substrate specificity; for example, the POX3 gene encodes an acyl-CoA oxidase that is active against short-chain fatty acids, whereas the POX2 gene encodes an acyl-CoA oxidase that is active against longer-chain fatty acids (Wang H. J., et al. J. Bacteriol., 181:5140-5148 (1999)). It is contempalted that the activity of any one of these genes could be reduced or eliminated, to thereby modify peroxisomal β-oxidation in the host cell of the invention in a manner that could be advantageous to the purposes herein. Finally, to avoid any confusion, the Applicants will refer to the acyl-CoA oxidases as described above as POX genes, although this terminology can be used interchangeably with the Aco gene nomeclature, according to some publicly available literature.
Similarly, several lipases (EC 3.1.1.3) have been detected in Y. lipolytica, including intracellular, membrane-bound and extracellular enzymes (Choupina, A., et al. Curr. Genet. 35:297 (1999); Pignede, G., et al. J. Bacteriol. 182:2802-2810 (2000)). For example, Lip1 (GenBank Accession No. Z50020) and Lip3 (GenBank Accession No. AJ249751) are intracellular or membrane bound, while Lip2 (GenBank Accession No. AJ012632) encodes an extracellular lipase. Each of these lipases are targets for disruption, since the enzymes catalyze the reaction wherein TAG and water are degraded directly to DAG and a fatty acid anion.
In a further alternate embodiment, the activity of several phospholipases can be manipulated in the preferred host strain of Yarrowia lipolytica. Phospholipases play a critical role in the biosynthesis and degradation of membrane lipids. More specifically, the term “phospholipase” refers to a heterogeneous group of enzymes that share the ability to hydrolyze one or more ester linkage in glycerophospholipids. Although all phospholipases target phospholipids as substrates, each enzyme has the ability to cleave a specific ester bond. Thus, phospholipase nomeclature differentitates individual phospholipases and indicates the specific bond targeted in the phospholipid molecule. For example, phospholipase A1 (PLA1) hydrolyzes the fatty acyl ester bond at the sn-1 position of the glycerol moiety, while phospholipase A2 (PLA2) removes the fatty acid at the sn-2 position of this molecule. The action of PLA1 (EC 3.1.1.32) and PLA2 (EC 3.1.1.4) results in the accumulation of free fatty acids and 2-acyl lysophospholipid or 1-acyl lysophospholipid, respectively. Phospholipase C (PLC) (EC 3.1.4.3) hydrolyzes the phosphodiester bond in the phospholipid backbone to yield 1,2-DAG and, depending on the specific phospholipid species involved, phosphatidylcholine, phosphatidylethanolamine, etc. (e.g., PLC, is responsible for the reaction: 1-phosphatidyl-1 D-myo-inositol 4,5-bisphosphate+H2O=1D-myo-inositol 1,4,5-trisphosphate+DAG; ISC1 encodes an inositol phosphosphingolipid-specific phospholipase C [Sawai, H., et al. J. Biol. Chem. 275:39793-39798 (2000)]). The second phosphodiester bond is cleaved by phospholipase D (PLD) (EC 3.1.4.4) to yield phosphatidic acid and choline or ethanolamine, again depending on the phospholipid class involved. Phospholipase B (PLB) has the capability of removing both sn-1 and sn-2 fatty acids and is unique in having both hydrolase (wherein the enzyme cleaves fatty acids from both phospholipids [PLB activity] and lysophospholipids [lysophospholipase activity] for fatty acid release) and lysophospholipase-transacylase activities (wherein the enzyme can produce phospholipid by transferring a free fatty acid to a lysophospholipid). It may be useful to overexpress one or more of these phopsholipases, in order to increase the concentration of ARA that accumulates in the total oil fraction of the transformant Yarrowia host cells. It is hypothesized that this result will be observed because the phospholipases release acyl groups from PC into the CoA pool either for elongation or incorporation into triglycerides.
In another alternate embodiment, those enzymes in the CDP-choline pathway responsible for phosphatidylcholine (PC) biosynthesis can also be manipulated in the preferred host strain of Yarrowia lipolytica, as a means to increase overall ARA biosynthesis. The utility of this technique has been demonstrated by the overexpression of the Y. lipolytica CPT1 gene encoding diacylglycerol cholinephosphotransferase (EC 2.7.8.2), thereby resulting in increased EPA biosynthesis in an engineered strain of Y. lipolytica. One skilled in the art will be familiar with the PC biosynthetic pathway and recognize other appropriate candidate enzymes.
Although methods for manipulating biochemical pathways such as those described above are well known to those skilled in the art, an overview of some techniques for reducing or eliminating the activity of a native gene will be briefly presented below. These techniques would be useful to down-regulate the activity of the native Yarrowia lipolytica Δ12 desaturase, GPAT, LPMT, DGAT1, DGAT2, PDAT, LPCAT, acyl-CoA oxidase 2 (Aco2 or Pox2), acyl-CoA oxidase 3 (Aco3 or Pox3) and/or lipase genes, as discussed above.
Although one skilled in the art will be well equipped to ascertain the most appropriate technique to be utilized to reduce or eliminate the activity of a native gene, in general, the endogenous activity of a particular gene can be reduced or eliminated by, for example: 1.) disrupting the gene through insertion, substitution and/or deletion of all or part of the target gene; 2.) providing a cassette for transcription of antisense sequences to the gene's transcription product; 3.) using a host cell which naturally has [or has been mutated to have] little or none of the specific gene's activity; 4.) over-expressing a mutagenized hereosubunit (i.e., in an enzyme that comprises two or more hereosubunits), to thereby reduce the enzyme's activity as a result of the “dominant negative effect”; and 5.) using iRNA technology. In some cases, inhibition of undesired gene pathways can also be accomplished through the use of specific inhibitors (e.g., desaturase inhibitors such as those described in U.S. Pat. No. 4,778,630).
For gene disruption, a foreign DNA fragment (typically a selectable marker gene, but optionally a chimeric gene or chimeric gene cluster conveying a desirable phenotype upon expression) is inserted into the structural gene to be disrupted in order to interrupt its coding sequence and thereby functionally inactivate the gene. Transformation of the disruption cassette into the host cell results in replacement of the functional native gene by homologous recombination with the non-functional disrupted gene (see, for example: Hamilton et al., J. Bacteriol. 171:4617-4622 (1989); Balbas et al., Gene, 136:211-213 (1993); Gueldener et al., Nucleic Acids Res. 24:2519-2524 (1996); and Smith et al., Methods Mol. Cell. Biol. 5:270-277(1996)).
Antisense technology is another method of down-regulating genes when the sequence of the target gene is known. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the anti-sense strand of RNA will be transcribed. This construct is then introduced into the host cell and the antisense strand of RNA is produced. Antisense RNA inhibits gene expression by preventing the accumulation of mRNA that encodes the protein of interest. The person skilled in the art will know that special considerations are associated with the use of antisense technologies in order to reduce expression of particular genes. For example, the proper level of expression of antisense genes may require the use of different chimeric genes utilizing different regulatory elements known to the skilled artisan.
Although targeted gene disruption and antisense technology offer effective means of down-regulating genes where the sequence is known, other less specific methodologies have been developed that are not sequence-based (e.g., mutagenesis via UV radiation/chemical agents or use of transposable elements/transposons; see WO 04/101757).
In alternate embodiments, the endogenous activity of a particular gene can be reduced by manipulating the regulatory sequences controlling the expression of the protein. As is well known in the art, the regulatory sequences associated with a coding sequence include transcriptional and translational “control” nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of the coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Thus, manipulation of a particular gene's regulatory sequences may refer to manipulation of the gene's promoters, translation leader sequences, introns, enhancers, initiation control regions, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures. Thus, for example, the promoter of a DAG AT could be deleted or disrupted, in order to down-regulate the DAG AT's expression and thereby achieve a reduced rate of lipid and oil biosynthesis. Alternatively, the native promoter driving expression of a DAG AT could be substituted with a heterologous promoter having diminished promoter activity with respect to the native promoter. Methods useful for manipulating regulatory sequences are well known to those skilled in the art.
In summary, using the teachings provided herein, transformant oleaginous microbial hosts will produce at least about 5% ARA in the total lipids, preferably at least about 10% ARA in the total lipids, more preferably at least about 15% ARA in the total lipids, more preferably at least about 20% ARA in the total lipids and most preferably at least about 25-30% ARA in the total lipids.
Fermentation Processes for ARA Production
The transformed microbial host cell is grown under conditions that optimize expression of chimeric genes (e.g., encoding desaturases, elongases, acyltransferases, etc.) and produce the greatest and the most economical yield of ARA. In general, media conditions that may be optimized include the type and amount of carbon source, the type and amount of nitrogen source, the carbon-to-nitrogen ratio, the oxygen level, growth temperature, pH, length of the biomass production phase, length of the oil accumulation phase and the time of cell harvest. Yarrowia lipolytica are generally grown in complex media (e.g., 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 in the present invention must contain a suitable carbon source. Suitable carbon sources may include, but are not limited to: monosaccharides (e.g., glucose, fructose), disaccharides (e.g., lactose, sucrose), oligosaccharides, polysaccharides (e.g., starch, cellulose or mixtures thereof), sugar alcohols (e.g., glycerol) or mixtures from renewable feedstocks (e.g., cheese whey permeate, cornsteep liquor, sugar beet molasses, barley malt). Additionally, carbon sources may include alkanes, fatty acids, esters of fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids and various commercial sources of fatty acids including vegetable oils (e.g., soybean oil) and animal fats. Additionally, the carbon source may include one-carbon sources (e.g., carbon dioxide, methanol, formaldehyde, formate and carbon-containing amines) for which metabolic conversion into key biochemical intermediates has been demonstrated. Hence it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon-containing sources. Although all of the above mentioned carbon sources and mixtures thereof are expected to be suitable in the present invention, preferred carbon sources are sugars 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 or glutamate) source. 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 oleaginous yeast and promotion of the enzymatic pathways necessary for ARA production. Particular attention is given to several metal ions (e.g., Mn+2, Co+2, Zn+2, Mg+2) that promote synthesis of lipids and PUFAs (Nakahara, T. et al., Ind. Appl. Single Cell Oils, D. J. Kyle and R. Colin, eds. pp 61-97 (1992)).
Preferred growth media in the present invention are common commercially prepared media, such as Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.). Other defined or synthetic growth media may also be used and the appropriate medium for growth of Yarrowia lipolytica 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 7.0 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.
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 ARA in Yarrowia lipolytica. This approach is described in WO 2004/101757, as are various suitable fermentation process designs (i.e., batch, fed-batch and continuous) and considerations during growth.
Purification and Processing of ARA
PUFAs, including ARA, may be found in the host microorganism as free fatty acids or in esterified forms such as acylglycerols, phospholipids, sulfolipids or glycolipids, and 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 ARA and other PUFAs may include extraction 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 WO 2004/101757 for additional details.
Oils containing ARA that have been refined and/or purified can be hydrogenated, to thereby result in fats with various melting properties and textures. Many processed fats, including spreads, confectionary fats, hard butters, margarines, baking shortenings, etc., require varying degrees of solidity at room temperature and can only be produced through alteration of the source oil's physical properties. This is most commonly achieved through catalytic hydrogenation.
Hydrogenation is a chemical reaction in which hydrogen is added to the unsaturated fatty acid double bonds with the aid of a catalyst such as nickel. Hydrogenation has two primary effects. First, the oxidative stability of the oil is increased as a result of the reduction of the unsaturated fatty acid content. Second, the physical properties of the oil are changed because the fatty acid modifications increase the melting point resulting in a semi-liquid or solid fat at room temperature.
There are many variables which affect the hydrogenation reaction and which, in turn, alter the composition of the final product. Operating conditions including pressure, temperature, catalyst type and concentration, agitation and reactor design are among the more important parameters which can be controlled. Selective hydrogenation conditions can be used to hydrogenate the more unsaturated fatty acids in preference to the less unsaturated ones. Very light or brush hydrogenation is often employed to increase stability of liquid oils. Further hydrogenation converts a liquid oil to a physically solid fat. The degree of hydrogenation depends on the desired performance and melting characteristics designed for the particular end product. Liquid shortenings, used in the manufacture of baking products, solid fats and shortenings used for commercial frying and roasting operations, and base stocks for margarine manufacture are among the myriad of possible oil and fat products achieved through hydrogenation. A more detailed description of hydrogenation and hydrogenated products can be found in Patterson, H. B. W., Hydrogenation of Fats and Oils: Theory and Practice. The American Oil Chemists' Society, 1994.
ARA-Producing Strains if Y. lipolytica for use in Foodstuffs
The market place currently supports a large variety of food and feed products, incorporating ω-3 and/or ω-6 fatty acids (particularly ARA, EPA and DHA). It is contemplated that the yeast microbial oils of the invention comprising ARA will function in food and feed products to impart the health benefits of current formulations.
Microbial oils containing ω-3 and/or ω-6 fatty acids produced by the yeast hosts described herein will be suitable for use in a variety of food and feed products including, but not limited to food analogs, meat products, cereal products, baked foods, snack foods, and a dairy products. Additionally the present microbial oils may be used in formulations to impart health benefit in medical foods including medical nutritionals, dietary supplements, infant formula as well as pharmaceutical products. One of skill in the art of food processing and food formulation will understand how the amount and composition of the microbial oil may be added to the food or feed product. Such an amount will be referred to herein as an “effective” amount and will depend on the food or feed product, the diet that the product is intended to supplement or the medical condition that the medical food or medical nutritional is intended to correct or treat.
Food analogs can be made using processes well known to those skilled in the art. There can be mentioned meat analogs, cheese analogs, milk analogs and the like. Meat analogs made from soybeans contain soy protein or tofu and other ingredients mixed together to simulate various kinds of meats. These meat alternatives are sold as frozen, canned or dried foods. Usually, they can be used the same way as the foods they replace. Examples of meat analogs include, but are not limited to: ham analogs, sausage analogs, bacon analogs, and the like.
Food analogs can be classified as imitation or substitutes depending on their functional and compositional characteristics. For example, an imitation cheese need only resemble the cheese it is designed to replace. However, a product can generally be called a substitute cheese only if it is nutritionally equivalent to the cheese it is replacing and meets the minimum compositional requirements for that cheese. Thus, substitute cheese will often have higher protein levels than imitation cheeses and be fortified with vitamins and minerals.
Milk analogs or nondairy food products include, but are not limited to: imitation milks and nondairy frozen desserts (e.g., those made from soybeans and/or soy protein products).
Meat products encompass a broad variety of products. In the United States “meat” includes “red meats” produced from cattle, hogs and sheep. In addition to the red meats there are poultry items which include chickens, turkeys, geese, guineas, ducks and the fish and shellfish. There is a wide assortment of seasoned and processed meat products: fresh, cured and fried, and cured and cooked. Sausages and hot dogs are examples of processed meat products. Thus, the term “meat products” as used herein includes, but is not limited to, processed meat products.
A cereal food product is a food product derived from the processing of a cereal grain. A cereal grain includes any plant from the grass family that yields an edible grain (seed). The most popular grains are barley, corn, millet, oats, quinoa, rice, rye, sorghum, triticale, wheat and wild rice. Examples of a cereal food product include, but are not limited to: whole grain, crushed grain, grits, flour, bran, germ, breakfast cereals, extruded foods, pastas, and the like.
A baked goods product comprises any of the cereal food products mentioned above and has been baked or processed in a manner comparable to baking, i.e., to dry or harden by subjecting to heat. Examples of a baked good product include, but are not limited to: bread, cakes, doughnuts, bars, pastas, bread crumbs, baked snacks, mini-biscuits, mini-crackers, mini-cookies, and mini-pretzels. As was mentioned above, oils of the invention can be used as an ingredient.
A snack food product comprises any of the above or below described food products.
A fried food product comprises any of the above or below described food products that has been fried.
The beverage can be in a liquid or in a dry powdered form.
For example, there can be mentioned non-carbonated drinks; fruit juices, fresh, frozen, canned or concentrate; flavored or plain milk drinks, etc. Adult and infant nutritional formulas are well known in the art and commercially available (e.g., Similac®, Ensure®, Jevity®, and Alimentum® from Ross Products Division, Abbott Laboratories). Infant formulas are liquids or reconstituted powders fed to infants and young children. They serve as substitutes for human milk. Infant formulas have a special role to play in the diets of infants because they are often the only source of nutrients for infants. Although breast-feeding is still the best nourishment for infants, infant formula is a close enough second that babies not only survive but thrive. Infant formula is becoming more and more increasingly close to breast milk.
A dairy product is a product derived from milk. A milk analog or nondairy product is derived from a source other than milk, for example, soymilk as was discussed above. These products include, but are not limited to: whole milk, skim milk, fermented milk products such as yogurt or sour milk, cream, butter, condensed milk, dehydrated milk, coffee whitener, coffee creamer, ice cream, cheese, etc.
Additional food products into which the ARA-containing oils of the invention could be included are, for example: chewing gums, confections and frostings, gelatins and puddings, hard and soft candies, jams and jellies, white granulated sugar, sugar substitutes, sweet sauces, toppings and syrups, and dry-blended powder mixes.
Health Food Products, and Pharmaceuticals
A health food product is any food product that imparts a health benefit and include functional foods, medical foods, medical nutritionals and dietary supplements. Additionally, microbial oils of the invention may be used in standard pharmaceutical compositions. The present engineered strains of Yarrowia lipolytica or the microbial oils produced therefrom comprising ARA could readily be incorporated into the any of the above mentioned food products, to thereby produce e.g., a functional or medical food. For example more concentrated formulations comprising ARA include capsules, powders, tablets, softgels, gelcaps, liquid concentrates and emulsions which can be used as a dietary supplement in humans or animals other than humans.
Use in Dietary Supplements
More concentrated formulations comprising ARA include capsules, powders, tablets, softgels, gelcaps, liquid concentrates and emulsions which can be used as a dietary supplement in humans or animals other than humans. In particular, the ARA-oil of the present invention is particularly suitable for incorporation into dietary supplements such as infant formulas or baby food.
Infant formulas are liquids or reconstituted powders fed to infants and young children. “Infant formula” is defined herein as an enteral nutritional product which can be substituted for human breast milk in feeding infants and typically is composed of a desired percentage of fat mixed with desired percentages of carbohydrates and proteins in an aquous solution (e.g., see U.S. Pat. No. 4,670,285). Based on the worldwide composition studies, as well as levels specified by expert groups, average human breast milk typically contains about 0.20% to 0.40% of total fatty acids (assuming about 50% of calories from fat); and, generally the ratio of DHA to ARA would range from about 1:1 to 1:2 (see, e.g., formulations of Enfamil LIPILTM [Mead Johnson & Company] and Similac AdvanceTM [Ross Products Division, Abbott Laboratories]). Infant formulas have a special role to play in the diets of infants because they are often the only source of nutrients for infants; and, although breast-feeding is still the best nourishment for infants, infant formula is a close enough second that babies not only survive but thrive.
Use in Animal Feeds
Animal feeds are generically defined herein as products intended for use as feed or for mixing in feed for animals other than humans. And, as was mentioned above, the ARA-comprising oils of the invention can be used as an ingredient in various animal feeds.
More specifically, although not limited therein, it is expected that the oils of the invention can be used within pet food products, ruminant and poultry food products and aquacultural food products. Pet food products are those products intended to be fed to a pet [e.g., a dog, cat, bird, reptile, rodent]; these products can include the cereal and health food products above, as well as meat and meat byproducts, soy protein products, grass and hay products (e.g., alfalfa, timothy, oat or brome grass, vegetables). Ruminant and poultry food products are those wherein the product is intended to be fed to e.g., turkeys, chickens, cattle and swine. As with the pet foods above, these products can include cereal and health food products, soy protein products, meat and meat byproducts, and grass and hay products as listed above. And, aquacultural food products (or “aquafeeds”) are those products intended to be used in aquafarming which concerns the propagation, cultivation or farming of aquatic organisms and/or animals in fresh or marine waters.
It is contemplated that the present engineered strains of Yarrowia lipolytica that are producing high concentrations of ARA, EPA and/or DHA will be especially useful to include in most animal feed formulations. In addition to providing necessary ω-3 and/or ω-6 PUFAs, the yeast itself is a useful source of protein and other feed nutrients (e.g., vitamins, minerals, nucleic acids, complex carbohydrates, etc.) that can contribute to overall animal health and nutrition, as well as increase a formulation's palatablility. More specifically, Yarrowia lipolytica (ATCC #20362) has the following approximate chemical composition, as a percent relative to the dry cell weight: 35% protein, 40% lipid, 10% carbohydrate, 5% nucleic acids, 5% ash and 5% moisture. Furthermore, within the carbohydrate fraction, β-glucans comprise approximately 45.6 mg/g, mannans comprise approximately 11.4 mg/g, and chitin comprises approximately 52.6 mg/g (while trehalose is a minor component [approximately 0.7 mg/g]).
A considerable body of literature has examined the immuno-modulating effects of β-glucans, mannans and chitin. The means by which β-glucans, the primary constituents of bacterial and fungal cell walls, stimulate non-specific immunity (i.e., “immunostimulant effects”) to thereby improve health of aquaculture species, pets and farm animals and humans are best studied, although both chitin and mannans are similarly recognized as useful immunostimulants. Simplistically, an overall enhancement of immune response can be achieved by the use of β-glucans, since these β-1,3-D-polyglucose molecules stimulate the production of white blood cells (e.g., macrophages, neutrophils and monocytes) in a non-specific manner to thereby enable increased sensitivity and defense against a variety of pathogenic antigens or environmental stressors. More specifically, numerous studies have demonstrated that β-glucans: convey enhanced protection against viral, bacterial, fungal and parasitic infections; exert an adjuvant effect when used in conjunction with antibiotics and vaccines; enhance wound healing; counter damage resulting from free radicals; enhance tumor regression; modulate toxicity of bacterial endotoxins; and strengthen mucosal immunity (reviewed in Raa, J. et al., Norwegian Beta Glucan Research, Clinical Applications of Natural Medicine. Immune: Depressions Dysfunction & Deficiency (1990)). A sample of current literature documenting the utility of yeast β-glucans, mannans and chitins in both traditional animal husbandry and within the aquacultural sector include: L. A. White et al. (J. Anim. Sci. 80:2619-2628 (2002)), supplementation in weanling pigs; K. S. Swanson et al. (J. Nutr. 132:980-989 (2002)), supplementation in dogs; J. Ortuno et al. (Vet. Immunol. Immonopath. 85:41-50 (2002)), whole Saccharomyces cerevisiae administered to gilthead seabream; A. Rodriguez et al. (Fish Shell. Immuno. 16:241-249 (2004)), whole Mucor circinelloides administered to gilthead seabream; M. Bagni et al. (Fish Shell. Immuno. 18:311-325 (2005)), supplementation of sea bass with a yeast extract containing β-glucans; J. Raa (In: Cruz-Suárez, L. E., Ricque-Marie, D., Tapia-Salazar, M., Olvera-Novoa, M. A. y Civera-Cerecedo, R., (Eds.). Avances en Nutrición Acuicola V. Memorias del V Simposium Internacional de Nutrición Acuícola. 19-22 Noviembre, 2000. Mérida, Yucatán, Mexico), a review of the use of immune-stimulants in fish and shellfish feeds.
Based on the unique protein:lipid:carbohydrate composition of Yarrowia lipolytica, as well as unique complex carbohydrate profile (comprising an approximate 1:4:4.6 ratio of mannan:β-glucans:chitin), it is contemplated that the genetically engineered yeast cells of the present invention (or portions thereof) would be a useful additive to animal feed formulations (e.g., as whole [lyophilized] yeast cells, as purified cells walls, as purified yeast carbohydrates or within various other fractionated forms).
With respect to the aquaculture industry, an increased understanding of the nutritional requirements for various fish species and technological advances in feed manufacturing have allowed the development and use of manufactured or artificial diets (formulated feeds) to supplement or to replace natural feeds in the aquaculture industry. In general, however, the general proportions of various nutrients included in aquaculture feeds for fish include (with respect to the percent by dry diet): 32-45% proteins, 4-28% fat (of which at least 1-2% are ω-3 and/or ω-6 PUFAs), 10-30% carbohydrates, 1.0-2.5% minerals and 1.0-2.5% vitamins. A variety of other ingredients may optionally be added to the formulation. These include: (1) carotenoids, particularly for salmonid and ornamental “aquarium” fishes, to enhance flesh and skin coloration, respectively; (2) binding agents, to provide stability to the pellet and reduce leaching of nutrients into the water (e.g., beef heart, starch, cellulose, pectin, gelatin, gum arabic, locust bean, agar, carageenin and other alginates); (3) preservatives, such as antimicrobials and antioxidants, to extend the shelf-life of fish diets and reduce the rancidity of the fats (e.g., vitamin E, butylated hydroxyanisole, butylated hydroxytoluene, ethoxyquin, and sodium and potassium salts of propionic, benzoic or sorbic acids); (4) chemoattractants and flavorings, to enhance feed palatability and its intake; and, (5) other feedstuffs. These other feedstuffs can include such materials as fiber and ash (for use as a filler and as a source of calcium and phosphorus, respectively) and vegetable matter and/or fish or squid meal (e.g., live, frozen or dried algae, brine shrimp, rotifers or other zooplankton) to enhance the nutritional value of the diet and increase its acceptance by the fish. Nutrient Requirements of Fish (National Research Council, National Academy: Washington D.C., 1993) provides detailed descriptions of the essential nutrients for fish and the nutrient content of various ingredients.
The manufacture of aquafeed formulations requires consideration of a variety of factors, since a complete diet must be nutritionally balanced, palatable, water stable, and have the proper size and texture. With regard to nutrient composition of aquafeeds, one is referred to: Handbook on Ingredients for Aquaculture Feeds (Hertrampf, J. W. and F. Piedad-Pascual. Kluwer Academic: Dordrecht, The Netherlands, 2000) and Standard Methods for the Nutrition and Feeding of Farmed Fish and Shrimp (Tacon, A. G. J. Argent Laboratories: Redmond, 1990). In general, feeds are formulated to be dry (i.e., final moisture content of 6-10%), semi-moist (i.e., 35-40% water content) or wet (i.e., 50-70% water content). Dry feeds include the following: simple loose mixtures of dry ingredients (i.e., “mash” or “meals”); compressed pellets, crumbles or granules; and flakes. Depending on the feeding requirements of the fish, pellets can be made to sink or float. Semi-moist and wet feeds are made from single or mixed ingredients (e.g., trash fish or cooked legumes) and can be shaped into cakes or balls.
It is contemplated that the present engineered strains of Yarrowia lipolytica that are producing high concentrations of ARA will be especially useful to include in most aquaculture feeds. In addition to providing necessary ω-6 PUFAs, the yeast itself is a useful source of protein that can increase the formulation's palatablility. In alternate embodiments, the oils produced by the present strains of Y. lipolytica could be introduced directly into the aquaculture feed formulations, following extraction and purification from the cell mass.
DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention demonstrates the synthesis of up to 10-14% ARA in the total lipid fraction of the oleaginous yeast, Yarrowia lipolytica. As shown in FIG. 4, numerous strains of Y. lipolytica were created by integrating various genes into wildtype ATCC #20362 Y. lipolytica, wherein each transformant strain was capable of producing different amounts of PUFAs (including ARA). The complete lipid profiles of strains Y2034 and Y2047 (expressing the Δ6 desaturase/Δ6 elongase pathway) and strain Y2214 (expressing the Δ9 elongase/Δ8 desaturase pathway) are shown below in Table 10. Fatty acids are identified as 16:0, 16:1, 18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA and ARA; and the composition of each is presented as a % of the total fatty acids.







TABLE 10







Lipid Profile Of Yarrowia lipolytica Strains Y2034, Y2047 And Y2214









Fatty Acid Content















Strain
16:0
16:1
18:0
18:1
18:2
GLA
DGLA
ARA


















Y2034
13.1
8.1
1.7
7.4
14.8
25.2
8.3
11.2


Y2047
15.9
6.6
0.7
8.9
16.6
29.7
0.0
10.9


Y2214
7.9
15.3
0.0
13.7
37.5
0.0
7.9
14.0









A more detailed summary of the genetic modifications contained within strain Y2047 is described below (wherein complete details are provided in the Examples):

    
    
        (1) Expression of 1 copy of a Fusarium moniliforme Δ12 desaturase, within a FBA::F.Δ12::LIP2 chimeric gene;
        (2) Expression of 1 copy of a synthetic Δ6 desaturase gene (codon-optimized for expression in Y. lipolytica) derived from a Mortierella alpina Δ6 desaturase, within a TEF::Δ6S::LIP1 chimeric gene;
        (3) Expression of 1 copy of a synthetic Δ5 desaturase gene (codon-optimized for expression in Y. lipolytica) derived from a Homo sapiens Δ5 desaturase, within a TEF::H.D5S::PEX16 chimeric gene;
        (4) Expression of 1 copy of a synthetic high affinity C18/20 elongase gene (codon-optimized for expression in Y. lipolytica) derived from a Mortierella alpina high affinity C18/20 elongase, within a FBAIN::EL1S::PEX20 chimeric gene;
        (5) Expression of 1 copy of a synthetic C18/20 elongase gene (codon-optimized for expression in Y. lipolytica) derived from a Thraustochytrium aureum C18/20 elongase, within a TEF::EL2S::XPR chimeric gene; and,
        (6) Disruption of a native Y. lipolytica Leu2 gene encoding β-isopropylmalate dehydrogenase.
    
    


Similarly, a more detailed summary of the genetic modifications contained within strain Y2214 is described below (wherein complete details are provided in the Examples):

    
    
        (1) Expression of 5 copies of a synthetic Δ9 elongase (codon-optimized for expression in Y. lipolytica) derived from a Isochrysis galbana Δ9 elongase, within GPAT:: IgD9e::PEX20, TEF::IgD9e::LIP1, and FBAINm:: D9e::OCT chimeric genes;
        (2) Expression of 3 copies of a synthetic Δ8 desaturase (codon-optimized for expression in Y. lipolytica) derived from a Euglena gracillis Δ8 desaturase, within FBAIN::D8SF::PEX16 and GPD::D8SF::PEX16 chimeric genes;
        (3) Expression of 2 copies of a Mortierella alpina Δ5 desaturase, within GPAT::MAΔ5::PEX20 and FBAIN::MAΔ5::PEX20 chimeric genes;
        (4) Expression of 2 copies of a synthetic Δ5 desaturase gene (codon-optimized for expression in Y. lipolytica) derived from a Isochrysis galbana Δ5 desaturase, within YAT1::I.D5S::LIP1 and GPM/FBAIN::I.D5S::OCT chimeric genes;
        (5) Expression of 1 copy of a Fusarium moniliforme Δ12 desaturase, within a FBAIN::F.D12S::PEX20 chimeric gene;
        (6) Expression of 1 copy of a synthetic C16/18 elongase gene (codon-optimized for expression in Y. lipolytica) derived from a Rattus norvegicus rELO gene, within a GPM/FBAIN::rELO2S::OCT chimeric gene; and,
        (7) Disruption of a native Y. lipolytica Lys5 gene encoding saccharopine dehydrogenase.
    
    


Although the Applicants demonstrate production of 11% and 14% ARA, respectively, in these particular recombinant strains of Yarrowia lipolytica, it is contemplated that the concentration of ARA in the host cells could be dramatically increased via additional genetic modifications, according to the invention herein. Furthermore, on the basis of the teachings and results described herein, it is expected that one skilled in the art will recognize the feasability and commercial utility created by using oleaginous yeast as a production platform for the synthesis of a variety of ω-3 and/or ω-6 PUFAs, using the Δ6 desaturase/Δ6 elongase pathway and/or the Δ9 elongase/Δ8 desaturase pathway.
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) (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.
E. coli (XL1-Blue) competent cells were purchased from the Stratagene Company (San Diego, Calif.). E. coli strains were typically grown at 37° C. on Luria Bertani (LB) plates.
General molecular cloning was performed according to standard methods (Sambrook et al., supra). Oligonucleotides were synthesized by Sigma-Genosys (Spring, Tex.). Individual PCR amplification reactions were carried out in a 50 μl total volume, comprising: PCR buffer (containing 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.75), 2 mM MgSO4, 0.1% Triton X-100), 100 μg/mL BSA (final concentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmole of each primer and 1 μl of Pfu DNA polymerase (Stratagene, San Diego, Calif.), unless otherwise specified. Site-directed mutagenesis was performed using Stratagene's QuickChangeTM Site-Directed Mutagenesis kit, per the manufacturers' instructions. When PCR or site-directed mutagenesis 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.).
DNA sequence was generated on an ABI Automatic sequencer using dye terminator technology (U.S. Pat. No. 5,366,860; EP 272,007) using a combination of vector and insert-specific primers. Sequence editing was performed in Sequencher (Gene Codes Corporation, Ann Arbor, Mich.). All sequences represent coverage at least two times in both directions. Comparisons of genetic sequences were accomplished using DNASTAR software (DNA Star, Inc.). Alternatively, 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.
BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993) and Nucleic Acids Res. 25:3389-3402 (1997)) searches were conducted to identity isolated sequences having similarity to sequences contained in the BLAST “nr” database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the SWISS-PROT protein sequence database, EMBL and DDBJ databases). Sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the “nr” database, using the BLASTX algorithm (Gish, W. and States, D. J. Nature Genetics 3:266-272 (1993)) provided by the NCBI.
The results of BLAST comparisons summarizing the sequence to which a query sequence had the most similarity are reported according to the % identity, % similarity, and Expectation value. “% Identity” is defined as the percentage of amino acids that are identical between the two proteins. “% Similarity” is defined as the percentage of amino acids that are identical or conserved between the two proteins. “Expectation 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.
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).
Transformation and Cultivation of Yarrowia lipolytica 
Yarrowia lipolytica strains ATCC #20362, #76982 and #90812 were purchased from the American Type Culture Collection (Rockville, Md.). Y. lipolytica strains were usually grown at 28° C. on YPD agar (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar). Alternatively, “SD” media comprises: 0.67% yeast nitrogen base with ammonium sulfate, without amino acids and 2% glucose.
Transformation of Y. lipolytica was performed according to the method of Chen, D. C. et al. (Appl. Microbiol Biotechnol. 48(2):232-235 (1997)), unless otherwise noted. Briefly, Yarrowia was streaked onto a YPD plate and grown at 30° C. for approximately 18 hr. Several large loopfuls of cells were scraped from the plate and resuspended in 1 mL of transformation buffer containing: 2.25 mL of 50% PEG, average MW 3350; 0.125 mL of 2 M Li acetate, pH 6.0; 0.125 mL of 2 M DTT; and 50 μg sheared salmon sperm DNA. Then, approximately 500 ng of linearized plasmid DNA was incubated in 100 μl of resuspended cells, and maintained at 39° C. for 1 hr with vortex mixing at 15 min intervals. The cells were plated onto selection media plates and maintained at 30° C. for 2 to 3 days.
For selection of transformants, SD medium or 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). Supplements of adenine, leucine, lysine and/or uracil were added as appropriate to a final concentration of 0.01% (thereby producing “MMA”, “MMLe”, “MMLy” and “MMU” selection media, each prepared with 20 g/L agar).
Alternatively, transformants were selected on 5-fluoroorotic acid (“FOA”; also 5-fluorouracil-6-carboxylic acid monohydrate) selection media, comprising: 0.17% yeast nitrogen base (DIFCO Laboratories) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, 75 mg/L uracil, 75 mg/L uridine, 900 mg/L FOA (Zymo Research Corp., Orange, Calif.) and 20 g/L agar.
Finally, for the “two-stage growth conditions” designed to promote conditions of oleaginy, High Glucose Media (“HGM”) was prepared as follows: 14 g/L KH2PO4, 4 g/LK2HPO4, 2 g/L MgSO4.7H2O, 80 g/L glucose (pH 6.5). Strains were cultured under “two-stage growth conditions” according to the following protocol: first, cells were grown in triplicate in liquid MM at 30° C. with shaking at 250 rpm/min for 48 hrs. The cells were collected by centrifugation and the liquid supernatant was extracted. The pelleted cells were resuspended in HGM and grown for either 72 hrs or 96 hrs at 30° C. with shaking at 250 rpm/min. The cells were again collected by centrifugation and the liquid supernatant was extracted.
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 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
Identification of Promoters for High Expression in Yarrowia lipolytica 
Comparative studies investigating the promoter activities of the TEF, GPD, GPDIN, GPM, GPAT, FBA, FBAIN and YAT1 promoters were performed, by synthesizing constructs comprising each promoter and the E. coli gene encoding β-glucuronidase (GUS) as a reporter gene (Jefferson, R. A. Nature. 14(342):837-838 (1989)). Then, GUS activity was measured by histochemical and fluorometric assays (Jefferson, R. A. Plant Mol. Biol. Reporter 5:387-405 (1987)) and/or by using Real Time PCR for mRNA quantitation.
Construction of Plasmids Comprising a Chimeric Promoter::GUS::XPR Gene
Plasmid pY5-30 (FIG. 5A; SEQ ID NO:113) contained: a Yarrowia autonomous replication sequence (ARS18); a ColE1 plasmid origin of replication; an ampicillin-resistance gene (AmpR), for selection in E. coli; a Yarrowia LEU2 gene, for selection in Yarrowia; and a chimeric TEF::GUS::XPR gene. Based on this plasmid, a series of plasmids were created wherein the TEF promoter was replaced with a variety of other native Y. lipolytica promoters.
The putative promoter regions were amplified by PCR, using the primers shown below in Table 11 and either genomic Y. lipolytica DNA as template or a fragment of genomic DNA containing an appropriate region of DNA cloned into the pGEM-T-easy vector (Promega, Madison, Wis.).







TABLE 11







Construction of Plasmids Comprising A Chimeric Promoter::GUS::XPR Gene













Location With Respect to

Plasmid


Promoter
Primers
Gene
RE Sites
Name





GPD
YL211,
−968 bp to the ‘ATG’
SalI and
pYZGDG



YL212
translation initiation site of
NcoI



(SEQ ID
the gpd gene



NOs: 169 and
(SEQ ID NO: 158)



170)


GPDIN
YL376, YL377
−973 bp to +201 bp
PstI/NcoI
pDMW222



(SEQ ID NOs:
around the the gpd gene
(for



171 and 172)
(thereby including a 146 bp
promoter)




intron wherein the intron is
and PstI/




located at position +49 bp
SalI (for




to +194 bp) (SEQ ID NO:
vector)




159)


GPM
YL203, YL204
−875 bp to the ‘ATG’
NcoI and
pYZGMG



(SEQ ID NOs:
translation initiation site of
SalI



173 and 174)
the gpm gene (SEQ ID




NO: 160)


GPAT
GPAT-5-1,
−1130 bp to the ‘ATG’
SalI and
pYGPAT-



GPAT-5-2
translation initiation site of
NcoI
GUS



(SEQ ID NOs:
the gpat gene (SEQ ID



175 and 176)
NO: 164)


FBA
ODMW314,
−1001 bp to −1 bp around
NcoI and
pDMW212



YL341
the fba gene
SalI



(SEQ ID NOs:
(SEQ ID NO: 161)



177 and 178)


FBAIN
ODMW320,
−804 bp to +169 bp
NcoI and
pDMW214



ODMW341
around the fba gene (thereby
SalI



(SEQ ID NOs:
including a 102 bp intron



179 and 180)
wherein the intron is located




at position +62 bp to +165 bp)




(SEQ ID NO: 162)


YAT1
27203-F,
−778 bp to −1 bp around
HindIII and
pYAT-GUS



27203-R
the yat1 gene
SalI; also



(SEQ ID NOs:
(SEQ ID NO: 165)
NcoI and



181 and 182)

HindIII





Note:


The ‘A’ nucleotide of the ‘ATG’ translation initiation codon was designated as +1.






The individual PCR amplification reactions for GPD, GPDIN, GPM, FBA and FBAIN were carried out in a 50 μl total volume, as described in the General Methods. The thermocycler conditions were set for 35 cycles at 95° C. for 1 min, 56° C. for 30 sec and 72° C. for 1 min, followed by a final extension at 72° C. for 10 min.
The PCR amplification for the GPAT promoter was carried out in a 50 μl total volume using a 1:1 dilution of a premixed 2×PCR solution (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 of each primer, 50 ng template and 1.25 U of TaKaRa Ex TaqTM DNA polymerase (Takara Mirus Bio, Madison, Wis.). The thermocycler conditions were set for 30 cycles at 94° C. for 2.5 min, 55° C. for 30 sec and 72° C. for 2.5 min, followed by a final extension at 72° C. for 6 min.
The PCR amplification for the YAT1 promoter was carried out in a composition comparable to that described above for GPAT. 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, followed by a final extension for 7 min at 72° C.
Each PCR product was purified using a Qiagen PCR purification kit and then digested with restriction enzymes (according to the Table above using standard conditions) and the digested products were purified following gel electrophoresis in 1% (w/v) agarose. The digested PCR products (with the exception of those from YAT1) were then ligated into similarly digested pY5-30 vector. Ligated DNA from each reaction was then used to individually transform E. coli Top10, E. coli DH10B or E. coli DH5α. Transformants were selected on LB agar containing ampicillin (100 μg/mL).
YAT1 required additional manipulation prior to cloning into pY5-30. Specifically, upon digestion of the YAT1 PCR product with HindIII and SalI, a  ̃600 bp fragment resulted; digestion with NcoI and HindIII resulted in a  ̃200 bp fragment. Both products were isolated and purified. Then, 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.
Analysis of the plasmid DNA from each transformation reaction confirmed the presence of the expected plasmid. These plasmids were designated as follows: PYZGDG (comprising a GPD::GUS::XPR chimeric gene), pDMW222 (comprising a GPDIN::GUS::XPR chimeric gene), pYZGMG (comprising a GPM::GUS::XPR chimeric gene), pYGPAT-GUS (comprising a GPAT::GUS::XPR chimeric gene), pDMW212 (comprising a FBA::GUS::XPR chimeric gene), pDMW214 (comprising a FBAIN::GUS::XPR chimeric gene) and pYAT-GUS (comprising a YAT1::GUS::XPR chimeric gene).
Each of the plasmids above, and additionally plasmid pY5-30 (comprising a TEF::GUS::XPR chimeric gene), was transformed separately into Y. lipolytica as described in the General Methods. The Y. lipolytica host was either Y. lipolytica ATCC #76982 or Y. lipolytica ATCC #20362, strain Y2034 (infra [Example 4], capable of producing 10% ARA via the Δ6 desaturase/Δ6 elongase pathway). All transformed cells were plated onto minimal media plates lacking leucine and maintained at 30° C. for 2 to 3 days.
Comparative Analysis of Yarrowia Promoters by Histochemical Analysis of GUS Expression
Yarrowia lipolytica ATCC #76982 strains containing plasmids pY5-30, pYZGDG, pYZGMG, pDMW212 and pDMW214 were grown from single colonies in 3 mL MM at 30° C. to an OD600 ̃1.0. Then, 100 μl of cells were collected by centrifugation, resuspended in 100 μl of histochemical staining buffer, and incubated at 30° C. Staining buffer was prepared by dissolving 5 mg of 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) in 50 μl dimethyl formamide, followed by the addition of 5 mL 50 mM NaPO4, pH 7.0. The results of histochemical staining (FIG. 5B) showed that the TEF promoter in construct pY5-30, the GPD promoter in construct pYZGDG, the GPM promoter in construct pYZGMG, the FBA promoter in construct pDMW212, and the FBAIN promoter in construct pDMW214 were all active. Both the FBA and FBAIN promoters appeared to be much stronger than all the other promoters, with the FBAIN promoter having the strongest promoter activity.
In a separate experiment, Y. lipolytica Y2034 strains containing plasmids pY5-30, pYGPAT-GUS, pYAT-GUS and pDMW214 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 (supra). 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 GPAT promoter in construct pYGPAT-GUS was active, as was the YAT1 promoter in construct pYAT-GUS, when grown in SD medium for 24 hrs (FIG. 5C, “24 hr in SD medium”). Comparatively, the GPAT promoter appeared to be much stronger than the TEF promoter and had diminished activity with respect to the FBAIN promoter. Likewise, the YAT1 promoter appeared to be stronger than the TEF promoter 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. 5C, “24 hr in HG medium”). This remained true after 120 hrs in HGM (FIG. 5C, “120 hr in HG medium”). Thus, the YAT1 promoter appeared to be induced in HGM, a medium that promotes oleaginous growth conditions due to nitrogen limitation.
Comparative Analysis of Yarrowia Promoters by Fluorometric Assay of GUS Expression
GUS activity was also assayed by fluorometric determination of the production of 4-methylumbelliferone (4-MU) from the corresponding substrate β-glucuronide (Jefferson, R. A. Plant Mol. Biol. Reporter 5:387-405 (1987)).
Yarrowia lipolytica ATCC #76982 strains containing plasmids pY5-30, PYZGDG, PYZGMG, pDMW212 and pDMW214 were grown from single colonies in 3 mL MM (as described above) at 30° C. to an OD600 ̃1.0. Then, the 3 mL cultures were each added to a 500 mL flask containing 50 mL MM and grown in a shaking incubator at 30° C. for about 24 hrs. The cells were collected by centrifugation, resuspended in Promega Cell Lysis Buffer and lysed using the BIO 101 Biopulverizer system (Vista, Calif.). After centrifugation, the supernatants were removed and kept on ice.
Similarly, Y. lipolytica strain Y2034 containing plasmids pY5-30, pYAT-GUS, pYGPAT-GUS and pDMW214 constructs, respectively, 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. 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 the 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, 100 μl of extract was added to 700 μl of GUS assay buffer (2 mM 4-methylumbelliferyl-β-D-glucuronide (“MUG”) in extraction buffer) or 200 μl of extract was added to 800 μl of GUS assay buffer. The mixtures were 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 CytoFluor Series 4000 Fluorescence Multi-Well Plate Reader (PerSeptive Biosystems, 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 10 μl of extract and 200 μl of BioRad Bradford reagent or 20 μl of extract and 980 μl of BioRad Bradford reagent (Bradford, M. M. Anal. Biochem. 72:248-254 (1976)). GUS activity was expressed as nmoles of 4-MU per minute per mg of protein.
Results of these fluorometric assays designed to compare the TEF, GPD, GPM, FBA and FBAIN promoters in Y. lipolytica ATCC #76982 strains are shown in FIG. 6A. Specifically, the FBA promoter was 2.2 times stronger than the GPD promoter in Y. lipolytica. Additionally, the GUS activity of the FBAIN promoter was about 6.6 times stronger than the GPD promoter.
Results of these fluorometric assays designed to compare the TEF, GPAT, YAT1 and FBAIN promoters in Y. lipolytica strain Y2034 are shown in the Table below.







TABLE 12







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.
Comparative Analysis of Yarrowia Promoters by Quantitative PCR Analyses of GUS Expression
The transcriptional activities of the TEF, GPD, GPDIN, FBA and FBAIN promoters were determined in Y. lipolytica containing the pY5-30, PYZGDG, pDMW222, pDMW212 and pDMW214 constructs by quantitative PCR analyses. This required isolation of RNA and real time RT-PCR.
More specifically, Y. lipolytica ATCC #76982 strains containing pY5-30, PYZGDG, pDMW222, pDMW212 and pDMW214 were grown from single colonies in 6 mL of MM in 25 mL Erlenmeyer flasks for 16 hrs at 30° C. Each of the 6 mL starter cultures was then added to individual 500 mL flasks containing 140 mL HGM and incubated at 30° C. for 4 days. In each interval of 24 hrs, 1 mL of each culture was removed from each flask to measure the optical density, 27 mL was removed and used for a fluorometric GUS assay (as described above), and two aliquots of 1.5 mL were removed for RNA isolation. The culture for RNA isolation was centrifuged to produce a cell pellet.
The RNA was isolated from Yarrowia strains according to the modified Qiagen RNeasy mini protocol (Qiagen, San Diego, Calif.). Briefly, at each time point for each sample, 340 μL of Qiagen's buffer RLT was used to resuspend each of the two cell pellets. The buffer RLT/cell suspension mixture from each of the two tubes was combined in a bead beating tube (Bio101, San Diego, Calif.). About 500 μL of 0.5 mL glass beads was added to the tube and the cells were disrupted by bead beating 2 min at setting 5 (BioPulverizer, Bio101 Company, San Diego, Calif.). The disrupted cells were then pelleted by centrifugation at 14,000 rpm for 1 min and 350 μl of the supernatent was transferred to a new microcentrifuge tube. Ethanol (350 μL of 70%) was added to each homogenized lysate. After gentle mixing, the entire sample was added to a RNeasy mini column in a 2 mL collection tube. The sample was centrifuged for 15 sec at 10,000 rpm. Buffer RW1 (350 μL) was added to the RNeasy mini column and the column was centrifuged for 15 sec at 10,000 rpm to wash the cells. The eluate was discarded. Qiagen's DNasel stock solution (10 μL) was added to 70 μl of Buffer RDD and gently mixed. This entire DNase solution was added to the RNeasy mini column and incubated at room temperature for 15 min. After the incubation step, 350 μL of Buffer RW1 was added to the mini column and the column was centrifuged for 15 sec at 10,000 rpm. The column was washed twice with 700 μL Buffer RW1. RNase-free water (50 μL) was added to the column. The column was centrifuged for 1 min at 10,000 rpm to elute the RNA.
A two-step RT-PCR protocol was used, wherein total Yarrowia RNA was first converted to cDNA and then cDNA was analyzed using Real Time PCR. The conversion to cDNA was performed using Applied Biosystems' High Capacity cDNA Archive Kit (PN#4322171; Foster City, Calif.) and Molecular Biology Grade water from MediaTech, Inc. (PN# 46-000-Con; Holly Hill, Fla.). Total RNA from Yarrowia (100 ng) was converted to cDNA by combining it with 10 μl of RT buffer, 4 μl of 25×dNTPs, 10 μl 10× Random Hexamer primers, 5 μl Multiscribe Reverse Transcriptase and 0.005 μl RNase Inhibitor, and brought to a total reaction volume of 100 μl with water. The reactions were incubated in a thermocycler for 10 min at 25° C. followed by 2 hrs at 37° C. The cDNA was stored at −20° C. prior to Real Time analysis.
Real Time analysis was performed using the SYBR Green PCR Master Mix from Applied Biosystems (PN# 4309155). The Reverse Transcription reaction (2 μl) was added to 10 μl of 2×SYBR PCR Mix, 0.2 μl of 100 μM Forward and Reverse primers for either URA (i.e., primers YL-URA-16F and YL-URA-78R [SEQ ID NOs:183 and 184]) or GUS (i.e., primers GUS-767F and GUS-891R [SEQ ID NO:185 and 186]) and 7.2 μl water. The reactions were thermocycled for 10 min at 95° C. followed by 40 cycles of 95° C. for 5 sec and 60° C. for 1 min in an ABI 7900 Sequence Detection System instrument. Real time fluorescence data was collected during the 60° C. extension during each cycle.
Relative quantitation was performed using the ΔΔCT method as per User Bulletin #2: “Relative Quantitation of Gene Expression”, Applied Biosystems, Updated 10/2001. The URA gene was used for normalization of GUS expression. In order to validate the use of URA as a normalizer gene, the PCR efficiency of GUS and URA were compared and they were found to be 1.04 and 0.99, respectively (where 1.00 equals 100% efficiency). Since the PCR efficiencies were both near 100%, the use of URA as a normalizer for GUS expression was validated, as was the use of the ΔΔCT method for expression quantitation. The normalized quantity is referred to as the ACT.
The GUS mRNA in each different strain (i.e., Y. lipolytica ATCC #76982 strains containing the pYZGDG, pDMW222, pDMW212 and pDMW214 constructs) was quantified to the mRNA level of the strain with pY5-30 (TEF::GUS). Thus, relative quantitation of expression was calculated using the mRNA level of the strain with TEF::GUS as the reference sample. The normalized value for GPD::GUS, GPDIN::GUS, FBA::GUS and FBAIN::GUS was compared to the normalized value of the TEF::GUS reference. This quantity is referred to as the ΔΔCT. The ΔΔCT values were then converted to absolute values by utilizing the formula 2−ΔCT. These values refer to the fold increase in the mRNA level of GUS in the strains comprising the chimeric GPD::GUS, GPDIN::GUS, FBA::GUS and FBAIN::GUS genes, as compared to the chimeric TEF::GUS gene. Using this methodology, it was possible to compare the activity of the TEF promoter to the GPD, GPDIN, FBA and FBAIN promoters.
The results of the relative quantitation of mRNA for each GUS chimeric gene are shown in FIG. 6B. More specifically, the assay showed that after 24 hrs in HGM, the transcription activity of FBA and FBAIN promoters was about 3.3 and 6 times stronger than the TEF promoter, respectively. Similarly, the transcription activity of the GPD and GPDIN promoters is about 2 and 4.4 times stronger than the TEF promoter, respectively. While the transcription activities of the FBA::GUS, FBAIN::GUS, GPD::GUS and GPDIN::GUS gene fusion decreased over the 4 day period of the experiment, the transcriptional activity of the FBAIN and GPDIN promoters was still about 3 and 2.6 times stronger than the TEF promoter in the final day of the experiment.
Example 2
Identification of Enhancers Useful to Increase Gene Transcription in Yarrowia lipolytica 
Based on the strong promoter activities of FBAIN and GPDIN (wherein activity was greater than that of the FBA and GPD promoters, respectively) and the identification of an intron within each promoter region, the present work was conducted to determine whether enhancers were present in each intron.
Specifically, two chimeric promoters consisting of a GPM::FBAIN promoter fusion and a GPM::GPDIN promoter fusion were generated to drive expression of the GUS reporter gene. The chimeric promoters (comprised of a “component 1” and a “component 2”) are described below in Table 13.







TABLE 13







Construction of Plasmids Comprising A Chimeric Promoter Within


A Chimeric Promoter::GUS::XPR Gene










Chimeric


Plasmid


Promoter
Component 1
Component 2
Name





GPM::FBAIN
−1 bp to
+1 bp to +171 bp region
pDMW224


(SEQ ID NO:
−843 bp
of FBAIN, wherein the


167)
region
intron is located at



of GPM
position +62 bp to +165




bp


GPM::GPDIN
−1 bp to
+1 bp to +198 bp region
pDMW225


(SEQ ID NO:
−843 bp
of GPDIN, wherein the


168)
region
intron is located at



of GPM
position +49 bp to +194




bp









The chimeric promoters were positioned such that each drove expression of the GUS reporter gene in plasmids pDMW224 and pDMW225.

The activities of the GPM::FBAIN promoter and the GPM::GPDIN promoter were compared with the TEF, FBAIN, GPDIN and GPM promoters by comparing the GUS activity in Y. lipolytica strains comprising pDMW224 and pDMW225 relative to the GUS activity in Y. lipolytica strains comprising pY5-30, PYZGDG, pYZGMG and pDMW214 constructs based on results from histochemical assays (as described in Example 1). As previously determined, the FBAIN promoter was the strongest promoter. However, the chimeric GPM::FBAIN promoter and the chimeric GPM::GPDIN promoter were both much stronger than the GPM promoter and appeared to be equivalent in activity to the GPDIN promoter. Thus, this confirmed the existence of an enhancer in both the GPDIN promoter and the FBAIN promoter.
One skilled in the art would readily be able to construct similar chimeric promoters, using either the GPDIN intron or the FBAIN intron.
Example 3
Sulfonylurea Selection
Genetic improvement of Yarrowia has been hampered by the lack of suitable non-antibiotic selectable transformation markers. The present Example describes the development of a dominant, non antibiotic marker for Y. lipolytica based on sulfonylurea resistance that is also generally applicable to industrial yeast strains that may be haploid, diploid, aneuploid or heterozygous.
Theory and Initial Sensitivity Screening
Acetohydroxyacid synthase (AHAS) is the first common enzyme in the pathway for the biosynthesis of branched-chain amino acids. It is the target of the sulfonylurea and imidazolinone herbicides. As such, sulfonyl urea herbicide resistance has been reported in both microbes and plants. For example, in Saccharomyces cerevisiae, the single W586L mutation in AHAS confers resistance to sulfonylurea herbicides (Falco, S. C., et al., Dev. Ind. Microbiol. 30:187-194 (1989); Duggleby, R. G., et. al. Eur. J. Biochem. 270:2895 (2003)).
When the amino acid sequences of wild type AHAS Y. lipolytica (GenBank Accession No. XP—501277) and S. cerevisiae (GenBank Accession No. P07342) enzymes were aligned, the Trp amino acid residue at position 586 of the S. cerevisiae enzyme was equivalent to the Trp residue at position 497 of the Y. lipolytica enzyme. It was therefore hypothesized that W497L mutation in the Y. lipolytica enzyme would likely confer sulfonylurea herbicide resistance, if the wild type cells were themselves sensitive to sulfonylurea. Using methodology well known to those of skill in the art, it was determined that sulfonylurea (chlorimuron ethyl) at a concentration of 100 μg/mL in minimal medium was sufficient to inhibit growth of wild type Y. lipolytica strains ATCC #20362 and ATCC #90812.
Synthesis of a Mutant W497L AHAS Gene
The Y. lipolytica AHAS gene containing the W497L mutation (SEQ ID NO:280) was created from genomic DNA in a two-step reaction. First, the 5′ portion of the AHAS gene was amplified from genomic DNA using Pfu UltraTM High-Fidelity DNA Polymerase (Stratagene, Catalog #600380) and primers 410 and 411 [SEQ ID NOs:365 and 366]; the 3′ portion of the gene was amplified similarly using primers 412 and 413 [SEQ ID NOs:367 and 368]. The two pairs of primers were overlapping such that the overlapping region contained the W497L mutation (wherein the mutation was a ‘CT’ change to ‘TG’).
The 5′ and 3′ PCR products of the correct size were gel purified and used as the template for the second round of PCR, wherein the entire mutant gene was amplified using primers 414 and 415 (SEQ ID NOs:369 and 370) and a mixture of the products from the two primary PCR reactions. This mutant gene carried its own native promoter and terminator sequences. The second round PCR product of the correct size was gel purified and cloned by an in-fusion technique into the vector backbone of plasmid pY35 [containing a chimeric TEF::Fusarium moniliforme Δ12 desaturase (Fm2) gene, the E. coli origin of replication, a bacterial ampicillin resistance gene, the Yarrowia Leu 2 gene and the Yarrowia autonomous replication sequence (ARS); see WO 2005/047485 for additional details], following its digestion with enzymes SalI/BsiWI. The in-fusion reaction mixture was transformed into TOP10 competent cells (Invitrogen, Catalog #C4040-10). After one day selection on LB/Amp plates, eight (8) colonies were analyzed by DNA miniprep. Seven clones were confirmed to be correct by restriction digest. One of them that contained the sulfonylurea resistance gene as well as the LEU gene was designated “pY57” (or “pY57.YI.AHAS.w4971”; FIG. 7A).
Wild type Y. lipolytica strains ATCC #90812 and #20362 were transformed with pY57 and ‘empty’ LEU by a standard Lithium Acetate method. Transformation controls comprising ‘No-DNA’ were also utilized. Transformants were plated onto either MM or MM+sulfonylurea (SU; 100 μg/mL) agar plates and the presence or absence of colonies was evaluated following four days of growth.







TABLE 14







AHAS Selection In Yarrowia lipolytica










ATCC #90812
ATCC #20362













MM + SU

MM + SU


Plasmid
MM
(100 μg/mL)
MM
(100 μg/mL)





pY57
colonies
colonies
colonies
colonies


Leu vector
colonies
No colonies
colonies
No colonies


control


No DNA control
No
No colonies
No colonies
No colonies



colonies









Based on the results shown above, AHAS W497L was a good non-antibiotic selection marker in both Y. lipolytica ATCC #90812 and #20362. Subsequently, Applicants used a sulfonylurea concentration of 150 μg/mL. This new marker is advantageous for transforming Y. lipolytica since it does not rely on a foreign gene but on a mutant native gene and it neither requires auxotrophy nor results in auxotrophy. The herbicide is non-toxic to humans and animals.
It is expected that this selection method will be generally applicable to other industrial yeast strains that may be haploid, diploid, aneuploid or heterozygous, if mutant AHAS enzymes were created in a manner analogous to that described herein.
Example 4
Δ6 Desaturase/Δ6 Elongase Pathway: Generation of Y2034 and Y2047 Strains to Produce about 10-11% ARA of Total Lipids
The present Example describes the construction of strains Y2034 and Y2047, derived from Yarrowia lipolytica ATCC #20362, capable of producing 10 and 11% ARA, respectively, relative to the total lipids (FIG. 4). These strains were both engineered to express the Δ6 desaturase/Δ6 elongase pathway; thus, it was not unexpected that analysis of the complete lipid profiles of strains Y2034 and Y2047 indicated co-synthesis of 25-29% GLA.
The development of strains Y2034 and Y2047 first required the construction of strain M4 (producing 8% DGLA).
Generation of M4 Strain to Produce about 8% DGLA of Total Lipids
Construct pKUNF12T6E (FIG. 7B; SEQ ID NO:114) was generated to integrate four chimeric genes (comprising a Δ12 desaturase, a Δ6 desaturase and two C18/20 elongases) into the Ura3 loci of wild type Yarrowia strain ATCC #20362, to thereby enable production of DGLA. The pKUNF12T6E plasmid contained the following components:







TABLE 15







Description of Plasmid pKUNF12T6E (SEQ ID NO: 114)








RE Sites



And


Nucleotides


Within


SEQ ID


NO: 114
Description Of Fragment And Chimeric Gene Components





AscI/BsiWI
784 bp 5′ part of Yarrowia Ura3 gene (GenBank Accession


(9420-8629)
No. AJ306421)


SphI/PacI
516 bp 3′ part of Yarrowia Ura3 gene (GenBank Accession


(12128-1)
No. AJ306421)


SwaI/BsiWI
FBAIN::EL1S::Pex20, comprising:


(6380-8629)
FBAIN: FBAIN promoter (SEQ ID NO: 162)



EL1S: codon-optimized elongase 1 gene (SEQ ID



NO: 19), derived from Mortierella alpina (GenBank



Accession No. AX464731)



Pex20: Pex20 terminator sequence from Yarrowia



Pex20 gene (GenBank Accession No. AF054613)


BglII/SwaI
TEF:: Δ6S::Lip1, comprising:


(4221-6380)
TEF: TEF promoter (GenBank Accession No.



AF054508)



Δ6S: codon-optimized Δ6 desaturase gene (SEQ ID



NO: 3), derived from Mortierella alpina (GenBank



Accession No. AF465281)



Lip1: Lip1 terminator sequence from Yarrowia Lip1



gene (GenBank Accession No. Z50020)


PmeI/ClaI
FBA::F. Δ12::Lip2, comprising:


(4207-1459)
FBA: FBA promoter (SEQ ID NO: 161)



F. Δ12: Fusarium moniliforme Δ12 desaturase gene



(SEQ ID NO: 27)



Lip2: Lip2 terminator sequence from Yarrowia Lip2



gene (GenBank Accession No. AJ012632)


ClaI/PacI
TEF::EL2S::XPR, comprising:


(1459-1)
TEF: TEF promoter (GenBank Accession No.



AF054508)



EL2S: codon-optimized elongase gene (SEQ ID



NO: 22), derived from Thraustochytrium aureum (U.S.



Pat. No. 6,677,145)



XPR: ~100 bp of the 3′ region of the Yarrowia Xpr gene



(GenBank Accession No. M17741)









The pKUNF12T6E plasmid was digested with AscI/SphI, and then used for transformation of wild type Y. lipolytica ATCC #20362 according to the General Methods. The transformant cells were plated onto FOA selection media plates and maintained at 30° C. for 2 to 3 days. The FOA resistant colonies were picked and streaked onto MM and MMU selection plates. The colonies that could grow on MMU plates but not on MM plates were selected as Ura− strains. Single colonies of Ura− strains were then inoculated into liquid MMU at 30° C. and shaken at 250 rpm/min for 2 days.
The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed the presence of DGLA in the transformants containing the 4 chimeric genes of pKUNF12T6E, but not in the wild type Yarrowia control strain. Most of the selected 32 Ura− strains produced about 6% DGLA of total lipids. There were 2 strains (i.e., strains M4 and 13-8) that produced about 8% DGLA of total lipids.
Generation of Y2034 and Y2047 Strains to Produce about 10% ARA of Total Lipids
Constructs pDMW232 (FIG. 7C; SEQ ID NO:115) and pDMW271 (FIG. 7D; SEQ ID NO:116) were generated to integrate either two or three Δ5 chimeric genes into the Leu2 gene of Yarrowia strain M4, respectively.
The plasmids pDMW232 and pDMW271 contained the following components, as described in Tables 16 and 17, respectively:







TABLE 16







Description of Plasmid pDMW232 (SEQ ID NO: 115)








RE Sites



And


Nucleotides


Within SEQ


ID NO: 115
Description Of Fragment And Chimeric Gene Components





AscI/BsiWI
788 bp 5′ part of Yarrowia Leu2 gene (GenBank Accession


(5550-4755)
No. AF260230)


SphI/PacI
703 bp 3′ part of Yarrowia Leu2 gene (GenBank Accession


(8258-8967)
No. AF260230)


SwaI/BsiWI
FBAIN::MAΔ5::Pex20, comprising:


(2114-4755)
FBAIN: FBAIN promoter (SEQ ID NO: 162)



MAΔ5: Mortierella alpina Δ5 desaturase gene (SEQ ID



NO: 6) (GenBank Accession No. AF067654)



Pex20: Pex20 terminator sequence of Yarrowia Pex20



gene (GenBank Accession No. AF054613)


SwaI/ClaI
TEF::MAΔ5::Lip1, comprising:


(2114-17)
TEF: TEF promoter (GenBank Accession No.



AF054508)



MAΔ5: SEQ ID NO: 6 (supra)



Lip1: Lip1 terminator sequence of Yarrowia Lip1 gene



(GenBank Accession No. Z50020)


PmeI/ClaI
Yarrowia Ura3 gene (GenBank Accession No. AJ306421)


(5550-4755)
















TABLE 17







Description of Plasmid pDMW271 (SEQ ID NO: 116)








RE Sites And



Nucleotides


Within SEQ
Description Of Fragment


ID NO: 116
And Chimeric Gene Components





AscI/BsiWI
788 bp 5′ part of Yarrowia Leu2 gene (GenBank


(5520-6315)
Accession No. AF260230)


SphI/PacI
703 bp 3′ part of Yarrowia Leu2 gene (GenBank


(2820-2109)
Accession No. AF260230)


SwaI/BsiWI
FBAIN::MAΔ5::Pex20: as described for pDMW232


(8960-6315)
(supra)


SwaI/ClaI
TEF::MAΔ5::Lip1: as described for pDMW232 (supra)


(8960-11055)


PmeI/ClaI
Yarrowia Ura3 gene (GenBank Accession No.


(12690-11055)
AJ306421)


ClaI/PacI
TEF::HΔ5S::Pex16, comprising:


(1-2109)
TEF: TEF promoter (GenBank Accession No.



AF054508)



HΔ5S: codon-optimized Δ5 desaturase gene (SEQ ID



NO: 13), derived from Homo sapiens (GenBank



Accession No. NP_037534)



Pex16: Pex16 terminator sequence of Yarrowia Pex16



gene (GenBank Accession No. U75433)









Plasmids pDMW232 and pDMW271 were each digested with AscI/SphI, and then used to transform strain M4 separately according to the General Methods. Following transformation, the cells were plated onto MMLe plates and maintained at 30° C. for 2 to 3 days. The individual colonies grown on MMLe plates from each transformation were picked and streaked onto MM and MMLe plates. Those colonies that could grow on MMLe plates but not on MM plates were selected as Leu2− strains. Single colonies of Leu2− strains were then inoculated into liquid MMLe media at 30° C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed the presence of ARA in pDMW232 and pDMW271 transformants, but not in the parental M4 strain. Specifically, among the 48 selected Leu2− transformants with pDMW232, there were 34 strains that produced less than 5% ARA, 11 strains that produced 6-8% ARA, and 3 strains that produced about 10% ARA of total lipids in the engineered Yarrowia. One of the strains that produced 10% ARA was named “Y2034”.
Meanwhile, of the 48 selected Leu2− transformants with pDMW271, there were 35 strains that produced less than 5% ARA of total lipids, 12 strains that produced 6-8% ARA, and 1 strain that produced about 11% ARA of total lipids in the engineered Yarrowia. The strain that produced 11% ARA was named “Y2047”.
Example 5
Generation of Intermediate Strain Y2031, having a Ura− Genotype and Producing 45% LA of Total Lipids
Strain Y2031 was generated by integration of the TEF::Y.Δ12::Pex20 chimeric gene of plasmid pKUNT2 (FIG. 8A) into the Ura3 gene locus of wild type Yarrowia strain ATCC #20362, to thereby to generate a Ura− genotype.
Specifically, plasmid pKUNT2 contained the following components:







TABLE 18







Description of Plasmid pKUNT2 (SEQ ID NO: 117)








RE Sites



And


Nucleotides


Within SEQ
Description Of Fragment And


ID NO: 117
Chimeric Gene Components





AscI/BsiWI
784 bp 5′ part of Yarrowia Ura3 gene (GenBank


(3225-3015)
Accession No. AJ306421)


SphI/PacI
516 bp 3′ part of Yarrowia Ura3 gene (GenBank


(5933-13)
Accession No. AJ306421)


EcoRI/BsiWI
TEF::Y.Δ12::Pex20, comprising:


(6380-8629)
TEF: TEF promoter (GenBank Accession No.



AF054508)



Y.Δ12: Yarrowia Δ12 desaturase gene (SEQ ID NO: 23)



Pex20: Pex20 terminator sequence from Yarrowia



Pex20 gene (GenBank Accession No. AF054613)









The pKUNT2 plasmid was digested with AscI/SphI, and then used for transformation of wild type Y. lipolytica ATCC #20362 according to the General Methods. The transformant cells were plated onto FOA selection media plates and maintained at 30° C. for 2 to 3 days. The FOA resistant colonies were picked and streaked onto MM and MMU selection plates. The colonies that could grow on MMU plates but not on MM plates were selected as Ura− strains. Single colonies (5) of Ura− strains were then inoculated into liquid MMU at 30° C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed that there were about 45% LA in two Ura− strains (i.e., strains #2 and #3), compared to about 20% LA in the wild type ATCC #20362. Transformant strain #2 was designated as strain “Y2031”.
Example 6
Synthesis and Functional Expression of a Codon-Optimized Δ9 Elongase Gene in Yarrowia lipolytica 
The codon usage of the Δ9 elongase gene of Isochrysis galbana (GenBank Accession No. AF390174) was optimized for expression in Y. lipolytica, in a manner similar to that described in WO 2004/101753. Specifically, according to the Yarrowia codon usage pattern, the consensus sequence around the ATG translation initiation codon, and the general rules of RNA stability (Guhaniyogi, G. and J. Brewer, Gene 265(1-2):11-23 (2001)), a codon-optimized Δ9 elongase gene was designed (SEQ ID NO:41), based on the DNA sequence of the I. galbana gene (SEQ ID NO:39). In addition to modification of the translation initiation site, 126 bp of the 792 bp coding region were modified, and 123 codons were optimized. None of the modifications in the codon-optimized gene changed the amino acid sequence of the encoded protein (GenBank Accession No. AF390174; SEQ ID NO:40).
In Vitro Synthesis of a Codon-Optimized Δ9 Elongase Gene for Yarrowia 
The codon-optimized Δ9 elongase gene was synthesized as follows. First, eight pairs of oligonucleotides were designed to extend the entire length of the codon-optimized coding region of the I. galbana Δ9 elongase gene (e.g., IL3-1A, IL3-1B, IL3-2A, IL3-2B, IL3-3A, IL3-3B, IL3-4A, IL34B, IL3-5A, IL3-5B, IL3-6A, IL3-6B, IL3-7A, IL3-7B, IL3-8A and IL3-8B, corresponding to SEQ ID NOs:187-202). Each pair of sense (A) and anti-sense (B) oligonucleotides were complementary, with the exception of a 4 bp overhang at each 5′-end. Additionally, primers IL3-1F, IL3-4R, IL3-5F and IL3-8R (SEQ ID NOs:203-206) also introduced NcoI, PstI, PstI and Not1 restriction sites, respectively, for subsequent subcloning.
Each oligonucleotide (100 ng) was phosphorylated at 37° C. for 1 hr in a volume of 20 μl containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM DTT, 0.5 mM spermidine, 0.5 mM ATP and 10 U of T4 polynucleotide kinase. Each pair of sense and antisense oligonucleotides was mixed and annealed in a thermocycler using the following parameters: 95° C. (2 min), 85° C. (2 min), 65° C. (15 min), 37° C. (15 min), 24° C. (15 min) and 4° C. (15 min). Thus, IL3-1A (SEQ ID NO:187) was annealed to IL3-1B (SEQ ID NO:188) to produce the double-stranded product “IL3-1AB”. Similarly, IL3-2A (SEQ ID NO:189) was annealed to IL3-2B (SEQ ID NO:190) to produce the double-stranded product “IL3-2AB”, etc.
Two separate pools of annealed, double-stranded oligonucleotides were then ligated together, as shown below: Pool 1 (comprising IL3-1AB, IL3-2AB, IL3-3AB and IL3-4AB); and, Pool 2 (comprising IL3-5AB, IL3-6AB, IL3-7AB and IL3-8AB). Each pool of annealed oligonucleotides was mixed in a volume of 20 μl with 10 U of T4 DNA ligase and the ligation reaction was incubated overnight at 16° C.
The product of each ligation reaction was then used as template to amplify the designed DNA fragment by PCR. Specifically, using the ligated “Pool 1” mixture (i.e., IL3-1AB, IL3-2AB, IL3-3AB and IL3-4AB) as template, and oligonucleotides IL3-1F and IL34R (SEQ ID NOs:203 and 204) as primers, the first portion of the codon-optimized Δ9 elongase gene was amplified by PCR. The PCR amplification was carried out in a 50 μl total volume, as described in the General Methods. Amplification was carried out as follows: initial denaturation at 95° C. for 3 min, followed by 35 cycles of the following: 95° C. for 1 min, 56° C. for 30 sec, 72° C. for 40 sec. A final extension cycle of 72° C. for 10 min was carried out, followed by reaction termination at 4° C. The 417 bp PCR fragment was subcloned into the pGEM-T easy vector (Promega) to generate pT9(1-4).
Using the ligated “Pool 2” mixture (i.e., IL3-5AB, IL3-6AB, IL3-7AB and IL3-8AB) as the template, and oligonucleotides IL3-5F and IL3-8R (SEQ ID NOs:205 and 206) as primers, the second portion of the codon-optimized Δ9 elongase gene was amplified similarly by PCR and cloned into pGEM-T-easy vector to generate pT9(5-8).
E. coli was transformed separately with pT9(1-4) and pT9(5-8) and the plasmid DNA was isolated from ampicillin-resistant transformants. Plasmid DNA was purified and digested with the appropriate restriction endonucleases to liberate the 417 bp NcoI/PstI fragment of pT9(14) (SEQ ID NO:207) and the 377 bp PstI/NotI fragment of pT9(5-8) (SEQ ID NO:208). These two fragments were then combined and directionally ligated together with Nco1/Not1 digested pZUF17 (SEQ ID NO:118; FIG. 8B) to generate pDMW237 (FIG. 8C; SEQ ID NO:119). The DNA sequence of the resulting synthetic Δ9 elongase gene (“IgD9e”) in pDMW237 was exactly the same as the originally designed codon-optimized gene (i.e., SEQ ID NO:41) for Yarrowia. 
Expression of the Codon-Optimized Δ9 Elongase Gene in Y. lipolytica 
Construct pDMW237 (FIG. 8C), an auto-replication plasmid comprising a chimeric FBAIN::1 g D9e::Pex20 gene, was transformed into Y. lipolytica Y2031 strain (Example 4) as described in the General Methods. Three transformants of Y2031 with pDMW237 were grown individually in MM media for two days and the cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
The GC results showed that there were about 7.1%, 7.3% and 7.4% EDA, respectively, produced in these transformants with pDMW237. These data demonstrated that the synthetic, codon-optimized IgD9e could convert C18:2 to EDA. The “percent (%) substrate conversion” of the codon-optimized gene was determined to be about 13%.
Example 7
Synthesis of a Codon-Optimized Δ8 Desaturase Gene in Yarrowia lipolytica 
The codon usage of the Δ8 desaturase gene of Euglena gracilis (GenBank Accession No. AAD45877) was optimized for expression in Y. lipolytica, in a manner similar to that described in WO 2004/101753 and Example 6 (supra). Despite synthesis of three different codon-optimized genes (i.e., “D8S-1”, “D8S-2” and “D8S-3”), none of the genes were capable of desaturating EDA to DGLA. It was therefore hypothesized that the previously published Δ8 desaturase sequences were incorrect and it was necessary to isolate the Δ8 desaturase from Euglena gracilis directly, following mRNA isolation, cDNA synthesis and PCR. This resulted in two similar sequences, identified herein as Eg5 (SEQ ID NOs:44 and 45) and Eg12 (SEQ ID NOs:46 and 47).
Functional analysis of each gene sequence was performed by cloning the genes into a Saccharomyces cerevisiae yeast expression vector and conducting substrate feeding trials. Although both Eg5 and Eg12 were able to desaturase EDA and ETrA to produce DGLA and ETA, respectively, Eg5 had significantly greater activity than Eg12.
Based on the confirmed Δ8 desaturase activity of Eg5, the sequence was codon-optimized for expression in Yarrowia lipolytica to thereby result in the synthesis of a synthetic, functional codon-optimized Δ8 desaturase designated as “D8SF” (SEQ ID NOs:48 and 49).
Preliminary In Vitro Synthesis of a Codon-Optimized Δ8 Desaturase Gene
A codon-optimized Δ8 desaturase gene (designated “D8S-1”; SEQ ID NO:209) was designed, based on the published sequence of Euglena gracilis (SEQ ID NOs:42 and 43), according to the Yarrowia codon usage pattern (WO 2004/101753), the consensus sequence around the ‘ATG’ translation initiation codon, and the general rules of RNA stability (Guhaniyogi, G. and J. Brewer, Gene 265(1-2):11-23 (2001)). In addition to modification of the translation initiation site, 200 bp of the 1260 bp coding region were modified (15.9%). None of the modifications in the codon-optimized gene changed the amino acid sequence of the encoded protein (SEQ ID NO:43) except the second amino acid from ‘K’ to ‘E’ to add a NcoI site around the translation initiation codon.
Specifically, the codon-optimized Δ8 desaturase gene was synthesized as follows. First, thirteen pairs of oligonucleotides were designed to extend the entire length of the codon-optimized coding region of the E. gracilis Δ8 desaturase gene (e.g., D8-1A, D8-1B, D8-2A, D8-2B, D8-3A, D8-3B, D84A, D8-4B, D8-5A, D8-5B, D8-6A, D8-6B, D8-7A, D8-7B, D8-8A, D8-8B, D8-9A, D8-9B, D8-10A, D8-10B, D8-11A, D8-11B, D8-12A, D8-12B, D8-13A and D8-13B, corresponding to SEQ ID NOs:210-235). Each pair of sense (A) and anti-sense (B) oligonucleotides were complementary, with the exception of a 4 bp overhang at each 5′-end. Additionally, primers D8-1A, D8-3B, D8-7A, D8-9B and D8-13B (SEQ ID NOs:210, 215, 222, 227 and 235) also introduced NcoI, BglII, Xho1, SacI and Not1 restriction sites, respectively, for subsequent subcloning.
Oligonucleotides (100 ng of each) were phosphorylated as described in Example 6, and then each pair of sense and antisense oligonucleotides was mixed and annealed together [e.g., D8-1A (SEQ ID NO:210) was annealed to D8-1B (SEQ ID NO:211) to produce the double-stranded product “D8-1AB” and D8-2A (SEQ ID NO:212) was annealed to D8-2B (SEQ ID NO:213) to produce the double-stranded product “D8-2AB”, etc.].
Four separate pools of annealed, double-stranded oligonucleotides were then ligated together, as shown below: Pool 1 (comprising D8-1AB, D8-2AB and D8-3AB); Pool 2 (comprising D84AB, D8-5AB and D8-6AB); Pool 3 (comprising D8-7AB, D8-8AB, and D8-9AB); and, Pool 4 (comprising D8-10AB, D8-11 AB, D8-12AB and D8-13AB). Each pool of annealed oligonucleotides was mixed in a volume of 20 μl with 10 U of T4 DNA ligase and the ligation reaction was incubated overnight at 16° C.
The product of each ligation reaction was then used as template to amplify the designed DNA fragment by PCR. Specifically, using the ligated “Pool 1” mixture (i.e., D8-1AB, D8-2AB and D8-3AB) as template, and oligonucleotides D8-1F and D8-3R (SEQ ID NOs:236 and 237) as primers, the first portion of the codon-optimized Δ8 desaturase gene was amplified by PCR. The PCR amplification was carried out in a 50 μl total volume, as described in Example 6. The 309 bp PCR fragment was subcloned into the pGEM-T easy vector (Promega) to generate pT8(1-3).
Using the ligated “Pool 2” mixture (i.e., D8-4AB, D8-5AB and D8-6AB) as the template, and oligonucleotides D8-4F and D8-6R (SEQ ID NOs:238 and 239) as primers, the second portion of the codon-optimized Δ8 desaturase gene was amplified similarly by PCR and cloned into pGEM-T-easy vector to generate pT8(4-6). Using the ligated “Pool 3” mixture (i.e., D8-7AB, D8-8AB and D8-9AB) as the template and oligonucleotides D8-7F and D8-9R (SEQ ID NOs:240 and 241) as primers, the third portion of the codon-optimized Δ8 desaturase gene was amplified similarly by PCR and cloned into pGEM-T-easy vector to generate pT8(7-9). Finally, using the “Pool 4” ligation mixture (i.e., D8-10AB, D8-11AB, D8-12AB and D8-13AB) as template, and oligonucleotides D8-10F and D8-13R (SEQ ID NOs:242 and 243) as primers, the fourth portion of the codon-optimized Δ8 desaturase gene was amplified similarly by PCR and cloned into pGEM-T-easy vector to generate pT8(10-13).
E. coli was transformed separately with pT8(1-3), pT8(4-6), pT8(7-9) and pT8(10-13) and the plasmid DNA was isolated from ampicillin-resistant transformants. Plasmid DNA was purified and digested with the appropriate restriction endonucleases to liberate the 309 bp NcoI/BglII fragment of pT8(1-3) (SEQ ID NO:244), the 321 bp BglII/XhoI fragment of pT8(4-6) (SEQ ID NO:245), the 264 bp XhoI/SacI fragment of pT8(7-9) (SEQ ID NO:246) and the 369 bp Sac1/Not1 fragment of pT8(10-13) (SEQ ID NO:247). These fragments were then combined and directionally ligated together with Nco1/Not1 digested pY54PC (SEQ ID NO:120; WO2004/101757) to generate pDMW240 (FIG. 8D). This resulted in a synthetic Δ8 desaturase gene (“D8S-1”, SEQ ID NO:209) in pDMW240.
Compared with the published Δ8 desaturase amino acid sequence (SEQ ID NO:43) of E. gracilis, the second amino acid of D8S-1 was changed from ‘K’ to ‘E’ in order to add the NcoI site around the translation initiation codon. Another version of the synthesized gene, with the exact amino acid sequence as the published E. gracilis Δ8 desaturase sequence (SEQ ID NO:43), was constructed by in vitro mutagenesis (Stratagene, San Diego, Calif.) using pDMW240 as a template and oligonucleotides ODMW390 and ODMW391 (SEQ ID NOs:248 and 249) as primers. The resulting plasmid was designated pDMW255. The synthetic Δ8 desaturase gene in pDMW255 was designated as “D8S-2” and the amino acid sequence was exactly the same as the sequence depicted in SEQ ID NO:43.
Nonfunctional Codon-Optimized Δ8 Desaturase Genes
Yarrowia lipolytica strain ATCC #76982(Leu-) was transformed with pDMW240 (FIG. 8D) and pDMW255, respectively, as described in the General Methods. Yeast containing the recombinant constructs were grown in MM supplemented with EDA [20:2(11,14)]. Specifically, single colonies of transformant Y. lipolytica containing either pDMW240 (containing D8S-1) or pDMW255 (containing D8S-2) were grown in 3 mL MM at 30° C. to an OD600 ̃1.0. For substrate feeding, 100 μl of cells were then subcultured in 3 mL MM containing 10 μg of EDA substrate for about 24 hr at 30° C. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
Neither transformant produced DGLA from EDA and thus D8S-1 and D8S-2 were not functional and could not desaturate EDA. The chimeric D8S-1::XPR and D8S-2::XPR genes are shown in SEQ ID NOs:250 and 251, respectively.
A three amino acid difference between the protein sequence of the Δ8 desaturase deposited in GenBank (Accession No. AAD45877 [SEQ ID NO:43]) and in WO 00/34439 or Wallis et al. (Archives of Biochem. Biophys, 365:307-316 (1999)) (SEQ ID NO:252 herein) was found. Specifically, three amino acids appeared to be missing in GenBank Accession No. MD45877. Using pDMW255 as template and ODMW392 and ODMW393 (SEQ ID NOs:253 and 254) as primers, 9 bp were added into the synthetic D8S-2 gene by in vitro mutagenesis (Stratagene, San Diego, Calif.), thus producing a protein that was identical to the sequence described in WO 00/34439 and Wallis et al. (supra) (SEQ ID NO:252). The resulting plasmid was called pDMW261. The synthetic Δ8 desaturase gene in pDMW261 was designated as “D8S-3” (SEQ ID NO:255). Following transformation of the pDMW261 construct into Yarrowia, a similar feeding experiment using EDA was conducted, as described above. No desaturation of EDA to DGLA was observed with D8S-3.
Isolation of a Euglena gracilis Δ8 Desaturase Gene
Euglena gracilis was obtained from Dr. Richard Triemer's lab at Michigan State University (East Lansing, Mich.). From 10 mL of actively growing culture, a 1 mL aliquot was transferred into 250 mL of Euglena gracilis (Eg) Medium in a 500 mL glass bottle. Eg medium was made by combining: 1 g of sodium acetate, 1 g of beef extract (Catalog #U126-01, Difco Laboratories, Detroit, Mich.), 2 g of Bacto® Tryptone (Catalog #0123-17-3, Difco Laboratories) and 2 g of Bacto® Yeast Extract (Catalog #0127-17-9, Difco Laboratories) in 970 mL of water. After filter sterilizing, 30 mL of Soil-Water Supernatant (Catalog #15-3790, Carolina Biological Supply Company, Burlington, N.C.) was aseptically added to produce the final Eg medium. E. gracilis cultures were grown at 23° C. with a 16 hr light, 8 hr dark cycle for 2 weeks with no agitation.
After 2 weeks, 10 mL of culture was removed for lipid analysis and centrifuged at 1,800×g for 5 min. The pellet was washed once with water and re-centrifuged. The resulting pellet was dried for 5 min under vacuum, resuspended in 100 μL of trimethylsulfonium hydroxide (TMSH) and incubated at room temperature for 15 min with shaking. After this, 0.5 mL of hexane was added and the vials were incubated for 15 min at room temperature with shaking. Fatty acid methyl esters (5 μL injected from hexane layer) were separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Catalog #24152, Supelco Inc.). The oven temperature was programmed to hold at 220° C. for 2.7 min, increase to 240° C. at 20° C./min and then hold for an additional 2.3 min. Carrier gas was supplied by a Whatman hydrogen generator. Retention times were compared to those for methyl esters of standards commercially available (Catalog #U-99-A, Nu-Chek Prep, Inc.) and the resulting chromatogram is shown in FIG. 9.
The remaining 2 week culture (240 mL) was pelleted by centrifugation at 1,800×g for 10 min, washed once with water and re-centrifuged. Total RNA was extracted from the resulting pellet using the RNA STAT-60TM reagent (TEL-TEST, Inc., Friendswood, Tex.) and following the manufacturer's protocol provided (use 5 mL of reagent, dissolved RNA in 0.5 mL of water). In this way, 1 mg of total RNA (2 mg/mL) was obtained from the pellet. The mRNA was isolated from 1 mg of total RNA using the mRNA Purification Kit (Amersham Biosciences, Piscataway, N.J.) following the manufacturer's protocol provided. In this way, 85 μg of mRNA was obtained.
cDNA was synthesized from 765 ng of mRNA using the SuperScriptTM Choice System for cDNA synthesis (InvitrogenTM Life Technologies, Carlsbad, Calif.) with the provided oligo(dT) primer according to the manufacturer's protocol. The synthesized cDNA was dissolved in 20 μL of water.
The E. gracilis Δ8 desaturase was amplified from cDNA with oligonucleotide primers Eg5-1 and Eg3-3 (SEQ ID NOs:256 and 257) using the conditions described below. Specifically, cDNA (1 μL) was combined with 50 pmol of Eg5-1, 50 pmol of Eg5-1, 1 μL of PCR nucleotide mix (10 mM, Promega, Madison, Wis.), 5 μL of 10× PCR buffer (Invitrogen), 1.5 μL of MgCl2 (50 mM, Invitrogen), 0.5 μL of Taq polymerase (Invitrogen) and water to 50 μL. The reaction conditions were 94° C. for 3 min followed by 35 cycles of 94° C. for 45 sec, 55° C. for 45 sec and 72° C. for 1 min. The PCR was finished at 72° C. for 7 min and then held at 4° C. The PCR reaction was analyzed by agarose gel electrophoresis on 5 μL and a DNA band with molecular weight around 1.3 kB was observed. The remaining 45 μL of product was separated by agarose gel electrophoresis and the DNA band was purified using the ZymocleanTM Gel DNA Recovery Kit (Zymo Research, Orange, Calif.) following the manufacturer's protocol. The resulting DNA was cloned into the pGEM®—T Easy Vector (Promega) following the manufacturer's protocol. Multiple clones were sequenced using T7, M13-28Rev, Eg3-2 and Eg5-2 (SEQ ID NOS:258-261, respectively).
Thus, two classes of DNA sequences were obtained, Eg5 (SEQ ID NO:44) and Eg12 (SEQ ID NO:46), that differed in only a few bp. Translation of Eg5 and Eg12 gave rise to protein sequences that differed in only one amino acid, SEQ ID NO:45 and 47, respectively. Thus, the DNA and protein sequences for Eg5 are set forth in SEQ ID NO:44 and SEQ ID NO:45, respectively; the DNA and protein sequences for Eg12 are set forth in SEQ ID NO:46 and SEQ ID NO:47, respectively.
Comparison of the Isolated E. gracilis Δ8 Desaturase Sequences to Published E. gracilis Δ8 Desaturase Sequences
An alignment of the protein sequences set forth in SEQ ID NO:45 (Eg5) and SEQ ID NO:47 (Eg12) with the protein sequence from GenBank Accession No. AAD45877 (gi: 5639724; SEQ ID NO:43 herein) and with the published protein sequences of Wallis et al. (Archives of Biochem. Biophys., 365:307-316 (1999); WO 00/34439) [SEQ ID NO:252 herein] is shown in FIG. 10. Amino acids conserved among all 4 sequences are indicated with an asterisk (*). Dashes are used by the program to maximize alignment of the sequences. The putative cytochrome b5 domain is underlined. A putative His box is shown in bold. Percent identity calculations revealed that the Eg5 Δ8 desaturase protein sequence is 95.5% identical to SEQ ID NO:43 and 96.2% identical to SEQ ID NO:252, wherein “% identity” is defined as the percentage of amino acids that are identical between the two proteins. Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp, CABIOS. 5:151-153 (1989)) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For a more complete analysis of the differences between the various E. gracilis Δ8 desaturase sequences, refer to co-pending U.S. patent application Ser. No. 11/166,993.
Functional Analysis of the Euglena gracilis Δ8 Desaturase Sequences in Saccharomyces cerevisiae 
The yeast episomal plasmid (YEp)-type vector pRS425 (Christianson et al., Gene, 110:119-22 (1992)) contains sequences from the Saccharomyces cerevisiae 2μ endogenous plasmid, a LEU2 selectable marker and sequences based on the backbone of a multifunctional phagemid, pBluescript II SK+. The S. cerevisiae strong, constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter was cloned between the SacII and SpeI sites of pRS425 in the same way as described in Jia et al. (Physiological Genomics, 3:83-92 (2000)) to produce pGPD-425. A NotI site was introduced into the BamHI site of pGPD-425 (thus producing a NotI site flanked by BamHI sites), thereby resulting in plasmid pY-75. Eg5 (SEQ ID NO:44) and Eg12 (SEQ ID NO:46) were released from the pGEM® T Easy vectors described above by digestion with NotI and cloned into the NotI site of pY-75 to produce pY89-5 (deposited as ATCC #PTA-6048) and pY89-12, respectively. In this way, the Δ8 desaturases (i.e., Eg5 [SEQ ID NO:44] and Eg12 [SEQ ID NO:46]) were cloned behind a strong constitutive promoter for expression in S. cerevisiae. A map of pY89-5 is shown in FIG. 8E.
Plasmids pY89-5, pY89-12 and pY-75 were transformed into Saccharomyces cerevisiae BY4741 (ATCC #201388) using standard lithium acetate transformation procedures. Transformants were selected on DOBA media supplemented with CSM-leu (Qbiogene, Carlsbad, Calif.). Transformants from each plate were inoculated into 2 mL of DOB medium supplemented with CSM-leu (Qbiogene) and grown for 1 day at 30° C., after which 0.5 mL was transferred to the same medium supplemented with either EDA or EtrA to 1 mM. These were incubated overnight at 30° C., 250 rpm, and pellets were obtained by centrifugation and dried under vacuum. Pellets were transesterified with 50 μL of TMSH and analyzed by GC as described in the General Methods. Two clones for pY-75 (i.e., clones 75-1 and 75-2) and pY89-5 (i.e., clones 5-6-1 and 5-6-2) were each analyzed, while two sets of clones for pY89-12 (i.e., clones 12-8-1, 12-8-2, 12-9-1 and 12-9-2) from two independent transformations were analyzed.
The lipid profile obtained by GC analysis of clones fed EDA are shown in Table 19; and the lipid profile obtained by GC analysis of clones fed EtrA are shown in Table 20. Fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid), 18:0, 18:1 (oleic acid), 20:2 [EDA], 20:3 (8,11,14) [DGLA], 20:3 (11,14,17) [ETrA] and 20:4 (8,11,14,17) [ETA]; and the composition of each is presented as a % of the total fatty acids.







TABLE 19







Lipid Analysis Of Transformant S. cerevisiae Overexpressing The


Euglena gracilis Δ8 Desaturases: EDA Substrate Feeding





















% 20:2








20:3
Con-


Clone
16:0
16:1
18:0
18:1
20:2
(8, 11, 14)
verted

















75-1 (control)
14
32
5
38
10
0
0


75-2 (control)
14
31
5
41
9
0
0


5-6-1 (Eg5)
14
32
6
40
6
2
24


5-6-2 (Eg5)
14
30
6
41
7
2
19


12-8-1 (Eg12)
14
30
6
41
9
1
7


12-8-2 (Eg12)
14
32
5
41
8
1
8


12-9-1 (Eg12)
14
31
5
40
9
1
8


12-9-2 (Eg12)
14
32
5
41
8
1
7
















TABLE 20







Lipid Analysis Of Transformant S. cerevisiae Overexpressing The


Euglena gracilis Δ8 Desaturases: ETrA Substrate Feeding



















20:3
20:4
% 20:3







(11,
(8, 11,
Con-


Clone
16:0
16:1
18:0
18:1
14, 17)
14, 17)
verted

















75-1 (control)
12
25
5
33
24
0
0


75-2 (control)
12
24
5
36
22
1
5


5-6-1 (Eg5)
13
25
6
34
15
7
32


5-6-2 (Eg5)
13
24
6
34
17
6
27


12-8-1 (Eg12)
12
24
5
34
22
2
8


12-8-2 (Eg12)
12
25
5
35
20
2
9


12-9-1 (Eg12)
12
24
5
34
22
2
9


12-9-2 (Eg12)
12
25
6
35
20
2
9









The data in Tables 19 and 20 showed that the cloned Euglena Δ8 desaturases were able to desaturate EDA and EtrA. The sequence set forth in SEQ ID NO:47 has one amino acid change compared to the sequence set forth in SEQ ID NO:45 and has reduced Δ8 desaturase activity.
The small amount of 20:4(8,11,14,17) generated by clone 75-2 in Table 20 had a slightly different retention time than a standard for 20:4(8,11,14,17). This peak was more likely a small amount of a different fatty acid generated by the wild-type yeast in that experiment.
Further Modification of the Δ8 Desaturase Gene Codon-Optimized for Yarrowia lipolytica 
The amino acid sequence of the synthetic D8S-3 gene in pDMW261 was corrected according to the amino acid sequence of the functional Euglena Δ8 desaturase (SEQ ID NOs:44 and 45). Using pDMW261 as a template and oligonucleotides ODMW404 (SEQ ID NO:262) and D8-13R (SEQ ID NO:243), the DNA fragment encoding the synthetic D8S-3 desaturase gene was amplified. The resulting PCR fragment was purified with Bio101 's Geneclean kit and subsequently digested with Kpn1 and NotI (primer ODMW404 introduced a KpnI site while primer D8-13R introduced a NotI site). The Kpn1/NotI fragment (SEQ ID NO:263) was cloned into Kpn1/NotI digested pKUNFmKF2 (FIG. 11A; SEQ ID NO:121) to produce pDMW277 (FIG. 11B).
Oligonucleotides YL521 and YL522 (SEQ ID NOs:264 and 265), which were designed to amplify and correct the 5′ end of the D8S-3 gene, were used as primers in another PCR reaction where pDMW277 was used as the template. The primers introduced into the PCR fragment a Nco1 site and BglII site at its 5′ and 3′ ends, respectively. The 318 bp PCR product was purified with Bio101's GeneClean kit and subsequently digested with Nco1 and BglII. The digested fragment, along with the 954 bp BglII/NotI fragment from pDMW277, was used to exchange the NcoI/NotI fragment of pZF5T-PPC (FIG. 11C; SEQ ID NO:122) to form pDMW287. In addition to correcting the 5′ end of the synthetic D8S-3 gene, this cloning reaction also placed the synthetic Δ8 desaturase gene under control of the Yarrowia lipolytica FBAIN promoter (SEQ ID NO:162).
The first reaction in a final series of site-directed mutagenesis reactions was then performed on pDMW287. The first set of primers, YL525 and YL526 (SEQ ID NOs:266 and 267), was designed to correct amino acid from F to S (position #50) of the synthetic D8S-3 gene in pDMW287. The plasmid resulting from this mutagenesis reaction then became the template for the next site-directed mutagenesis reaction with primers YL527 and YL528 (SEQ ID NOs:268 and 269). These primers were designed to correct the amino acid from F to S (position #67) of the D8S-3 gene and resulted in creation of plasmid pDMW287/YL527.
To complete the sequence corrections within the second quarter of the gene, the following reactions were carried out concurrently with the mutations on the first quarter of the gene. Using pDMW287 as template and oligonucleotides YL529 and YL530 (SEQ ID NOs:270 and 271) as primers, an in vitro mutagenesis reaction was carried out to correct the amino acid from C to W (position #177) of the synthetic D8S-3 gene. The product (i.e., pDMW287/Y529) of this mutagenesis reaction was used as the template in the following reaction using primers YL531 and YL532 (SEQ ID NOs:272 and 273) to correct the amino acid from P to L (position #213). The product of this reaction was called pDMW287/YL529-31.
Concurrently with the mutations on the first and second quarter of the gene, reactions were similarly carried out on the 3′ end of the gene. Each subsequent mutagenesis reaction used the plasmid product from the preceding reaction. Primers YL533 and YL534 (SEQ ID NOs:274 and 275) were used on pDMW287 to correct the amino acid from C to S (position #244) to create pDMW287/YL533. Primers YL535 and YL536 (SEQ ID NOs:276 and 277) were used to correct the amino acid A to T (position #280) in the synthetic D8S-3 gene of pDMW287/YL533 to form pDMW287/YL533-5. Finally, the amino acid P at position #333 was corrected to S in the synthetic D8S-3 gene using pDMW287/YL533-5 as the template and YL537 and YL538 (SEQ ID NOs:278 and 279) as primers. The resulting plasmid was named pDMW287/YL533-5-7.
The BglII/XhoI fragment of pDMW287/YL529-31 and the XhoI/NotI fragment of pDMW287/YL533-5-7 were used to change the BglII/NotI fragment of pDMW287/YL257 to produce pDMW287F (FIG. 11D) containing the completely corrected synthetic Δ8 desaturase gene, designated D8SF and set forth in SEQ ID NO:48. SEQ ID NO:49 sets forth the amino acid sequence encoded by nucleotides 2-1270 of SEQ ID NO:48, which is essentially the same as the sequence set forth in SEQ ID NO:45, except for an additional valine following the start methionine.
Example 8
Functional Expression of the Codon-Optimized Δ9 Elongase Gene and Codon-Optimized Δ8 Desaturase in Yarrowia lipolytica 
The present Example describes DGLA biosynthesis and accumulation in Yarrowia lipolytica that was transformed to co-express the codon-optimized Δ9 elongase and codon-optimized Δ8 desaturase from Examples 6 and 7. This experiment thereby confirmed both genes' activity and Y. lipolytica's ability to express the Δ9 elongase/Δ8 desaturase pathway.
Specifically, the ClaI/PacI fragment comprising a chimeric FBAIN::D8SF::Pex16 gene of construct pDMW287F (Example 7) was inserted into the ClaI/PacI sites of pDMW237 (Example 6) to generate the construct pDMW297 (FIG. 11E; SED ID NO:123).
Plasmid pDMW297 contained the following components:







TABLE 21







Description of Plasmid pDMW297 (SEQ ID NO: 123)








RE Sites And



Nucleotides


Within SEQ
Description Of Fragment


ID NO: 123
And Chimeric Gene Components





EcoRI/ClaI
ARS18 sequence (GenBank Accession No. A17608)


(9053-10448)


ClaI/PacI
FBAIN::D8SF::Pex16, comprising:


(1-2590)
FBAIN: FBAIN promoter (SEQ ID NO: 162)



D8SF: codon-optimized Δ8 desaturase gene (SEQ ID



NO: 48), derived from Euglena gracilis (GenBank



Accession No. AF139720)



Pex16: Pex16 terminator sequence of Yarrowia Pex16



gene (GenBank Accession No. U75433)


PacI/SalI
Yarrowia Ura3 gene (GenBank Accession No.


(2590-4082)
AJ306421)


SalI/BsiWI
FBAIN::IgD9e::Pex20, comprising:


(4082-6257)
FBAIN: FBAIN promoter (SEQ ID NO: 162)



IgD9e: codon-optimized Δ9 elongase gene (SEQ ID



NO: 41), derived from Isochrysis galbana (GenBank



Accession No. 390174)



Pex20: Pex20 terminator sequence of Yarrowia Pex20



gene (GenBank Accession No. AF054613)









Construct pDMW297 was then used for transformation of strain Y2031 (Example 5) according to the General Methods. The transformant cells were plated onto MM selection media plates and maintained at 30° C. for 2 to 3 days. A total of 8 transformants grown on the MM plates were picked and re-streaked onto fresh MM plates. Once grown, these strains were individually inoculated into liquid MM at 30° C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed that DGLA was produced in all of the transformants analyzed. One strain produced about 3.2%, 4 strains produced 4.3-4.5%, two strains produced 5.5-5.8% and one strain produced 6.4% DGLA (designated herein as strain “Y0489”). The “percent (%) substrate conversion” of the codon-optimized D8SF gene in strain Y0489 was determined to be 75%.
Example 9
Δ9 Elongase/Δ8 Desaturase Pathway: Generation of Y2214 Strain to Produce about 14% ARA of Total Lipids
The present Example describes the construction of strain Y2214, derived from Yarrowia lipolytica ATCC #20362, capable of producing 14% ARA relative to the total lipids (FIG. 4). This strain was engineered to express the Δ9 elongase/Δ8 desaturase pathway; thus, analysis of the complete lipid profiles of strain Y2214 indicating no GLA co-synthesis in the final ARA-containing oil was expected.
The development of strain Y2214 herein required the construction of strains Y2152 and Y2153 (producing  ̃3.5% DGLA), strains Y2173 and Y2175 (producing 14-16% DGLA), and strains Y2183 and 2184 (producing 5% ARA).
Generation of Strains Y2152 and Y2153 to Produce about  ̃3.5% DGLA of Total Lipids
Construct pZP2C16M899 (FIG. 12A, SEQ ID NO:124) was used to integrate a cluster of four chimeric genes (comprising two Δ9 elongases, a synthetic C16/18 fatty acid elongase and a Δ8 desaturase), as well as a Yarrowia AHAS gene (acetohydroxy-acid synthase) containing a single amino acid mutation. The mutated AHAS enzyme in Yarrowia conferred resistance to sulfonylurea, which was used as a positive screening marker. Plasmid pZP2C16M899 was designed to integrate into the Pox2 gene site of Yarrowia strain ATCC #20362 and thus contained the following components:







TABLE 22







Description of Plasmid pZP2C16M899 (SEQ ID NO: 124)








RE Sites And



Nucleotides


Within SEQ ID
Description Of Fragment


NO: 124
And Chimeric Gene Components





BsiWI/AscI
810 bp 5′ part of Yarrowia Aco2 gene (GenBank


(6152-6962)
Accession No. AJ001300)


SphI/EcoRI
655 bp 3′ part of Yarrowia Aco2 gene (GenBank


(9670-10325)
Accession No. AJ001300)


BsiWI/PmeI
GPM/FBAintron::rELO2S::Oct, comprising:


with EcoRV
GPM/FBAIN: GPM::FBAIN chimeric promoter (SEQ ID


(929-3195)
NO: 167)



rELO2S: codon-optimized rELO2 elongase gene (SEQ



ID NO: 52), derived from rat (GenBank Accession No.



AB071986)



OCT: OCT terminator sequence of Yarrowia OCT gene



(GenBank Accession No. X69988)


BsiWI/EcoRI
GPAT::IgD9e::Pex20, comprising:


(929-14447,
GPAT: GPAT promoter (SEQ ID NO: 164)


reverse)
IgD9e: codon-optimized Δ9 elongase gene (SEQ ID



NO: 41), derived from I. galbana



Pex20: Pex20 terminator sequence of Yarrowia Pex20



gene (GenBank Accession No. AF054613)


EcoRI/SwaI
TEF::IgD9e::Lip1, comprising:


(14447-12912)
TEF: TEF promoter (GenBank Accession No.



AF054508)



IgD9e: codon-optimized Δ9 elongase gene (SEQ ID



NO: 41), derived from I. galbana



Lip1: Lip1 terminator sequence of Yarrowia Lip1 gene



(GenBank Accession No. Z50020)


SwaI/PacI
FBAIN::D8SF::Pex16, comprising:


(12912-10325)
FBAIN: FBAIN promoter (SEQ ID NO: 162)



D8SF: codon-optimized Δ8 desaturase gene (SEQ ID



NO: 48), derived from Euglena gracilis (GenBank



Accession No. AF139720)



Pex16: Pex16 terminator sequence of Yarrowia Pex16



gene (GenBank Accession No. U75433) gene


PmeI with
Yarrowia lipolytica AHAS gene comprising a W497L


EcoRV/
mutation (SEQ ID NO: 280)


BsiWI


(3195-6152)









Plasmid pZP2C16M899 was digested with SphI/AscI, and then used to transform ATCC #20362 according to the General Methods. Following transformation, cells were plated onto MM plates containing 150 mg sulfonylurea and maintained at 30° C. for 2 to 3 days. The sulfonylurea resistant colonies were picked and streaked onto MM with sulfonylurea selection plates. A total of 96 transformants were then inoculated into liquid MM with sulfonylurea at 30° C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed the presence of DGLA in the transformants containing the 4 chimeric genes of pZP2C16M899, but not in the wild type Yarrowia control strain. Most of the selected 96 strains produced less than 2% DGLA of total lipids. There were 28 strains that produced 2-2.9% DGLA of total lipids. There were 2 strains that produced about 3.5% DGLA of total lipids. Strains #65 and #73 were designated herein as strains “Y2152” and “Y2153”, respectively.
Generation of Strains Y2173 and Y2175 to Produce about 14-16% DGLA of Total Lipids
Construct pDMW314 (FIG. 12B, SEQ ID NO:125) was used to integrate a cluster of four chimeric genes (comprising two Δ9 elongases, a Δ8 desaturase and a Δ12 desaturase) into the Ura3 gene site of Yarrowia strains Y2152 and Y2153, to thereby enhance production of DGLA. Plasmid pDMW314 contained the following components:







TABLE 23







Description of Plasmid pDMW314 (SEQ ID NO: 125)








RE Sites And



Nucleotides


Within SEQ ID
Description Of Fragment And


NO: 125
Chimeric Gene Components





AscI/BsiWI
784 bp 5′ part of Yarrowia Ura3 gene (GenBank


(10066-9275)
Accession No. AJ306421)


SphI/PacI
516 bp 3′ part of Yarrowia Ura3 gene (GenBank


(12774-1)
Accession No. AJ306421)


SwaI/BsiWI
FBAIN::F.D12S::Pex20, comprising:


(6582-9275)
FBAIN: FBAIN promoter (SEQ ID NO: 162)



F.Δ12: Fusarium moniliforme Δ12 desaturase gene



(SEQ ID NO: 27)



Pex20: Pex20 terminator sequence from Yarrowia



Pex20 gene (GenBank Accession No. AF054613)


ClaI/EcoRI
GPAT::IgD9E::Pex20: as described for pZP2C16M899


(6199-4123)
(supra)


EcoRI/SwaI
TEF::IgD9E::Lip1: as described for pZP2C16M899


(4123-2588)
(supra)


SwaI/PacI
FBAIN::D8SF::Pex16: as described for pZP2C16M899


(2588-1)
(supra)









Plasmid pDMW314 was digested with AscI/SphI, and then used for transformation of Y. lipolytica strains Y2152 and Y2153 according to the General Methods. The transformant cells were plated onto FOA selection media plates and maintained at 30° C. for 2 to 3 days. The FOA resistant colonies were picked and streaked onto MM and MMU selection plates. The colonies that could grow on MMU plates but not on MM plates were selected as Ura− strains. Single colonies of Ura− strains were then inoculated into liquid MMU at 30° C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.

GC analyses showed increased production of DGLA in almost all transformants containing the 4 chimeric genes of pDMW314. Most of the selected 48 Ura− strains of Y2152 with pDMW314 produced about 6-8% DGLA of total lipids. There was one strain (i.e., #47, designated herein as “Y2173”) that produced about 13.9% DGLA of total lipids.
Most of the selected 24 Ura− strains of Y2153 with pDMW314 produced about 6-8% DGLA of total lipids. There were two strains (i.e., #6 and #11, designated herein as strains “Y2175” and “Y2176”) that produced about 16.3% and 17.2% DGLA of total lipids, respectively.
Generation of Strains Y2183 and Y2184 to Produce about 5% ARA of Total Lipids
Construct pDMW322 (FIG. 12C, SEQ ID NO:126) was used to integrate a cluster of two chimeric Δ5 desaturase genes into the Leu2 gene site of Yarrowia Y2173 and Y2175 strains to thereby enable production of ARA. Plasmid pDMW322 contained the following components:







TABLE 24







Description of Plasmid pDMW232 (SEQ ID NO: 126)








RE Sites



And


Nucleotides


Within SEQ


ID NO: 126
Description Of Fragment And Chimeric Gene Components





AscI/BsiWI
788 bp 5′ part of Yarrowia Leu2 gene (GenBank Accession


(3437-2642)
No. AF260230)


SphI/PacI
703 bp 3′ part of Yarrowia Leu2 gene (GenBank Accession


(6854-6145)
No. AF260230)


SwaI with
FBAIN::MAΔ5::Pex20, comprising:


PmeI/BsiWI
FBAIN: FBAIN promoter (SEQ ID NO: 162)


(1-2642)
MAΔ5: Mortierella alpina Δ5 desaturase gene (SEQ ID



NO: 6) (GenBank Accession No. AF067654)



Pex20: Pex20 terminator sequence of Yarrowia Pex20



gene (GenBank Accession No. AF054613)


EcoRI/SwaI
GPM/FBAIN::I.Δ5S::Oct, comprising:


with PmeI
GPM/FBAIN: GPM::FBAIN chimeric promoter (SEQ ID


(8833-1)
NO: 167)



I.Δ5S: codon-optimized Δ5 desaturase gene (SEQ ID



NO: 10), derived from Isochrysis galbana (WO 2002/



081668)



OCT: OCT terminator sequence of Yarrowia OCT gene



(GenBank Accession No. X69988)


EcoRI/PmeI
Yarrowia Ura3 gene (GenBank Accession No. AJ306421)


(8833-7216)









Plasmid pDMW322 was digested with AscI/SphI, and then used to transform strains Y2173 and Y2175 separately according to the General Methods. Following transformation, the cells were plated onto MMLe plates and maintained at 30° C. for 2 to 3 days. The individual colonies grown on MMLe plates from each transformation were picked and streaked onto MM and MMLe plates. Those colonies that could grow on MMLe plates but not on MM plates were selected as Leu2− strains. Single colonies of Leu2− strains were then inoculated into liquid MMLe media at 30° C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed the presence of ARA in pDMW322 transformants, but not in the parental Y2173 and Y2175 strains. Specifically, among the 48 selected Leu2− transformants of Y2173 with pDMW322, most strains produced less than 4.4% ARA of total lipids; however, there were two strains (i.e., #1 and #42, designated herein as strains “Y2181” and “Y2182”) that produced about 4.5 and 5.8% ARA of total lipids, respectively.
In parallel, among the 48 selected Leu2− transformants of Y2175 with pDMW322, most strains produced less than 4.5% ARA of total lipids. There were three strains (i.e., #22, #42 and #47, designated herein as strains “Y2183”, “Y2184” and “Y2185”), that produced about 4.9%, 4.6% and 4.7% ARA of total lipids, respectively, in the engineered Yarrowia. 
Generation of Strain Y2214 to Produce about 14% ARA of Total Lipids
Construct pZKSL5598 (FIG. 12D, SEQ ID NO:127) was used to integrate a cluster of four chimeric genes (comprising a Δ9 elongase, a Δ8 desaturase and two Δ5 desaturases) into the Lys5 gene (GenBank Accession No. M34929) site of Yarrowia Y2183 and Y2185 strains to thereby enhance production of ARA. Plasmid pZKSL5598 contained the following components:







TABLE 25







Description of Plasmid pZKSL5598 (SEQ ID NO: 127)








RE Sites And



Nucleotides


Within SEQ ID
Description Of Fragment And


NO: 127
Chimeric Gene Components





AscI/BsiWI
794 bp 5′ part of Yarrowia Lys5 gene (GenBank


(10409-9573)
Accession No. M34929)


SphI/PacI
687 bp 3′ part of Yarrowia Lys5 gene (GenBank


(13804-13117)
Accession No. M34929)


BsiWI/SwaI
NT::I.D5S::Lip1, comprising:


(7150-9573)
NT: YAT1 promoter (SEQ ID NO: 165)



I.Δ5S: codon-optimized Δ5 desaturase gene (SEQ ID



NO: 10), derived from Isochrysis galbana (WO 2002/



081668)



Lip1: Lip1 terminator sequence of Yarrowia Lip1 gene



(GenBank Accession No. Z50020)


SalI/BsiWI
GPAT::MAΔ5::Pex20, comprising:


(4537-7150)
GPAT: GPAT promoter (SEQ ID NO: 164)



MAΔ5: Mortierella alpina Δ5 desaturase gene (SEQ ID



NO: 6) (GenBank Accession No. AF067654)



Pex20: Pex20 terminator sequence from Yarrowia



Pex20 gene (GenBank Accession No. AF054613)


SwaI/PmeI
FBAINm::IgD9e::OCT, comprising:


(2381-348)
FBAINm: FBAINm promoter (SEQ ID NO: 163)



IgD9e: codon-optimized Δ9 elongase gene (SEQ ID



NO: 41), derived from I. galbana



OCT: OCT terminator sequence of Yarrowia OCT gene



(GenBank Accession No. X69988)


ClaI/PacI
GPD::D8SF::Pex16, comprising:


(1-13804)
GPD: GPD promoter (SEQ ID NO: 158)



D8SF: codon-optimized Δ8 desaturase gene (SEQ ID



NO: 48), derived from Euglena gracilis (GenBank



Accession No. AF139720)



Pex16: Pex16 terminator sequence of Yarrowia Pex16



gene (GenBank Accession No. U75433)


SalII/PmeI
Yarrowia Leu2 gene (GenBank Accession No.


(4537-2417)
AF260230)









Plasmid pZKSL5598 was digested with AscI/SphI, and then used to transform strains Y2183 and Y2184 separately according to the General Methods. Following transformation, the cells were plated onto MMLys plates and maintained at 30° C. for 2 to 3 days. The individual colonies grown on MMLys plates from each transformation were picked and streaked onto MM and MMLys plates. Those colonies that could grow on MMLys plates but not on MM plates were selected as Lys− strains. Single colonies of Lys− strains were then inoculated into liquid MMLys media at 30° C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed increased production of ARA in pZKSL5598 transformants. Among the 48 selected Lys− transformants of Y2183 with pZKSL5598, most strains produced between 4-9.5% ARA of total lipids. Three strains (i.e., #7, #12 and #37, designated herein as strains “Y2209”, “Y2210” and “Y2211”) produced about 9.9%, 10.3% and 9.6% ARA of total lipids, respectively.
Among the 48 selected Lys− transformants of Y2184 with pZKSL5598, most strains produced between 4-11% ARA of total lipids. Two strains (i.e., #3 and #22, designated herein as strains “Y2213” and “Y2214”) produced about 11.9% and 14% ARA of total lipids, respectively.
Example 10
Generation of Intermediate Strain Y2067U, Producing 14% EPA of Total Lipids
The present Example describes the construction of strain Y2067U, derived from Yarrowia lipolytica ATCC #20362, capable of producing significant concentrations of EPA relative to the total lipids (FIG. 4). The affect of M. alpina LPAAT1, DGAT1 and DGAT2 gene over-expression and Y. lipolytica CPT1 gene over-expression were examined in this EPA producing strain based on analysis of TAG content and/or composition, as described in Examples 14, 15, 16 and 21, respectively (infra).
The development of strain Y2067U (producing 14% EPA) herein required the construction of strain M4 (producing 8% DGLA and described in Example 4), strain Y2034 (producing 10% ARA and described in Example 4), strain E (producing 10% EPA), strain EU (producing 10% EPA) and strain Y2067 (producing 15% EPA).
Generation of E Strain to Produce about 10% EPA of Total Lipids in Engineered Yarrowia 
Construct pZP3L37 (FIG. 13A; SEQ ID NO:128) was created to integrate three synthetic Δ17 desaturase chimeric genes into the acyl-CoA oxidase 3 gene of the Y2034 strain described in Example 4. The plasmid pZP3L37 contained the following components:







TABLE 26







Description of Plasmid pZP3L37 (SEQ ID NO: 128)








RE Sites And



Nucleotides


Within SEQ ID
Description Of


NO: 128
Fragment And Chimeric Gene Components





AscI/BsiWI
763 bp 5′ part of Yarrowia Pox3 gene (GenBank


(6813-6043)
Accession No. AJ001301)


SphI/PacI
818 bp 3′ part of Yarrowia Pox3 gene (GenBank


(9521-10345)
Accession No. AJ001301)


ClaI/BsiWI
TEF::Δ17S::Pex20, comprising:


(4233-6043)
TEF: TEF promoter (GenBank Accession No.



AF054508)



Δ17S: codon-optimized Δ17 desaturase gene (SEQ ID



NO: 16), derived from S. diclina (US 2003/0196217 A1)



Pex20: Pex20 terminator sequence of Yarrowia Pex20



gene (GenBank Accession No. AF054613)


ClaI/PmeI
FBAIN::Δ17S::Lip2, comprising:


(4233-1811)
FBAIN: FBAIN promoter (SEQ ID NO: 162)



Δ17S: SEQ ID NO: 16 (supra)



Lip2: Lip2 terminator sequence of Yarrowia Lip2 gene



(GenBank Accession No. AJ012632)


PmeI/SwaI
Yarrowia Leu2 gene (GenBank Accession


(1811-1)
No. AF260230)


PacI/SwaI
FBAINm::Δ17S::Pex16, comprising:


(10345-1)
FBAINm: FBAINm promoter (SEQ ID NO: 163)



Δ17S: SEQ ID NO: 16 (supra)



Pex16: Pex16 terminator sequence of Yarrowia Pex16



gene (GenBank Accession No. U75433)









Plasmid pZP3L37 was digested with AscI/SphI, and then used to transform strain Y2034 according to the General Methods. Following transformation, the cells were plated onto MM plates and maintained at 30° C. for 2 to 3 days. A total of 48 transformants grown on the MM plates were picked and re-streaked onto fresh MM plates. Once grown, these strains were individually inoculated into liquid MM at 30° C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed the presence of EPA in most of the transformants with pZP3L37, but not in the parental strain (i.e., Y2034). Among the 48 selected transformants with pZP3L37, there were 18 strains that produced less than 2% EPA, 14 strains that produced 2-3% EPA, and 1 strain that produced about 7% EPA of total lipids in the engineered Yarrowia. 
The strain that produced 7% EPA was further analyzed after culturing the strain using “two-stage growth conditions”, as described in the General Methods (i.e., 48 hrs MM, 72 hrs HGM). GC analyses showed that the engineered strain produced about 10% EPA of total lipids after the two-stage growth. The strain was designated as the “E” strain.
Generation of EU Strain to Produce about 10% EPA of Total Lipids with Ura− Phenotype
Strain EU (Ura−) was created by identifying mutant cells of strain E that were 5-FOA resistant. Specifically, one loop of Yarrowia E strain cells were inoculated into 3 mL YPD medium and grown at 30° C. with shaking at 250 rpm for 24 hrs. The culture was diluted with YPD to an OD600 of 0.4 and then incubated for an additional 4 hrs. The culture was plated (100 μl/plate) onto MM+FOA plates and maintained at 30° C. for 2 to 3 days. A total of 16 FOA resistant colonies were picked and streaked onto MM and MM+FOA selection plates. From these, 10 colonies grew on FOA selection plates but not on MM plates and were selected as potential Ura− strains.
One of these strains was used as host for transformation with pY37/F15, comprising a chimeric GPD::Fusarium moniliforme Δ15::XPR2 gene and a Ura3 gene as a selection marker (FIG. 13B; SEQ ID NO:129). After three days of selection on MM plates, hundreds of colonies had grown on the plates and there was no colony growth of the transformation control that carried no plasmid. This experiment confirmed that the 5-FOA resistant host strain was Ura−, and this strain was designated as strain “EU”. Single colonies of the EU strain were then inoculated into liquid MMU additionally containing 0.1 g/L uridine and cultured for 2 days at 30° C. with shaking at 250 rpm/min. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification and subsequently analyzed with a Hewlett-Packard 6890 GC. GC analyses showed that the EU strain produced about 10% EPA of total lipids.
Generation of Y2067 Strain to Produce about 15% EPA of Total Lipids
Plasmid pKO2UF2PE (FIG. 13C; SEQ ID NO:130) was created to integrate a cluster containing two chimeric genes (comprising a heterologous Δ12 desaturase and a C18/20 elongase) and a Ura3 gene into the native Yarrowia Δ12 desaturase gene of strain EU. Plasmid pKO2UF2PE contained the following components:







TABLE 27







Description of Plasmid pKO2UF2PE (SEQ ID NO: 130)








RE Sites And



Nucleotides


Within SEQ ID
Description Of Fragment


NO: 130
And Chimeric Gene Components





AscI/BsiWI
730 bp 5′ part of Yarrowia Δ12 


(3382-2645)
desaturase gene (SEQ ID NO: 23)


SphI/EcoRI
556 bp 3′ part of Yarrowia Δ12 desaturase


(6090-6646)
gene (SEQ ID NO: 23)


SwaI/BsiWI/
FBAINm::F.Δ12DS::Pex20, comprising:


(1-2645)
FBAINm: FBAINm promoter (SEQ ID NO: 163)



F.Δ12: Fusarium moniliforme Δ12 desaturase gene



(SEQ ID NO: 27)



Pex20: Pex20 terminator sequence of Yarrowia Pex20



gene (GenBank Accession No. AF054613)


SwaI/PmeI
GPAT::EL1S::OCT, comprising:


(1-8525)
GPAT: GPAT promoter (SEQ ID NO: 164)



EL1S: codon-optimized elongase 1 gene (SEQ ID



NO: 19), derived from Mortierella alpina (GenBank



Accession No. AX464731)



OCT: OCT terminator sequence of Yarrowia OCT gene



(GenBank Accession No. X69988)


EcoRI/PacI
Yarrowia Ura3 gene (GenBank Accession


(6646-8163)
No. AJ306421)









Plasmid pKO2UF2PE was digested with AscI/SphI and then used to transform strain EU according to the General Methods (although strain EU was streaked onto a YPD plate and grown for approximately 36 hr prior to suspension in transformation buffer [versus 18 hrs]). Following transformation, cells were plated onto MM plates and maintained at 30° C. for 2 to 3 days. A total of 72 transformants grown on MM plates were picked and re-streaked separately onto fresh MM plates. Once grown, these strains were individually inoculated into liquid MM at 30° C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed the presence of EPA in almost all of the transformants with pKO2UF2PE. More specifically, among the 72 selected transformants, there were 17 strains that produced 8-9.9% EPA, 27 strains that produced 10-10.9% EPA, 16 strains that produced 11-11.9% EPA, and 7 strains that produced 12-12.7% EPA of total lipids in the engineered Yarrowia. The strain that produced 12.7% EPA was further analyzed by using the two-stage growth conditions, as described in the General Methods (i.e., 48 hrs MM, 72 hrs HGM). GC analyses showed that the engineered strain produced about 15% EPA of total lipids after the two-stage growth. The strain was designated as strain “Y2067”.
Generation of Y2067U Strain to Produce about 14% EPA of Total Lipids with Ura− Phenotype
In order to disrupt the Ura3 gene in strain Y2067, construct pZKUT16 (FIG. 13D; SEQ ID NO:131) was created to integrate a TEF::rELO2S::Pex20 chimeric gene into the Ura3 gene of strain Y2067. rELO2S is a codon-optimized rELO gene encoding a rat hepatic enzyme that elongates 16:0 to 18:0 (i.e., a C16/18 elongase). The plasmid pZKUT16 contained the following components:







TABLE 28







Description of Plasmid pZKUT16 (SEQ ID NO: 131)








RE Sites And



Nucleotides


Within SEQ ID
Description Of Fragment


NO: 131
And Chimeric Gene Components





BsiWI/PacI
721 bp 5′ part of Yarrowia Ura3 gene (GenBank


(1-721)
Accession No. AJ306421)


SalI/ClaI
724 bp 3′ part of Yarrowia Ura3 gene (GenBank


(3565-4289)
Accession No. AJ306421)


ClaI/BsiWI
TEF::rELO2S::Pex20, comprising:


(4289-1)
TEF: TEF promoter (GenBank Accession No.



AF054508)



rELO2S: codon-optimized rELO2 elongase gene (SEQ



ID NO: 52), derived from rat (GenBank Accession No.



AB071986)



Pex20: Pex20 terminator sequence of Yarrowia Pex20



gene (GenBank Accession No. AF054613)









The plasmid pZKUT16 was digested with SalI/PacI, and then used to transform Y2067 strain according to the General Methods. Following transformation, cells were plated onto MM+5-FOA selection plates and maintained at 30° C. for 2 to 3 days.
A total of 24 transformants grown on MM+5-FOA plates were picked and re-streaked onto MM plates and MM+5-FOA plates, separately. The strains that could grow on MM+5-FOA plates, but not on MM plates, were selected as Ura− strains. A total of 10 Ura− strains were individually inoculated into liquid MMU media at 30° C. and grown with shaking at 250 rpm/min for 1 day. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed the presence of 5 to 7% EPA in all of the transformants with pZKUT16 after one day growth in MMU media. The strain that produced 6.2% EPA was further analyzed using the two-stage growth conditions (i.e., 48 hrs MM, 96 hrs HGM). GC analyses showed that the engineered strain produced about 14% EPA of total lipids. The strain was designated as strain “Y2067U”.
The final genotype of this strain with respect to wildtype Yarrowia lipolytica ATCC #20362 was as follows: Ura3−, Pox3-, Y.Δ12-, FBA::F.Δ12::Lip2, FBAINm::F. Δ12::Pex20, TEF::Δ6S::Lip1, FBAIN::E1S::Pex20, GPAT::E1S::Oct, TEF::E2S::Xpr, FBAIN::Δ5::Pex20, TEF::Δ5::Lip1, FBAIN::Δ17S::Lip2, FBAINm::Δ17S::Pex16, TEF::Δ17S and TEF::rELO2S::Pex20.
Example 11
Generation of Intermediate Strain Y2107U1. Producing 16% EPA of Total Lipids
The present Example describes the construction of strain Y2107U1, derived from Yarrowia lipolytica ATCC #20362, capable of producing significant concentrations of EPA relative to the total lipids (FIG. 4). The affect of M. alpina GPAT gene over-expression was examined in this EPA producing strain based on analysis of TAG content and/or composition, as described in Example 17 (infra).
The development of strain Y2107U1 (producing 16% EPA and possessing a Ura− phenotype) herein required the construction of strain M4 (producing 8% DGLA and described in Example 4), strain Y2047 (producing 11% ARA and described in Example 4), strain Y2048 (producing 11% EPA), strain Y2060 (producing 13% EPA), strain Y2072 (producing 15% EPA), strain Y2072U1 (producing 14% EPA) and Y2089 (producing 18% EPA).
Generation of Y2048 Strain to Produce about 11% EPA of Total Lipids
Construct pZP3L37 (FIG. 13A; SEQ ID NO:128; Example 10) was utilized to integrate three synthetic Δ17 desaturase chimeric genes into the acyl-CoA oxidase 3 gene of strain Y2047 (Example 4). Specifically, plasmid pZP3L37 was digested with AscI/SphI, and then used to transform strain Y2047 according to the General Methods. Following transformation, the cells were plated onto MM plates and maintained at 30° C. for 2 to 3 days. A total of 96 transformants grown on the MM plates were picked and re-streaked onto fresh MM plates. Once grown, these strains were individually inoculated into liquid MM at 30° C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed the presence of EPA in most of the transformants with pZP3L37, but not in the parental strain (i.e., Y2047). Among the 96 selected transformants with pZP3L37, there were 20 strains that produced less than 2% EPA, 23 strains that produced 2-3% EPA, 5 strains that produced 3-4% EPA, and 2 strains (i.e., strain #71 and strain #94) that produced about 6% EPA of total lipids in the engineered Yarrowia. 
Strain #71 (which produced 6% EPA) was further analyzed by culturing it in two-stage growth conditions (i.e., 48 hrs MM, 72 hrs HMG). GC analyses showed that strain #71 produced about 11% EPA of total lipids. The strain was designated as “Y2048”.
Generation of Y2060 Strain to Produce about 13% EPA of Total Lipids with Ura− Phenotype
In order to disrupt the Ura3 gene in strain Y2048, construct pZKUT16 (FIG. 13D; SEQ ID NO:131; Example 10) was utilized to integrate a TEF::rELO2S::Pex20 chimeric gene into the Ura3 gene of strain Y2048. Specifically, plasmid pZKUT16 was digested with SalI/PacI, and then used to transform strain Y2048 according to the General Methods. Following transformation, cells were plated onto MM+5-FOA selection plates and maintained at 30° C. for 2 to 3 days.
A total of 40 transformants grown on MM+5-FOA plates were picked and re-streaked onto MM plates and MM+5-FOA plates, separately. Those strains that could grow on MM+5-FOA plates, but not on MM plates, were selected as Ura− strains. Each of these 40 Ura− strains were individually inoculated into liquid MMU and grown at 30° C. with shaking at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed that there were 14 strains that produced less than 5% EPA, 9 strains that produced 5-5.9% EPA, 15 strains that produced 6-6.9% EPA, and 7 strains that produced 7-8% EPA of total lipids after two day growth in MMU media. The strains that produced 7-8% EPA were further analyzed using two-stage growth conditions (i.e., 48 hrs MM, 96 hrs HGM). GC analyses showed that all these strains produced more than 10% EPA; and, one of them produced about 13% EPA of the total lipids. That strain was designated as strain “Y2060”.
Generation of Y2072 Strain to Produce about 15% EPA of Total Lipids
Construct pKO2UM25E (FIG. 14A; SEQ ID NO:132) was used to integrate a cluster of three chimeric genes (comprising a C18/20 elongase, a Δ12 desaturase and a Δ5 desaturase) and a Ura3 gene into the native Yarrowia Δ12 desaturase gene site of strain Y2060. Plasmid pKO2UM25E contained the following components:







TABLE 29







Description of Plasmid pKO2UM25E (SEQ ID NO: 132)








RE Sites And



Nucleotides


Within SEQ ID
Description Of Fragment


NO: 132
And Chimeric Gene Components





HindIII/AscI
728 bp 5′ part of Yarrowia Δ12 desaturase


(1-728)
gene (SEQ ID NO: 23)


SphI/EcoRI
556 bp 3′ part of Yarrowia Δ12 desaturase


(3436-3992)
gene (SEQ ID NO: 23)


BsiWI/HindIII
GPAT::EL1S::XPR, comprising:


(10437-1)
GPAT: GPAT promoter (SEQ ID NO: 164)



EL1S: codon-optimized elongase 1 gene (SEQ ID



NO: 19), derived from Mortierella alpina (GenBank



Accession No. AX464731)



XPR: ~100 bp of the 3′ region of the Yarrowia Xpr gene



(GenBank Accession No. M17741)


BgIII/BsiWI
FBAIN::M.Δ12::Pex20, comprising:


(7920-10437)
FBAIN: FBAIN promoter (SEQ ID NO: 162)



M.Δ12: Mortierella isabellina Δ12 desaturase gene



(GenBank Accession No. AF417245; SEQ ID NO: 25)



Pex20: Pex20 terminator sequence of Yarrowia Pex20



gene (GenBank Accession No. AF054613)


SalI/PacI
Yarrowia Ura3 gene (Gene Bank Accession


(6046-7544)
No. AJ306421)


EcoRI/SalI
TEF::I.Δ5S::Pex20, comprising:


(3992-6046)
TEF: TEF promoter (GenBank Accession No.



AF054508)



I.Δ5S: codon-optimized Δ5 desaturase gene (SEQ ID



NO: 10), derived from Isochrysis galbana (WO 2002/



081668)



Pex20: Pex20 terminator sequence of Yarrowia Pex20



gene (GenBank Accession No. AF054613)









Plasmid pKO2UM25E was digested with SphI/AscI, and then used to transform Y2060 according to the General Methods. Following transformation, cells were plated onto MM plates and maintained at 30° C. for 2 to 3 days.
A total of 63 transformants grown on MM plates were picked and re-streaked onto fresh MM plates. Once grown, these strains were individually inoculated into liquid MM at 30° C. and cultured with shaking at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed the presence of EPA in almost all transformants with pKO2UM25E after one-day growth in MM media. Among the 63 selected transformants, there were 26 strains that produced 6-8.9% EPA and 46 strains that produced more than 9% EPA. The strains that produced more than 9% EPA were selected for further analysis using two-stage growth conditions (i.e., 48 hrs MM, 96 hrs HGM). GC analyses showed that 45 out of the 46 selected strains produced 11-14.5% EPA while culture #2 produced 15.1% EPA of total lipids after the two-stage growth. This strain (i.e., #2) was designated as strain “Y2072”.
Generation of Y2072U1 Strain to Produce about 14% EPA of Total Lipids with Ura− Phenotype
The construct pZKUGPI5S (FIG. 14B; SEQ ID NO:133) was created to integrate a GPAT::I.Δ5S::Pex20 chimeric gene into the Ura3 gene of Y2072 strain. More specifically, plasmid pZKUGPI5S contained the following components:







TABLE 30







Description of Plasmid pZKUGPI5S (SEQ ID NO: 133)








RE Sites And



Nucleotides


Within SEQ ID
Description Of Fragment


NO: 133
And Chimeric Gene Components





BsiWI/PacI
721 bp 5′ part of Yarrowia Ura3 gene (GenBank


(318-1038)
Accession No. AJ306421)


SalI/ClaI
724 bp 3′ part of Yarrowia Ura3 gene (GenBank


(3882-4606)
Accession No. AJ306421)


ClaI/BsiWI
GPAT::I.Δ5S::Pex20, comprising:


(4606-318)
GPAT: GPAT promoter (SEQ ID NO: 164)



I.Δ5S: codon-optimized Δ5 desaturase gene (SEQ ID



NO: 10), derived from Isochrysis galbana (WO 2002/



081668)



Pex20: Pex20 terminator sequence of Yarrowia Pex20



gene (GenBank Accession No. AF054613)









Plasmid pZKUGPI5S was digested with SalI/PacI, and then used to transform strain Y2072 according to the General Methods. Following transformation, cells were plated onto MM+5-FOA selection plates and maintained at 30° C. for 3 to 4 days.
A total of 24 transformants grown on MM+5-FOA plates were picked and re-streaked onto MM plates and MM+5-FOA plates, separately. Those strains that could grow on MM+5-FOA plates, but not on MM plates, were selected as Ura− strains. Each of these 24 Ura− strains were individually inoculated into liquid MMU and grown at 30° C. with shaking at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed that there were 8 strains that produced 7.3-8.9% EPA, 14 strains that produced 9-9.9% EPA, 1 strain that produced 10.5% EPA (i.e., #1) and 1 strain that produced 10.7% EPA (i.e., #23) of total lipids after two day growth in MMU. Strains #1 and #23 were further analyzed using two-stage growth conditions (i.e., 48 hrs MM, 96 hrs HGM). GC analyses showed that these two strains produced about 14% EPA of total lipids after the two-stage growth. Strain #1 was designated as strain “Y2072U1”.
Generation of Y2089 Strain to Produce about 18% EPA of Total Lipids
Construct pDMW302T16 (FIG. 14C; SEQ ID NO:134) was created to integrate a cluster of four chimeric genes (comprising a C16/18 elongase, a C18/20 elongase, a Δ6 desaturase and a Δ12 desaturase) and a Ura3 gene into the Yarrowia lipase 1 gene site of Y2072U1 strain. Plasmid pDMW302T16 contained the following components:







TABLE 31







Description of Plasmid pDMW302T16 (SEQ ID NO: 134)








RE Sites And



Nucleotides


Within SEQ ID
Description Of Fragment


NO: 134
And Chimeric Gene Components





BsiWI/AscI
817 bp 5′ part of Yarrowia lipase1 gene


(1-817)
(GenBank Accession No. Z50020)


SphI/PacI
769 bp 3′ part of Yarrowia lipase1 gene


3525-4294
(GenBank Accession No. Z50020)


EcoRI/BsiWI
TEF::rELO2S::Pex20, comprising:


(13328-1)
TEF: TEF promoter (GenBank Accession No.



AF054508)



rELO2S: codon-optimized rELO2 elongase gene (SEQ



ID NO: 52), derived from rat (GenBank Accession No.



AB071986)



Pex20: Pex20 terminator sequence of Yarrowia Pex20



gene (GenBank Accession No. AF054613)


BgIII/EcoRI
FBAIN::D6S::Lip1, comprising:


(10599-13306)
FBAIN: FBAIN promoter (SEQ ID NO: 162)



Δ6S: codon-optimized Δ6 desaturase gene (SEQ ID



NO: 3), derived from Mortierella alpina (GenBank



Accession No. AF465281)



Lip1: Lip1 terminator sequence from Yarrowia Lip1



gene (GenBank Accession No. Z50020)


ClaI/PmeI
GPDIN::EL1S::Lip2, comprising:


(8078-10555)
GPDIN: GPDIN promoter (SEQ ID NO: 159)



EL1S: codon-optimized elongase 1 gene (SEQ ID



NO: 19), derived from Mortierella alpina (GenBank



Accession No. AX464731)



Lip2: Lip2 terminator of Yarrowia lipase2 gene



(GenBank Accession No. AJ012632)


EcoRI/ClaI
Yarrowia Ura 3 gene (Gene Bank Accession


(6450-8078)
No. AJ306421)


PacI/EcoRI
TEF::F.Δ12::Pex16, comprising:


(4294-6450)
TEF: TEF promoter (GenBank Accession No.



AF054508)



F.Δ12: Fusarium moniliforme Δ12 desaturase gene



(SEQ ID NO: 27)



Pex16: Pex16 terminator of Yarrowia Pex16 gene



(GenBank Accession No. U75433)









Plasmid pDMW302T16 was digested with SphI/AscI, and then used to transform strain Y2072U1 according to the General Methods. Following transformation, cells were plated onto MM plates and maintained at 30° C. for 3 to 4 days. A total of 48 transformants grown on MM plates were picked and re-streaked onto fresh MM plates. Once grown, these strains were individually inoculated into liquid MM and grown at 30° C. with shaking at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed that EPA was produced in almost all transformants of Y2072U1 with pDMW302T16 after two-day growth in MM media. Among the 48 selected transformants, there were 27 strains that produced less than 10% EPA, 14 strains that produced 10-12.9% EPA and 5 strains that produced 13-13.9% EPA. Strain #34 (producing 13.9% EPA) was selected for further analysis using the two-stage growth procedure (i.e., 48 hrs MM, 96 hrs HGM). GC analyses showed that strain #34 produced about 18% EPA of total lipids. Strain #34 was designated as strain “Y2089”.
The genotype of strain Y2089 with respect to wildtype Yarrowia lipolytica ATCC #20362 was as follows: Pox3-, LIP1-, Y.Δ12-, FBA::F.Δ2::Lip2, TEF::F.Δ12::Pex16, FBAIN::MΔ12::Pex20, TEF::Δ6S::Lip1, FBAIN::Δ6S::Lip1, FBAIN::E1S::Pex20, GPAT::E1S::Oct, GPDIN::E1S::Lip2, TEF::E2S::Xpr, FBAIN::MAΔ5::Pex20, TEF::MAΔ5::Lip1, TEF::HΔ5S::Pex16, TEF::IΔ5S::Pex20, GPAT::IΔ5S::Pex20, FBAIN::Δ17S::Lip2, FBAINm::Δ17S::Pex16, TEF::Δ17S::Pex16 and 2×TEF::rELO2S::Pex20.
Generation of Y2107U1 Strain to Produce about 16% EPA of Total Lipids with Ura− phenotype
Construct pZKUGPE1S (FIG. 14D; SEQ ID NO:135) was created to integrate a GPAT::EL1S::Pex20 chimeric gene into the Ura3 gene of strain Y2089. More specifically, plasmid pZKUGPE1S contained the following components:







TABLE 32







Description of Plasmid pZKUGPE1S (SEQ ID NO: 135)








RE Sites And



Nucleotides


Within SEQ ID
Description Of Fragment


NO: 135
And Chimeric Gene Components





BsiWI/PacI
721 bp 5′ part of Yarrowia Ura3 gene


(318-1038)
(GenBank Accession No. AJ306421)


SalI/ClaI
724 bp 3′ part of Yarrowia Ura3 gene


(3882-4606)
(GenBank Accession No. AJ306421)


ClaI/BsiWI
GPAT::E1S::Pex20, comprising:


(4606-318)
GPAT: GPAT promoter (SEQ ID NO: 164)



EL1S: codon-optimized elongase 1 gene (SEQ ID



NO: 19), derived from Mortierella alpina (GenBank



Accession No. AX464731)



Pex20: Pex20 terminator sequence of Yarrowia Pex20



gene (GenBank Accession No. AF054613)









Plasmid pZKUGPE1S was digested with PstI/PacI, and then used to transform strain Y2089 according to the General Methods. Following transformation, cells were plated onto MM+5-FOA selection plates and maintained at 30° C. for 3 to 4 days.
A total of 8 transformants grown on MM+5-FOA plates were picked and re-streaked onto MM plates and MM+5-FOA plates, separately. Those strains that could grow on MM+5-FOA plates, but not on MM plates, were selected as Ura− strains. Each of these 8 Ura− strains were individually inoculated into liquid MMU and grown at 30° C. with shaking at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed that there were 6 strains that produced 6.6-8.7% EPA and 2 strains that produced 9.4-10% EPA (i.e., #4 and #5) of total lipids after two day growth in MMU. Strains #4 and #5 were further analyzed using the two-stage growth conditions (i.e., 48 hrs MM, 96 hrs HGM). GC analyses showed that these two strains produced about 16% EPA of total lipids after the two-stage growth. Strain #4 was designated as strain “Y2107U1” and strain #5 was designated as strain “Y2107U2”.
Example 12
Generation of Intermediate Strain MU, Producing 9-12% EPA of Total Lipids
The present Example describes the construction of strain MU, derived from Yarrowia lipolytica ATCC #20362, capable of producing significant concentrations of EPA relative to the total lipids (FIG. 4). The affect of various native Y. lipolytica acyltransferase knockouts were examined in this EPA producing strain based on analysis of TAG content and/or composition, as described in Example 24 (infra).
The development of strain MU (producing 9-12% EPA herein) required the construction of strain M4 (producing 8% DGLA and described in Example 4), strain Y2034 (producing 10% ARA and described in Example 4), strain E (producing 10% EPA and described in Example 10), strain EU (producing 10% EPA and described in Example 10) and strain M26 (producing 14% EPA).
Generation of M26 Strain to Produce about 14% EPA of Total Lipids
Construct pKO2UM26E (SEQ ID NO:136; FIG. 15A) was used to integrate a cluster of three chimeric genes (comprising a C18/20 elongase, a Δ6 desaturase and a Δ12 desaturase) and a Ura3 gene into the Yarrowia Δ12 desaturase gene site of EU strain (Example 10). Plasmid pKO2UM26E contained the following components:
Table 33
Description of Plasmid pKO2UM26E (SEQ ID NO:136)







TABLE 33







Description of Plasmid pKO2UM26E (SEQ ID NO: 136)








RE Sites And



Nucleotides


Within SEQ ID
Description Of Fragment


NO: 136
And Chimeric Gene Components





HindIII/AscI
728 bp 5′ part of Yarrowia Δ12 desaturase


(1-728)
gene (SEQ ID NO: 23)


SphI/EcoRI
556 bp 3′ part of Yarrowia Δ12 desaturase


(3436-3992)
gene (SEQ ID NO: 23)


BsiWI/HindIII
GPAT::EL1S::XPR, comprising:


(11095-1)
GPAT: GPAT promoter (SEQ ID NO: 164)



EL1S: codon-optimized elongase 1 gene (SEQ ID



NO: 19), derived from Mortierella alpina (GenBank



Accession No. AX464731)



XPR: ~100 bp of the 3′ region of the Yarrowia Xpr gene



(GenBank Accession No. M17741)


BglII/BsiWI
FBAIN::M.Δ12::Pex20, comprising:


(8578-11095)
FBAIN: FBAIN promoter (SEQ ID NO: 162)



M.Δ12: Mortieralla isabellina Δ12 desaturase gene



(GenBank Accession No. AF417245; SEQ ID NO: 25)



Pex20: Pex20 terminator sequence of Yarrowia Pex20



gene (GenBank Accession No. AF054613)


SalI/PacI
Yarrowia Ura3 gene (GenBank Accession


(6704-8202)
No. AJ306421)


EcoRI/SalI
FBAIN::M.Δ6B::Pex20, comprising:


(3992-6704)
TEF: TEF promoter (GenBank Accession No.



AF054508)



M.Δ6B: Mortieralla alpina Δ6 desaturase gene “B”



(GenBank Accession No. AB070555; SEQ ID NO: 4)



Pex20: Pex20 terminator sequence of Yarrowia Pex20



gene (GenBank Accession No. AF054613)









The plasmid pKO2UM26E was digested with SphI/AscI, and then used to transform EU strain (Example 10) according to the General Methods. Following transformation, cells were plated onto MM plates and maintained at 30° C. for 2 to 3 days.
A total of 48 transformants grown on MM plates were picked and re-streaked onto fresh MM plates. Once grown, these strains were individually inoculated into liquid MM at 30° C. and grown with shaking at 250 rpm/min for 1 day. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed that EPA was produced in almost all transformants with pKO2UM26E after one-day growth in MM media. Among the 48 selected transformants, 5 strains produced less than 4% EPA, 23 strains produced 4-5.9% EPA, 9 strains produced 6-6.9% EPA and 11 strains produced 7-8.2% EPA of total lipids in the engineered Yarrowia. The strain that produced 8.2% EPA was selected for further analysis using the two-stage growth procedure (i.e., 48 hrs MM, 96 hrs HGM). GC analyses showed that the engineered strain produced about 14% EPA of total lipids. The strain was designated as strain “M26”.
The genotype of the M26 strain with respect to wildtype Yarrowia lipolytica ATCC #20362 was as follows: Pox3-, Y.Δ12-, FBA::F.Δ12::Lip2, FBAIN::MΔ12::Pex20, TEF::Δ6S::Lip1, FBAIN::Δ6B::Pex20, FBAIN::E1 S::Pex20, GPAT::E1 S::Xpr, TEF::E2S::Xpr, FBAIN::MAΔ5::Pex20, TEF::MAΔ5::Lip1, TEF::HΔ5S::Pex16, FBAIN::Δ17S::Lip2, FBAINm::Δ17S::Pex16, TEF::Δ17S::Pex16 and TEF::rELO2S::Pex20.
Generation of MU Strain to Produce about 14% EPA of Total Lipids
Strain MU was a Ura auxotroph of strain M26. This strain was made by transforming strain M26 with 5 μg of plasmid pZKUM (SEQ ID NO:137) that had been digested with PacI and HincII. Transformation was performed using the Frozen-EZ Yeast Transformation kit (Zymo Research Corporation, Orange, Calif.) and transformants were selected by plating 100 μl of the transformed cell mix on an agar plate with the following medium: 6.7 g/L yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.), 20 g/L dextrose, 50 mg/L uracil and 800 mg/L FOA. After 7 days, small colonies appeared that were plated on MM and MMU agar plates. All were URA auxotrophs. One of the strains was designated “MU”.
Example 13
Preparation of Mortierella alpina Genomic DNA and cDNA
The present Example describes the preparation of genomic DNA and cDNA from Mortierella alpina (ATCC #16266). This enabled isolation of the M. alpina LPMT2, DGAT1, DGAT2, GPAT and ELO3, as described in Examples 14, 15, 16, 17 and 18, respectively.
Preparation of Genomic DNA from Mortierella alpina 
Genomic DNA was isolated from Mortierella alpina (ATCC #16266) using a QiaPrep Spin Miniprep Kit (Qiagen, Catalog #627106). Cells grown on a YPD agar plate (2% Bacto-yeast extract, 3% Bacto-peptone, 2% glucose, 2.5% bacto-agar) were scraped off and resuspended in 1.2 mL of kit buffer P1. The resuspended cells were placed in two 2.0 mL screw cap tubes, each containing 0.6 mL glass beads (0.5 mm diameter). The cells were homogenized at the HOMOGENIZE setting on a Biospec (Bartlesville, Okla.) mini bead beater for 2 min. The tubes were then centrifuged at 14,000 rpm in an Eppendorf microfuge for 2 min. The supernatant (0.75 mL) was transferred to three 1.5 mL microfuge tubes. Equal volumes of kit buffer P2 were added to each tube. After mixing the tubes by inversion three times, 0.35 mL of buffer N3 was added to each tube. The contents of each tube were again mixed by inversion for a total of five times. The mixture was centrifuged at 14,000 rpm in an Eppendorf microfuge for 5 min. The supernatant from each tube was transferred individually into 3 separate kit spin columns. The columns were then subjected to the following steps: centrifugation (1 min at 14,000 rpm), wash once with buffer PE, centrifugation (1 min at 14,000 rpm), and then a final centrifugation (1 min at 14,000 rpm). Buffer EB (50 μl) was added to each column and let stand for 1 min. The genomic DNA was then eluted by centrifugation at 14,000 rpm for 1 min.
Preparation of cDNA from Mortierella alpina 
cDNA of Mortierella alpina was prepared using the BD-Clontech Creator Smart® cDNA library kit (Mississauga, ON, Canada), according to the manufacturer's protocol. Specifically, M. alpina strain ATCC #16266 was grown in 60 mL YPD medium (2% Bacto-yeast extract, 3% Bactor-peptone, 2% glucose) for 3 days at 23° C. Cells were pelleted by centrifugation at 3750 rpm in a Beckman GH3.8 rotor for 10 min and resuspended in 6×0.6 mL Trizole reagent (Invitrogen). 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 mini bead beater for 2 min. The tubes were briefly spun to settle the beads. Liquid was transferred to 4 fresh 1.5 mL microfuge tubes and 0.2 mL chloroform/isoamyl alcohol (24:1) was added to each tube. The tubes were shaken by hand for 1 min and let stand for 3 min. The tubes were then spun at 14,000 rpm for 10 min at 4° C. The upper layer was transferred to 4 new tubes. Isopropyl alcohol (0.5 mL) was added to each tube. Tubes were incubated at room temperature for 15 min, followed by centrifugation at 14,000 rpm and 4° C. for 10 min. The pellets were washed with 1 mL each of 75% ethanol, made with RNase-free water and air-dried. The total RNA sample was then redissolved in 500 μl of water, and the amount of RNA was measured by A260 nm using 1:50 diluted RNA sample. A total of 3.14 mg RNA was obtained.
This total RNA sample was further purified with the Qiagen RNeasy total RNA Midi kit following the manufacturer's protocol. Thus, the total RNA sample was diluted to 2 mL and mixed with 8 mL of buffer RLT with 80 μl of β-mercaptoethanol and 5.6 mL 100% ethanol. The sample was divided into 4 portions and loaded onto 4 RNeasy midid columns. The columns were then centrifuged for 5 min at 4500×g. To wash the columns, 2 mL of buffer RPE was loaded and the columns centrifuged for 2 min at 4500×g. The washing step was repeated once, except that the centrifugation time was extended to 5 min. Total RNA was eluted by applying 250 μl of RNase free water to each column, waiting for 1 min and centrifuging at 4500×g for 3 min.
PolyA(+)RNA was then isolated from the above total RNA sample, following the protocol of Amersham Biosciences' mRNA Purification Kit. Briefly, 2 oligo-dt-cellulose columns were used. The columns were washed twice with 1 mL each of high salt buffer. The total RNA sample from the previous step was diluted to 2 mL total volume and adjusted to 10 mM Tris/HCl, pH 8.0, 1 mM EDTA. The sample was heated at 65° C. for 5 min, then placed on ice. Sample buffer (0.4 mL) was added and the sample was then loaded onto the two oligo-dT-cellulose columns under gravity feed. The columns were centrifuged at 350×g for 2 min, washed 2× with 0.25 mL each of high salt buffer, each time followed by centrifugation at 350×g for 2 min. The columns were further washed 3 times with low salt buffer, following the same centrifugation routine. Poly(A)+ RNA was eluted by washing the column 4 times with 0.25 mL each of elution buffer preheated to 65° C., followed by the same centrifugation procedure. The entire purification process was repeated once. Purified poly(A)+ RNA was obtained with a concentration of 30.4 ng/μl.
cDNA was generated, using the LD-PCR method specified by BD-Clontech and 0.1 μg 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:281) and 1 μl of CDSIII/3′ PCR primer (SEQ ID NO:282). 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 first 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:283), 2 μl CDSIII/3′-PCR primer (SEQ ID NO:282), 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 14-20 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 2×106 colonies was obtained. Sequencing of the cDNA library was carried out by Agencourt Bioscience Corporation (Beverly, Mass.), using an M13 forward primer (SEQ ID NO:284).
Example 14
Mortierella alpina LPMT2 Expression Increases Percent PUFAs
The present Example describes increased EPA biosynthesis and accumulation in Yarrowia lipolytica strain Y2067U (Example 10) that was transformed to co-express the M. alpina LPMT2 (SEQ ID NOs:69 and 70). It is contemplated art that a Y. lipolytica host strain engineered to produce ARA via either the Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8 desaturase pathway could demonstrate increased ARA biosynthesis and accumulation, if the M. alpina LPAAT2 was similarly co-expressed therein (e.g., in strains Y2034, Y2047 and/or Y2214).
The M. alpina LPAAT2 ORF was cloned as follows. Primers MLPAT-F and MLPAT-R (SEQ ID NO:285 and 286) were used to amplify the LPAAT2 ORF from the cDNA of M. alpina (Example 13) by PCR. The reaction mixture contained 1 μl of the cDNA, 1 μl each of the primers, 22 μl water and 25 μl ExTaq premix 2×Taq PCR solution (TaKaRa Bio Inc., Otsu, Shiga, 520-2193, Japan). Amplification was carried out as follows: initial denaturation at 94° C. for 150 sec, followed by 30 cycles of denaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec and elongation at 72° C. for 90 sec. A final elongation cycle at 72° C. for 10 min was carried out, followed by reaction termination at 4° C. A  ̃950 bp DNA fragment was obtained from the PCR reaction. It was purified using a Qiagen (Valencia, Calif.) PCR purification kit according to the manufacturer's protocol. The purified PCR product was digested with NcoI and NotI, and cloned into Nco I-Not I cut pZUF17 vector (SEQ ID NO:118; FIG. 8B), such that the gene was under the control of the Y. lipolytica FBAIN promoter and the PEX20-3′ terminator region in the auto-replicating vector for expression in Y. lipolytica. Correct transformants were confirmed by restriction analysis of miniprep DNA and the resultant plasmid was designated as “pMLPAT-17” (SEQ ID NO:138).
To integrate the M. alpina LPMT2 into the genome of Yarrowia lipolytica, plasmid pMLPAT-Int was created. Primers LPAT-Re-5-1 and LPAT-Re-5-2 (SEQ ID NOs:287 and 288) were used to amplify a 1129 bp DNA fragment, YLPAT-5′ (SEQ ID NO:289), containing a 1103 bp fragment of Y. lipolytica genome immediately upstream of the AUG of the Y. lipolytica LPMT1 (SEQ ID NO:71). The reaction mixture contained 1 μl of Y. lipolytica genomic DNA, 1 μl each of the primers, 22 μl water and 25 μl ExTaq premix 2×Taq PCR solution (TaKaRa). Amplification was carried out as described above. A  ̃1130 bp DNA fragment was obtained from the PCR reaction. It was purified using Qiagen's PCR purification kit according to the manufacturer's protocol. The purified PCR product was digested with SalI and ClaI, and cloned into SalI-ClaI cut pBluescript SK (−) vector, resulting in plasmid “pYLPAT-5′”.
Primers LPAT-Re-3-1 and LPAT-Re-3-2 (SEQ ID NOs:290 and 291) were then used to amplify a 938 bp fragment, YLPAT-3′ (SEQ ID NO:292), containing a 903 bp fragment of Y. lipolytica genome immediately after the stop codon of Y. lipolytica LPAAT1, using the same conditions as above. The purified PCR product was digested with ClaI and XhoI, and cloned into ClaI-XhoI digested pYLPAT-5′. Correct transformants were confirmed by miniprep analysis and the resultant plasmid was designated “pYLPAT-5′-3′”.
pMLPAT-17 (SEQ ID NO:138) was then digested with ClaI and NotI, and a  ̃3.5 kb fragment containing the Y. lipolytica URA3 gene, the Y. lipolytica FBAIN promoter and the M. alpina LPAAT2 gene was isolated using a Qiagen Qiaexll gel purification kit according to the manufacturer's protocol. This fragment was cloned into ClaI-NotI digested pYLPAT-5′-3′. Correct transformants were confirmed by miniprep and restriction analysis. The resulting plasmid was named “pMLPAT-Int” (SEQ ID NO:139).
“Control” vector pZUF-MOD-1 (SEQ ID NO:140; FIG. 15B) was prepared as follows. First, primers pzuf-mod1 and pzuf-mod2 (SEQ ID NOs:293 and 294) were used to amplify a 252 bp “stuffer” DNA fragment using PDNR-LIB (ClonTech, Palo Alto, Calif.) as template. The amplified fragment was purified with a Qiagen QiaQuick PCR purification kit, digested with NcoI and NotI using standard conditions, and then purified again with a QiaQuick PCR purification kit. This fragment was ligated into similarly digested NcoI-/NotI-cut pZUF17 vector (SEQ ID NO:118; FIG. 8B) and the resulting ligation mixture was used to transform E. coli Top 10 cells (Invitrogen). Plasmid DNA was purified from 4 resulting colonies, using a Qiagen QiaPrep Spin Miniprep kit. The purified plasmids were digested with NcoI and NotI to confirm the presence of the  ̃250 bp fragment. The resulting plasmid was named “pZUF-MOD-1” (SEQ ID NO:140).
Y. lipolytica strain Y2067U (from Example 10, producing 14% EPA of total lipids) was transformed with plasmid pMLPAT-17, plasmid PZUF-MOD-1 (control) and SpeI/XbaI digested plasmid pMLPAT-Int, individually, according to the General Methods. Transformants were grown for 2 days in synthetic MM supplemented with amino acids, followed by 4 days in HGM. The fatty acid profile of two transformants containing pZUF-MOD-1, two transformants containing pMLPAT-17, and two transformants having pMLPAT-Int integrated into the genome are shown below in the Table, based on GC analysis (as described in the General Methods). Fatty acids are identified as 18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA and EPA; and the composition of each is presented as a % of the total fatty acids.







TABLE 34







Lipid Composition In Yarrowia Strain Y2067U Engineered To Overexpress


M. alpina LPAAT2









Total Fatty Acids















Strain
18:0
18:1
18:2
GLA
DGLA
ARA
ETA
EPA


















Y2067U + pZUF-MOD-1 #1
1.1
4.7
10.9
19.4
6.3
0.9
3.9
13.8


Y2067U + pZUF-MOD-1 #2
0.9
4.4
9.5
19.3
6.6
0.9
4.0
14.1


Y2067U + pMLPAT-17 #1
1.0
4.4
9.8
18.6
5.9
0.8
3.4
15.5


Y2067U + pMLPAT-17 #2
0.7
3.5
8.4
16.7
6.2
1.0
2.9
16.0


Y2067U + pMLPAT-Int #1
1.9
4.9
13.9
21.1
4.8
1.1
2.7
16.6


Y2067U + pMLPAT-Int #2
1.7
4.2
12.1
21.3
5.2
1.2
2.9
17.3









As demonstrated above, expression of the M. alpina LPMT2 from pMLPAT-17 increased the % EPA from −14% in the “control” strains to 15.5-16%. An additional increase in EPA to 16.6-17.3% was achieved when M. alpina LPAAT2 was integrated into the genome with pMLPAT-Int. Further increase would be expected, if the native Yarrowia lipolytica LPMT1 (SEQ ID NOs:71 and 72) and/or LPAAT2 (SEQ ID NOs:74 and 75) were knocked-out in e.g., strain Y2067U+pMLPAT-Int.
Example 15
Mortierella alpina DGAT1 Expression Increases Percent PUFAs
The present Example describes increased EPA biosynthesis and accumulation in Yarrowia lipolytica strain Y2067U (Example 10) that was transformed to co-express the M. alpina DGAT1 cDNA (SEQ ID NO:83). It is contemplated that a Y. lipolytica host strain engineered to produce ARA via either the Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8 desaturase pathway could demonstrate increased ARA biosynthesis and accumulation, if the M. alpina DGAT1 was similarly co-expressed therein (e.g., in strains Y2034, Y2047 and/or Y2214).
The M. alpina DGAT1 ORF was cloned as follows. First, to aid the cloning of the cDNA, the sequence of the second codon of the DGAT1 was changed from ‘ACA’ to ‘GCA’, resulting in an amino acid change of threonine to alanine. This was accomplished by amplifying the complete coding region of the M. alpina DGAT1 ORF with primers MACAT-F1 and MACAT-R (SEQ ID NOs:295 and 296). Specifically, the PCR reaction mixture contained 1 μl each of a 20 μM solution of primers MACAT-F1 and MACAT-R, 1 μl of M. alpina cDNA (supra, Example 13), 22 μl water and 25 μl ExTaq premix 2×Taq PCR solution (TaKaRa Bio Inc., Otsu, Shiga, 520-2193, Japan). Amplification was carried out as follows: initial denaturation at 94° C. for 150 sec, followed by 30 cycles of denaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec, and elongation at 72° C. for 90 sec. A final elongation cycle at 72° C. for 10 min was carried out, followed by reaction termination at 4° C. A  ̃1600 bp DNA fragment was obtained from the PCR reaction. It was purified using Qiagen's PCR purification kit according to the manufacturer's protocol.
The M. alpina DGAT1 ORF was to be inserted into Nco I- and Not I-digested plasmid pZUF17 (SEQ ID NO:118; FIG. 8B), such that the ORF was cloned under the control of the FBAIN promoter and the PEX20-3′ terminator region. However, since the DGAT1 ORF contained an internal NcoI site, it was necessary to perform two separate restriction enzyme digestions for cloning. First,  ̃2 μg of the purified PCR product was digested with BamHI and Nco I. The reaction mixture contained 20 U of each enzyme (Promega) and 6 μl of restriction buffer D in a total volume of 60 μl. The mixture was incubated for 2 hrs at 37° C. A  ̃320 bp fragment was separated by agarose gel electrophoresis and purified using a Qiagen Qiaex II gel purification kit. Separately,  ̃2 μg of the purified PCR product was digested with BamHI and Not I using identical reaction conditions to those above, except Nco I was replaced by Not I. A  ̃1280 bp fragment was isolated and purified as above. Finally,  ̃3 μg of pZUF17 was digested with Nco I and Not I and purified as described above, generating a  ̃7 kB fragment.
The  ̃7 kB Nco I/Not I pZUF17 fragment, the  ̃320 bp Nco I/BamHI DGAT1 fragment and the  ̃1280 bp BamHI/Not I DGAT1 fragment were ligated together in a three-way ligation incubated at room temperature overnight. The ligation mixture contained 100 ng of the 7 kB fragment and 200 ng each of the 320 bp and 1280 bp fragments, 2 μl ligase buffer, and 2 U T4 DNA ligase (Promega) in a total volume of 20 μl. The ligation products were used to transform E. coli Top 10 chemical competent cells (Invitrogen) according to the manufacturer's protocol.
Individual colonies (12 total) from the transformation were used to inoculate cultures for miniprep analysis. Restriction mapping and sequencing showed that 5 out of the 12 colonies harbored the desired plasmid, which was named “pMDGAT1-17” (FIG. 15C; SEQ ID NO:141).
Y. lipolytica strain Y2067U (from Example 10) was transformed with pMDGAT1-17 and pZUF-MOD-1 (supra, Example 14), respectively, according to the General Methods. Transformants were grown for 2 days in synthetic MM supplemented with amino acids, followed by 4 days in HGM. The fatty acid profile of two transformants containing pMDGAT1-17 and two transformants containing pZUF-MOD-1 are shown below in Table 35, based on GC analysis (as described in the General Methods). Fatty acids are identified as 18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA and EPA; and the composition of each is presented as a % of the total fatty acids.







TABLE 35







Lipid Composition In Yarrowia Strain Y2067U Engineered To Overexpress


M. alpina DGAT1









Total Fatty Acids















Strain
18:0
18:1
18:2
GLA
DGLA
ARA
ETA
EPA





Y2067U + pZUF-MOD-1 #1
1.31
6.92
12.03
23.11
5.72
1.05
3.80
13.20


Y2067U + pZUF-MOD-1 #2
1.39
6.83
12.15
21.99
5.83
1.07
3.82
13.47


Y2067U + pMDGAT1-17 #1
0.89
7.13
10.87
24.88
5.82
1.19
3.97
14.09


Y2067U + pMDGAT1-17 #2
0.86
7.20
10.25
22.42
6.35
1.26
4.38
15.07









As demonstrated above, expression of the M. alpina DGAT1 from plasmid pMDGAT1-17 increased the % EPA from  ̃13.3% in the “control” strains to  ̃14.1% (“Y2067U+pMDGAT1-17 #1”) and  ̃15.1% (“Y2067U+pMDGAT1-17 #2”), respectively. An additional increase in EPA would be expected, if the native Yarrowia lipolytica DGAT1 (SEQ ID NOs:81 and 82) were knocked-out in e.g., strain Y2067U+pMDGAT1-17.
Example 16
Mortierella alpina DGAT2 Increases Percent PUFAs
The present Example describes increased EPA biosynthesis and accumulation in Yarrowia lipolytica strain Y2067U (Example 10) that was transformed to co-express the M. alpina DGAT2 cDNA (SEQ ID NO:95). It is contemplated art that a Y. lipolytica host strain engineered to produce ARA via either the Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8 desaturase pathway could demonstrate increased ARA biosynthesis and accumulation, if the M. alpina DGAT2 was similarly co-expressed therein (e.g., in strains Y2034, Y2047 and/or Y2214).
The M. alpina DGAT2 ORF was cloned into plasmid pZUF17 as follows. First, the ORF was PCR-amplified using primers MDGAT-F and MDGAT-R1 (SEQ ID NOs:297 and 298) from the M. alpina cDNA (supra, Example 13). The expected 1015 bp fragment was isolated, purified, digested with Nco I and Not I and cloned into Nco I-Not I cut pZUF17 vector (SEQ ID NO:118; FIG. 8B), such that the gene was under the control of the Y. lipolytica FBAIN promoter and the PEX20-3′ terminator region. Correct transformants were confirmed by restriction analysis of miniprep DNA and the resultant plasmid was designated as “pMDGAT2-17” (SEQ ID NO:142).
Y. lipolytica strain Y2067U (from Example 10) was transformed with pMDGAT2-17 and pZUF-MOD-1 (supra, Example 14), respectively, according to the General Methods. Transformants were grown for 2 days in synthetic MM supplemented with amino acids, followed by 4 days in HGM. The fatty acid profile of two transformants containing pMDGAT2-17 and two transformants containing pZUF-MOD-1 are shown below based on GC analysis (as described in the General Methods). Fatty acids are identified as 18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA and EPA; and the composition of each is presented as a % of the total fatty acids.







TABLE 36







Lipid Composition In Yarrowia strain Y2067U Engineered To Overexpress


M. alpina DGAT2









Total Fatty Acids















Strain
18:0
18:1
18:2
GLA
DGLA
ARA
ETA
EPA





Y2067U + pZUF-MOD-1 #1
1.31
6.92
12.03
23.11
5.72
1.05
3.80
13.20


Y2067U + pZUF-MOD-1 #2
1.39
6.83
12.15
21.99
5.83
1.07
3.82
13.47


Y2067U + pMDGAT2-17 #1
0.00
7.47
10.77
25.30
5.70
1.43
3.45
15.12


Y2067U + pMDGAT2-17 #2
1.45
7.79
9.96
25.16
6.06
1.25
3.99
15.37









Expression of the M. alpina DGAT2 from plasmid pMDGAT2-17 increased the % EPA from  ̃13.3% in the “control” strains to  ̃15.25% (“Y2067U+pMDGAT2-17”). An additional increase in EPA would be expected, if the native Yarrowia lipolytica DGAT2 (SEQ ID NOs:89-94) were knocked-out in e.g., strain Y2067U+pMDGAT2-17.
Example 17
Mortierella alpina GPAT Increases Percent PUFAs
The present Example describes increased DGLA biosynthesis and accumulation (and reduced quantities of 18:1) in Yarrowia lipolytica strain Y2107U1 (Example 11) that was transformed to co-express the M. alpina GPAT ORF (SEQ ID NO:97). It is contemplated that a Y. lipolytica host strain engineered to produce ARA via either the Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8 desaturase pathway could demonstrate increased ARA biosynthesis and accumulation, if the M. alpina GPAT was similarly co-expressed therein (e.g., in strains Y2034, Y2047 and/or Y2214).
Identification of a M. alpina GPAT using Degenerate PCR Primers
Based on sequences of GPAT from Aspergillus nidulans (GenBank Accession No. EAA62242) and Neurospora crassa (GenBank Accession No. XP—325840), the following primers were designed for degenerate PCR:










MGPAT-N1
CCNCAYGCNAAYCARTTYGT
(SEQ ID NO:299)



 


MGPAT-NR5
TTCCANGTNGCCATNTCRTC
(SEQ ID NO:300)







[Note:


The nucleic acid degeneracy code used for SEQ ID


NOs:299 and 300 was as follows: R = A/G;


Y = C/T; and N = A/C/T/G.]






PCR amplification was carried out in a Perkin Elmer GeneAmp 9600 PCR machine using TaKaRa ExTaq premix Taq polymerase (TaKaRa Bio Inc., Otsu, Shiga, Japan). Amplification was carried out as follows: 30 cycles of denaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec and elongation at 72° C. for 90 sec, followed by a final elongation cycle at 72° C. for 7 min.
A fragment of  ̃1.2 kB was obtained (SEQ ID NO:99). This fragment was purified with a Qiagen QiaQuick PCR purification kit, cloned into the TOPO® cloning vector pCR2.1-TOPO (Invitrogen), and sequenced. The resultant sequence, when translated, had homology to known GPATs, based on BLAST program analysis.
Based on the sequence of the 1212 bp cDNA fragment, the 5′ and 3′ end regions of the M. alpina GPAT were cloned by PCR amplification and genome walking techniques. This enabled assembly of a contig, corresponding to the −1050 bp to +2885 bp region of the M. alpina GPAT (SEQ ID NO:100). This contig included the entire coding region of GPAT and four introns (SEQ ID NOs:104, 105, 106 and 107).
Specifically, the M. alpina cDNA sample described in Example 13 (1 μl) was used as a template for amplification of the 3′-end of the GPAT. MGPAT-5N1 (SEQ ID NO:301) and CDSIII/3′ (SEQ ID NO:282) were used as primers. PCR amplification was carried out in a Perkin Elmer GeneAmp 9600 PCR machine using TaKaRa ExTaq premix Taq polymerase (TaKaRa Bio Inc., Otsu, Shiga, Japan). Amplification was carried out as follows: 30 cycles of denaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec and elongation at 72° C. for 120 sec, followed by a final elongation cycle at 72° C. for 7 min.
The PCR product was diluted 1:10, and 1 μl of diluted PCR product was used as template for the second round of amplification, using MGPAT-5N2 (SEQ ID NO:302) and CDSIII/3′ as primers. The conditions were exactly the same as described above. The second round PCR product was again diluted 1:10 and 1 μl of the diluted PCR product used as template for a third round of PCR, using MGPAT-5N3 (SEQ ID NO:303) and CDSIII/3′ as primers. The PCR conditions were again the same.
A  ̃1 kB fragment was generated in the third round of PCR. This fragment was purified with a Qiagen PCR purification kit and cloned into pCR2.1-TOPO vector for sequence analysis. Results from sequence analysis showed that this 965 bp fragment (SEQ ID NO:101) corresponded with the 3′-end of the GPAT gene.
A Clontech Universal GenomeWalkerTM kit was used to obtain a piece of genomic DNA corresponding to the 5′-end region of the M. alpina GPAT. Briefly, 2.5 μg each of M. alpina genomic DNA was digested with DraI, EcoRV, PvuII or StuI individually, the digested DNA samples were purified using Qiagen Qiaquick PCR purification kits and eluted with 30 μl each of kit buffer EB, and the purified samples were then ligated with Genome Walker adaptor (SEQ ID NOs:304 [top strand] and 305 [bottom strand]), as shown below:








5′-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTG



GT-3′


 


3′-H2N-CCCGACCA-5′






Each ligation reaction mixture contained 1.9 μl of 25 μM Genome Walker adaptor, 1.6 μl 10× ligation buffer, 0.5 μl T4 DNA ligase and 4 μl of one of the purified digested genomic DNA samples. The reaction mixtures were incubated at 16° C. overnight. The reaction was terminated by incubation at 70° C. for 5 min. Then, 72 μl of 10 mM Tris HCl, 1 mM EDTA, pH 7.4 buffer was added to each ligation reaction mix.

Four separate PCR reactions were performed, each using one of the four ligation mixtures as template. The PCR reaction mixtures contained 1 μl of ligation mixture, 0.5 μl of 20 μM MGPAT-5-1A (SEQ ID NO:306), 1 μl of 10 μM kit primer AP1 (SEQ ID NO:307), 22.5 μl water, and 25 μl ExTaq premix Taq 2×PCR solution (TaKaRa). The PCR reactions were carried out for 32 cycles using the following conditions: denaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec, and elongation at 72° C. for 180 sec. A final elongation cycle at 72° C. for 7 min was carried out, followed by reaction termination at 4° C.
The products of each PCR reaction were diluted 1:50 individually and used as templates for a second round of PCR. Each reaction mixture contained 1 μl of one of the diluted PCR product as template, 0.5 μl of 20 μM MGPAT-3N1 (SEQ ID NO:308), 21 μl of 10 μM kit primer AP2 (SEQ ID NO:309), 22.5 μl water and 25 μl of ExTaq premix Taq 2×PCR solution (TaKaRa). PCR reactions were carried out for 32 cycles using the same thermocycler conditions described above.
A DNA fragment was obtained from the second round of PCR. This fragment was purified and cloned into pCR2.1-TOPO and sequenced. Sequence analysis showed that the 1908 bp fragment (SEQ ID NO:102) was the 5′-end of the M. alpina GPAT gene.
Similarly, a 966 bp fragment (SEQ ID NO:103) was obtained by two rounds of genome walking as described above, except using primer MGPAT-5N1 as the gene specific primer for the first round of PCR and primer MGPAT-5N2 as the gene specific primer for the second round. This fragment was also purified, cloned into pCR2.1-TOPO and sequenced. Sequence analysis showed that it contained a portion of the GPAT gene; however, the fragment was not long enough to extend to either end of the gene. Comparison with the 3′ cDNA sequence (SEQ ID NO:101) showed that the last 171 bp of the ORF was not included.
Assembly of the Full-Length GPAT Sequence from Mortierella alpina 
A 3935 bp sequence (SEQ ID NO:100) containing the complete GPAT gene (comprising a region extending 1050 bases upstream of the GPAT translation initiation ‘ATG’ codon and extending 22 bases beyond the GPAT termination codon) was assembled from the sequences of the original partial cDNA fragment (SEQ ID NO:99), the 3′ cDNA fragment (SEQ ID NO:101), the internal genomic fragment (SEQ ID NO:103), and the 5′ genomic fragment (SEQ ID NO:102) described above (graphically illustrated in FIG. 16). Included in this region is the 2151 bp GPAT ORF. The complete nucleotide sequence of the M. alpina GPAT ORF from ‘ATG’ to the stop codon ‘TAG’ is provided as SEQ ID NO:97 (corresponding to bases 1050 to 2863 of SEQ ID NO:100, excluding the four introns (i.e., intron 1 [SEQ ID NO:104], corresponding to bases 1195 to 1469 of SEQ ID NO:100; intron 2 [SEQ ID NO:105], corresponding to bases 1585 to 1839 of SEQ ID NO:100; intron 3 [SEQ ID NO:106], corresponding to bases 2795 to 2877 of SEQ ID NO:100 and intron 4 [SEQ ID NO:107], corresponding to bases 2940 to 3038 of SEQ ID NO:100). The translated amino acid sequence (SEQ ID NO:98) showed homology with a number of fungal, plant and animal GPATs.
More specifically, identity of the sequence was determined by conducting BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993)) searches. The amino acid fragment described herein as SEQ ID NO:98 had 47% identity and 65% similarity with the protein sequence of the putative GPAT of Ustilago maydis (GenBank Accession No. EAK84237), with an expectation value of 1 e-152; additionally, SEQ ID NO:98 had 47% identity and 62% similarity with the GPAT of Aspergillus fumigatus (GenBank Accession No. EAL20089), with an expectation value of 1e-142.
Construction of Plasmid pMGPAT-17, Comprising a FBAIN::MGPAT::PEX20-3′Chimeric Gene
The M. alpina GPAT ORF was cloned as follows. Primers mgpat-cdna-5 and mgpat-cdna-R (SEQ ID NOs:310 and 311) were used to amplify the GPAT ORF from the cDNA of M. alpina by PCR. The reaction mixture contained 1 μl of the cDNA, 1 μl each of the primers, 22 μl water and 25 μl ExTaq premix 2×Taq PCR solution (TaKaRa Bio Inc., Otsu, Shiga, 520-2193, Japan). Amplification was carried out as follows: initial denaturation at 94° C. for 150 sec, followed by 30 cycles of denaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec and elongation at 72° C. for 120 sec. A final elongation cycle at 72° C. for 10 min was carried out, followed by reaction termination at 4° C. An  ̃2.2 kB DNA fragment was obtained from the PCR reaction. It was purified using a Qiagen PCR purification kit according to the manufacturer's protocol.
The purified PCR product was digested with BamHI and EcoRI, and a  ̃470 bp fragment was isolated by gel agarose electrophoresis and purified using a Qiagen gel purification kit. Separately, the PCR product was also cut with EcoRI and NotI, and a 1.69 kB fragment isolated and purified as above. The two fragments were ligated into BamHI and NotI cut pZUF-MOD-1 vector (SEQ ID NO:140; FIG. 15B), such that the gene was under the control of the Y. lipolytica FBAIN promoter and the PEX20-3′ terminator region in the auto-replicating vector for expression in Y. lipolytica. Correct transformants were confirmed by restriction analysis of miniprep DNA and the resultant plasmid was designated as “pMGPAT-17” (SEQ ID NO:143; FIG. 15D).
Analysis of Lipid Composition in Transformant Y. lipolytica Over-Expressing M. alpina GPAT
Y. lipolytica strain Y2107U1 (from Example 11) was transformed with plasmid pMGPAT-17 and plasmid pZUF-MOD-1 (supra, Example 14), respectively, according to the General Methods. Transformants were grown for 2 days in synthetic MM supplemented with amino acids, followed by 4 days in HGM. The fatty acid profile of two transformants containing pZUF-MOD-1 and four transformants containing pMGPAT-17, are shown below in the Table, based on GC analysis (as described in the General Methods). Fatty acids are identified as 18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA and EPA; and the composition of each is presented as a % of the total fatty acids.







TABLE 37







Lipid Composition In Yarrowia Strain Y2107U1 Engineered To Over-


Express M. alpina GPAT









Total Fatty Acids















Strain
18:0
18:1
18:2
GLA
DGLA
ARA
ETA
EPA


















Y2107U1 + pZUF-MOD-1 #1
2.8
22.7
9.8
28.5
2.7
1.7
0.4
17.4


Y2107U1 + pZUF-MOD-1 #2
2.5
23.4
10.3
28.7
2.5
1.5
0.3
16.8


Y2107U1 + pMGPAT-17 #1
3.2
14.8
11.7
29.8
5.6
2.0
0.3
18.4


Y2107U1 + pMGPAT-17 #2
2.9
16.3
11.7
28.3
6.1
1.8
0.4
16.9


Y2107U1 + pMGPAT-17 #3
2.1
14.3
10.8
27.5
7.2
1.4
0.4
17.4


Y2107U1 + pMGPAT-17 #4
2.7
15.7
11.5
29.1
6.3
1.7
0.4
17.3









As demonstrated above, expression of the M. alpina GPAT from pMGPAT-17 increased the % DGLA from  ̃2.5% in the “control” strains to 6.5%. The level of 18:1 decreased from  ̃23% to  ̃16%. An additional increase in DGLA (or any other downstream PUFAs) would be expected, if the native Yarrowia lipolytica GPAT was knocked-out in a transformant strain expressing pMGPAT-17.
Example 18
Mortierella alpina Fatty Acid Elongase “ELO3” Increases Percent PUFAs Content
The present Example describes 35% more C18 fatty acids (18:0, 18:1, 18:2 and GLA) and 31% less C16 fatty acids in Yarrowia lipolytica strain Y2031 (Example 5) that was transformed to co-express the M. alpina C16/18 fatty acid elongase (“ELO3”; SEQ ID NOs:53 and 54), relative to control strains. It is contemplated that ELO3 (which could optionally be codon-optimized for increased expression), could push carbon flux into either the engineered Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8 desaturase pathway as a means to increase production of the desired PUFA, i.e., ARA. For example, a chimeric gene comprising this C16/18 fatty acid elongase could readily be introduced into e.g., strains Y2034, Y2047 or Y2214.
Sequence Identification of a M. alpina C16/18 Fatty Acid Elongase
A cDNA fragment (SEQ ID NO:55) encoding a portion of a M. alpina fatty acid elongase was identified from among 9,984 M. alpina cDNA sequences (Example 13). This cDNA fragment bore significant homology to a number of fatty acid elongases and thus was tentatively identified as an elongase.
The results of the BLAST comparison summarizing the sequence to which SEQ ID NO:55 had the most similarity are reported according to the % identity, % similarity, and Expectation value. Specifically, the translated amino acid sequence of SEQ ID NO:55 had 32% identity and 46% similarity with the protein sequence of a potential fatty acid elongase from Candida albicans SC5314 (GenBank Accession No. EAL04510.1, annotated therein as one of three potential fatty acid elongase genes similar to S. cerevisiae EUR4, FEN1 and ELO1), with an expectation value of 4e-13. Additionally, SEQ ID NO:55 had 35% identity and 53% similarity with ELO1 from Saccharomyces cerevisiae (GenBank Accession No. NC—001142, bases 67849-68781 of chromosome X). The S. cerevisiae ELO1 is described as a medium-chain acyl elongase, that catalyzes carboxy-terminal elongation of unsaturated C12-C16 fatty acyl-CoAs to C16-C18 fatty acids.
On the basis of the homologies reported above, the Yarrowia lipolytica gene product of SEQ ID NO:55 was designated herein as “elongase 3” or “ELO3”.
Analysis of the partial fatty acid elongase cDNA sequence (SEQ ID NO:55) indicated that the 5′ and 3′-ends were both incomplete. To obtain the missing 3′ region of the M. alpina ELO3, a Clontech Universal GenomeWalkerTM kit was used (as described in Example 17). Specifically, the same set of four ligation mixtures were used for a first round of PCR, using the same components and conditions as described previously, with the exception that MA Elong 3′1 (SEQ ID NO:312) and AP1 were used as primers (i.e., instead of primers MGPAT-5-1A and AP1). The second round of PCR used MA Elong 3′2 (SEQ ID NO:313) and AP2 as primers. A 1042 bp DNA fragment was obtained from the second round of PCR (SEQ ID NO:56). This fragment was purified and cloned into pCR2.1-TOPO and sequenced. Sequence analysis showed that the fragment contained the 3′-end of ELO3, including  ̃640 bp downstream of the ‘TAA’ stop codon of the gene.
The same set of four ligation mixtures used in the Clontech 3′-end RACE (supra) were also used to obtain the 5′-end region of the M. alpina ELO3. Specifically, a first round of PCR using the same components and conditions as described above was conducted, with the exception that MA Elong 5′1 (SEQ ID NO:314, nested at the 5′ end) and AP1 were used as primers (i.e., instead of primers MA Elong 3′1 and AP1). The second round of PCR used MA Elong 5′2 (SEQ ID NO:315, nested at the 5′ end) and AP2 as primers. A 2223 bp DNA fragment (SEQ ID NO:57) was obtained. It was purified and cloned into pCR2.1-TOPO and sequenced. Analysis of the sequence showed that it contained the 5′-region of the ELO3 gene.
Thus, the entire cDNA sequence of the M. alpina ELO3 (SEQ ID NO:53) was obtained by combining the original partial cDNA sequence (SEQ ID NO:55) with the overlapping 5′ and 3′ sequences obtained by genome walking (SEQ ID NOs:57 and 56, respectively; graphically illustrated in FIG. 17). This yielded a sequence of 3557 bp, identified herein as SEQ ID NO:58, comprising: 2091 bp upstream of the putative ‘ATG’ translation initiation codon of ELO3; the 828 bp ELO3 ORF (i.e., SEQ ID NO:53, corresponding to bases 2092-2919 of SEQ ID NO:58); and, 638 bp downstream of the ELO3 stop codon (corresponding to bases 2920-3557 of SEQ ID NO:58).
The corresponding genomic sequence of the M. alpina ELO3 is longer than the cDNA fragment provided as SEQ ID NO:58. Specifically, a 542 bp intron (SEQ ID NO:59) was found in the genomic DNA containing the ELO3 gene at 318 bp of the ORF; thus, the genomic DNA fragment, provided herein as SEQ ID NO:60, is 4,099 bp (FIG. 17).
The translated ELO3 protein sequence (SEQ ID NO:54) had the following homology, based on BLAST program analysis: 37% identity and 51% similarity to the potential fatty acid elongase from Candida albicans SC5314 (GenBank Accession No. EAL04510.1), with an expectation value of 4e43. Additionally, the translated ELO3 shared 33% identity and 44% similarity with the protein sequence of XP—331368 (annotated therein as a “hypothetical protein”) from Neurospora crassa, with an expectation value of 3e-44.
Construction of Plasmid pZUF6S-E3WT, Comprising A FBAIN::ELO3::PEX16-3′Chimeric Gene
The M. alpina fatty acid ELO3 ORF was cloned as follows. Primers MA Elong 5′ Ncol 3 and MA elong 3′ Notl (SEQ ID Nos ;316 and 317) were used to amplify the ELO3 ORF from the cDNA of M. alpina (Example 13) by PCR. The reaction mixture contained 1 μl of the cDNA, 1 μl each of the primers, 22 μl water and 25 μl ExTaq premix 2×Taq PCR solution (TaKaRa). Amplification was carried out as follows: initial denaturation at 94° C. for 30 sec, followed by 32 cycles of denaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec and elongation at 72° C. for 120 sec. A final elongation cycle at 72° C. for 7 min was carried out, followed by reaction termination at 4° C. An  ̃830 bp DNA fragment was obtained from the PCR reaction. It was purified using a Qiagen (Valencia, Calif.) PCR purification kit according to the manufacturer's protocol. The purified PCR product was divided into two aliquots, wherein one was digested with NcoI and NspI, while the other with NspI and NotI. The  ̃270 bp NcoI-NspI and  ̃560 bp NspI-NotI fragments were cloned into Nco I-Not I cut pZF5T-PPC vector (FIG. 11C; SEQ ID NO:122) by three-piece ligation, such that the gene was under the control of the Y. lipolytica FBAIN promoter and the PEX16-3′ terminator region (GenBank Accession No. U75433) in the auto-replicating vector for expression in Y. lipolytica. Correct transformants were confirmed by miniprep analysis and the resultant plasmid was designated as “pZF5T-PPC-E3” (SEQ ID NO:144).
Plasmid pZF5T-PPC-E3 was digested with ClaI and PacI and the  ̃2.2 kB band (i.e., the FBAIN::ELO 3::PEX16-3′ fragment) was purified from an agarose gel using a Qiagen gel extraction kit. The fragment was cloned into ClaI-PacI cut pZUF6S (FIG. 18A; SEQ ID NO:145), an auto-replication plasmid containing the Mortierella alpina Δ6 desaturase ORF (“D6S”; GenBank Accession No. AF465281) under the control of the FBAIN promoter with a Pex20-3′ terminator (i.e., a FBAIN::D6S::Pex20 chimeric gene) and a Ura3 gene. Correct transformants were confirmed by miniprep analysis and the resultant plasmid was designated as “pZUF6S-E3WT” (FIG. 18B; SEQ ID NO:146).
Analysis of Lipid Composition in Transformant Y. lipolytica Over-Expressing the M. alpina ELO3
Y. lipolytica strain Y2031 (Example 5) was transformed with plasmid pZUF6S (control, comprising a FBAIN::D6S::Pex20 chimeric gene) and plasmid pZUF6S-E3WT (comprising a FBAIN::D6S::Pex20 chimeric gene and the FBAIN::ELO 3::PEX16 chimeric gene) according to the General Methods. Transformants were grown for 2 days in synthetic MM supplemented with amino acids, followed by 4 days in HGM. The fatty acid profile of six clones containing pZUF6S (clones #1-6, from a single transformation) and 22 clones (from four different transformations [i.e., #3, 5, 6, and 7]) containing pZUF6S-E3WT are shown below in Table 38, based on GC analysis (as described in the General Methods). Fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid), 18:0, 18:1 (oleic acid), 18:2 (LA) and GLA; and the composition of each is presented as a % of the total fatty acids.







TABLE 38







Lipid Composition In Yarrowia Strain Y2031 Engineered To


Over-Express M. alpina ELO3








Y. lipolytica Strain



Y2031 Transformant
Fatty Acid Composition (% Of Total Fatty Acids)













And/Or Clone No.
16:0
16:1
18:0
18:1
18:2
GLA
















pZUF6S #1 (control)
9.0
23.2
1.2
38.2
19.8
6.9


pZUF6S #2 (control)
10.1
23.4
1.4
39.0
17.5
7.1


pZUF6S #3 (control)
9.7
22.7
1.4
39.0
20.2
7.0


pZUF6S #4 (control)
8.5
24.1
0.0
40.8
19.8
6.9


pZUF6S #5 (control)
9.8
22.4
1.7
39.1
20.2
6.8


pZUF6S #6 (control)
9.1
22.7
1.9
39.9
19.7
6.6


pZUF6S-E3WT #3-1
8.9
17.3
4.1
36.5
21.6
11.6


pZUF6S-E3WT #3-2
8.8
17.8
3.7
36.9
21.3
11.5


 
 
 
 
 
 
 


pZUF6S-E3WT #3-6
8.5
19.9
4.4
37.8
17.1
12.3


pZUF6S-E3WT #5-1
8.6
17.6
4.0
37.6
21.1
11.1


pZUF6S-E3WT #5-2
8.8
17.1
3.9
37.6
21.3
11.2


pZUF6S-E3WT #5-3
9.1
17.1
3.5
37.6
21.5
11.1


pZUF6S-E3WT #5-4
8.8
17.9
4.3
38.0
19.3
11.7


pZUF6S-E3WT #5-5
9.2
16.1
4.4
37.0
21.6
11.7


 
 
 
 
 
 
 


pZUF6S-E3WT #6-1
9.4
16.9
4.6
36.6
21.5
11.0


pZUF6S-E3WT #6-2
9.8
16.2
4.1
36.5
21.9
11.6


pZUF6S-E3WT #6-3
9.4
17.0
4.4
36.2
21.8
11.3


pZUF6S-E3WT #6-4
8.3
16.6
4.2
36.9
21.9
12.2


pZUF6S-E3WT #6-5
8.8
18.5
5.5
36.0
17.8
13.4


pZUF6S-E3WT #6-6
8.7
19.5
5.2
35.4
18.1
13.2


 
 
 
 
 
 
 


pZUF6S-E3WT #7-2
8.0
17.7
4.0
37.7
20.9
11.7


 
 
 
 
 
 
 


 
 
 
 
 
 
 


pZUF6S-E3WT #7-5
8.3
17.0
4.7
36.7
21.2
12.1


pZUF6S-E3WT #7-6
8.0
18.0
4.8
36.3
20.8
12.1









Some of the samples (labeled in bold and italics) deviated from expected readings. Specifically, neither Y2031+pZUF6S-E3WT #3-3 nor Y2031+pZUF6S-E3WT #5-6 produced GLA. Similarly, Y2031+pZUF6S-E3WT#7-1, #7-3 and #7-4 had GC errors, wherein the 16:0 and 16:1 peaks were read by the GC as a single peak. As a result of these variant results, Table 39 reports the average lipid in the control and transformant strains expressing ELO3. Specifically, Table 39 shows the averages from the fatty acid profiles in Table 38, although the lines indicated by bold and italics as being incorrect in Table 38 were not included when calculating these averages. “Total C16” represents the sum of the average areas of 16:0 and 16:1, while “Total C18” reflects the sum of the average areas of 18:0, 18:1, 18:2 and GLA.







TABLE 39







Average Lipid Composition In Yarrowia Strain Y2031


Engineered To Over-Express M. alpina ELO3










Y. lipolytica
Average Fatty Acid Composition




Strain Y2031
(% Of Total Fatty Acids)
Total
Total















Transformant
16:0
16:1
18:0
18:1
18:2
GLA
C16
C18


















pZUF6S
9.4
23.1
1.3
39.3
19.5
6.9
32.4
67.1


(control)


pZUF6S-
8.7
18.3
4.1
37.1
20.0
11.8
27.0
73.0


E3WT #3


pZUF6S-
8.9
17.2
4.0
37.6
21.0
11.4
26.1
73.9


E3WT #5


pZUF6S-
9.1
17.5
4.6
36.3
20.5
12.1
26.5
73.5


E3WT #6


pZUF6S-
8.1
17.6
4.5
36.9
21.0
12.0
25.6
74.4


E3WT #7









Based on the data reported above, overexpression of the M. alpina ELO3 resulted in an increased percentage of C18 and a reduced percentage of C16 when co-expressed with a M. alpina Δ6 desaturase in Yarrowia lipolytica strain Y2031, relative to a control strain of Y2031 overexpressing the M. alpina Δ6 desaturase only. This indicated that the M. alpina ELO3 was indeed a C16/18 fatty acid elongase.
Example 19
Yarrowia C16/18 Fatty Acid Elongase “YE2” Increases Percent PUFAs
The present Example describes increased GLA biosynthesis and accumulation in Yarrowia lipolytica strain Y2031 (Example 5) that was transformed to co-express the Y. lipolytica C16/18 fatty acid elongase (“YE2”; SEQ ID NO:62). It is contemplated that the YE2 elongase could push carbon flux into either the engineered Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8 desaturase pathway as a means to increase production of the desired PUFA, i.e., ARA. For example, a chimeric gene comprising this C16/18 fatty acid elongase could readily be introduced into e.g., strains Y2034, Y2047 or Y2214.
Sequence Identification of a Yarrowia lipolytica C16/18 Fatty Acid Elongase
A novel fatty acid elongase candidate from Y. lipolytica was identified by sequence comparison using the rat Elo2 C16/18 fatty acid elongase protein sequence (GenBank Accession No. AB071986; SEQ ID NO:51) as a query sequence. Specifically, this rElo2 query sequence was used to search GenBank and the public Y. lipolytica protein database of the “Yeast project Genolevures” (Center for Bioinformatics, LaBR1, Talence Cedex, France) (see also Dujon, B. et al., Nature 430 (6995):35-44 (2004)). This resulted in the identification of a homologous sequence, GenBank Accession No. CAG77901 (SEQ ID NOs:61 and 62), annotated as an “unnamed protein product”). This gene was designated as YE2.
Comparison of the Yarrowia YE2 amino acid sequences to public databases, using a BLAST algorithm (Altschul, S. F., et al., Nucleic Acids Res. 25:3389-3402 (1997)), revealed that the most similar known amino acid sequence was that from Candida albicans SC5314 (SEQ ID NO:63, GenBank Accession No. EAL04510), annotated as a probable fatty acid elongase. The proteins shared about 40% identity and scored at 236 with an E value of 7e-61.
Isolation of Yarrowia YE2 Gene
The coding region of the YE2 gene was amplified by PCR using Yarrowia genomic DNA as template and oligonucleotides YL597 and YL598 (SEQ ID NOs:318 and 319) as primers. The PCR reaction was carried out in a 50 μl total volume, as described in the General Methods. The thermocycler conditions were set for 35 cycles at 95° C. for 1 min, 56° C. for 30 sec, 72° C. for 1 min, followed by a final extension at 72° C. for 10 min. The PCR products of the YE2 coding region were purified, digested with NcoI/NotI, and then ligated with NcoI/NotI digested pZKUGPYE1-N (infra, Example 20; see also FIG. 18C, SEQ ID NO:147) to generate pZKUGPYE2 (FIG. 18D, SEQ ID NO:148). The addition of a NcoI site around the ‘ATG’ translation initiation codon changed the second amino acid of YE2 from L to V.
The ClaI/NotI fragment of pZKUGPYE2 (containing the GPAT promoter and YE2 coding region) and a NotI/PacI fragment containing the Aco terminator (prepared by PCR amplifying the ACO 3′ terminator with primers YL325 and YL326 [SEQ ID NOs:371 and 372] and then digesting with NotI/PacI), were directionally ligated with ClaI/PacI digested vector pZUF6S to produce pZUF6YE2. The ClaI/NcoI fragment of pZKUT16 (containing the TEF promoter) and the NcoI/PacI fragment of pZUF6YE2 (containing the coding region of YE2 and the Aco terminator) were subsequently directionally ligated with ClaI/PacI digested vector pZUF6S to produce pZUF6TYE2 (SEQ ID NO:149).
Analysis of Lipid Composition in Transformant Y. lipolytica Over-Expressing YE2
Plasmid pZUF6S (FIG. 18A, SEQ ID NO:145) and pZUF6TYE2 (SEQ ID NO:149) were used to separately transform Yarrowia strain Y2031. The components of these two plasmids are described in Tables 40 and 41.







TABLE 40







Description of Plasmid pZUF6S (SEQ ID NO: 145)








RE Sites And
Description Of


Nucleotides Within
Fragment And Chimeric Gene


SEQ ID NO: 145
Components





EcoRI/ClaI
Yarrowia autonomous replicating sequence


(3114-4510)
18 (ARS18; GenBank Accession No. M91600)


SalI/PacI
Yarrowia Ura3 gene (GenBank Accession No.


(6022-4530)
AJ306421)


EcoRI/BsiWI
FBAIN::Δ6S::Pex20, comprising:


(6063-318)
FBAIN: FBAIN promoter (SEQ ID NO: 162)



Δ6S: codon-optimized Δ6 desaturase gene (SEQ ID



NO: 3), derived from Mortierella alpina (GenBank



Accession No. AF465281)



Pex20: Pex20 terminator sequence from Yarrowia



Pex20 gene (GenBank Accession No. AF054613)
















TABLE 41







Description of Plasmid pZUF6TYE2 (SEQ ID NO: 149)








RE Sites And



Nucleotides Within
Description Of Fragment And Chimeric Gene


SEQ ID NO: 149
Components





EcoRI/ClaI
Yarrowia autonomous replicating sequence 18


(7461-8857)
(ARS18; GenBank Accession No. M91600)


SalI/PacI
Yarrowia Ura3 gene (GenBank Accession No.


(1907-415)
AJ306421)


EcoRI/BsiWI
FBAIN::Δ6S::Pex20: as described for pZUF6


(1948-4665)
(supra)


ClaI/PacI
TEF::YE2::Aco, comprising:


(8857-415)
TEF: TEF promoter (GenBank Accession No.



AF054508)



YE2: coding region of Yarrowia YE2 gene (SEQ ID



NO: 61; GenBank Accession No. CAG77901)



Aco: Aco3 terminator sequence of Yarrowia Aco3



gene (GenBank Accession No. AJ001301)









Y. lipolytica strain Y2031 (Example 5) was transformed with plasmid pZUF6S (control) and plasmid pZUF6TYE2 according to the General Methods. Transformants were grown for 2 days in liquid MM. The fatty acid profile of eight colonies each containing pZUF6S or pZUF6YE2 are shown below in Table 42, based on GC analysis (as described in the General Methods). Fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid), 18:0, 18:1 (oleic acid), 18:2 (LA) and GLA; and the composition of each is presented as a % of the total fatty acids.







TABLE 42







Comparison Of Fatty Acid Composition In Yarrowia Strain


Y2031 Transformed With pZUF6S And pZUF6TYE2








Y. lipolytica
Fatty Acid Composition


Strain Y2031
(% Of Total Fatty Acids)













Transformants
16:0
16:1
18:0
18:1
18:2
GLA
















pZUF6S #1 (control)
15.4
13.8
2.5
34.1
16.8
8.3


pZUF6S #2 (control)
15.2
12.8
3.0
36.5
16.4
8.3


pZUF6S #3 (control)
15.1
12.2
3.2
36.5
17.1
8.5


pZUF6S #4 (control)
15.2
12.8
3.1
36.3
16.6
8.4


pZUF6S #5 (control)
14.9
10.9
3.6
37.4
18.0
8.7


pZUF6S #6 (control)
14.8
10.1
4.2
37.6
18.7
8.6


pZUF6S #7 (control)
14.7
11.9
3.0
36.0
17.8
9.1


pZUF6S #8 (control)
14.9
12.6
2.9
35.9
17.3
8.8


Average
15.0
12.1
3.2
36.3
17.3
8.6


pZUF6TYE2 #1
13.1
8.4
4.4
42.4
16.8
9.7


pZUF6TYE2 #2
13.1
7.6
5.3
40.8
18.6
9.8


pZUF6TYE2 #3
13.5
8.1
4.6
39.2
19.0
10.6


pZUF6TYE2 #4
13.4
7.4
5.7
39.9
18.7
9.8


pZUF6TYE2 #5
13.4
8.4
5.5
45.2
14.3
7.6


pZUF6TYE2 #6
13.4
7.4
5.5
39.3
19.2
10.5


pZUF6TYE2 #7
13.4
8.6
4.4
40.6
17.9
9.9


pZUF6TYE2 #8
13.2
7.5
5.4
41.2
18.0
9.7


Average
13.3
8.0
5.0
41.1
17.8
9.7









GC analyses showed that there were about 27.1% C16 (C16:0 and C16:1) and 62.2% C18 (C18:0, C18:1, C18:2 and GLA) of total lipids produced in the Y2031 transformants with pZUF6S; there were about 21.3% C16 and 73.6% C18 produced in the Y2031 transformants with pZUF6TYE2. Thus, the total amount of C16 was reduced about 21.4%, and the total amount of C18 was increased about 18% in the pZUF6TYE2 transformants (as compared with the transformants with pZUF6S). These data demonstrated that YE2 functions as a C16/18 fatty acid elongase to produce C18 fatty acids in Yarrowia. Additionally, there was about 12.8% more GLA produced in the pZUF6TYE2 transformants relative to the GLA produced in pZUF6S transformants. These data suggested that the YE2 elongase could push carbon flux into the engineered PUFA pathway to produce more final product (i.e., GLA).
Example 20
Yarrowia C14/16 Fatty Acid Elongase “YE1” Increases Percent PUFAs
The present Example describes increased GLA biosynthesis and accumulation in Y. lipolytica strain Y2031 (Example 5) that was transformed to co-express the Y. lipolytica C14/16 fatty acid elongase (“YE1”; SEQ ID NO:65). It is contemplated that the YE1 elongase could push carbon flux into either the engineered Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8 desaturase pathway as a means to increase production of the desired PUFA, i.e., ARA. Specifically, a chimeric gene comprising this C14/16 fatty acid elongase could readily be introduced into strains Y2034, Y2047 or Y2214.
Sequence Identification of a Yarrowia lipolytica C14/16 Fatty Acid Elongase
A novel fatty acid elongase candidate from Yarrowia lipolytica was identified by sequence comparison using the rat Elo2 C16/18 fatty acid elongase protein sequence (GenBank Accession No. AB071986; SEQ ID NO:51) as a query sequence, in a manner similar to that used in Example 19. This resulted in the identification of a homologous sequence, GenBank Accession No. CAG83378 (SEQ ID NOs:64 and 65), annotated as an “unnamed protein product”. This gene was designated as “YE1”.
Comparison of the Yarrowia YE1 amino acid sequences to public databases, using a BLAST algorithm (Altschul, S. F., et al., Nucleic Acids Res. 25:3389-3402 (1997)), revealed that the most similar known sequence was FEN1 from Neurospora crassa (GenBank Accession No. CAD70918; SEQ ID NO:66), a probable fatty acid elongase sharing about 60% identity to YE1.
Isolation of Yarrowia YE1 Gene
The DNA sequence of YE1 gene (SEQ ID NO:64) possesses an internal NcoI site. In order to incorporate the Yarrowia translation motif around the ‘ATG’ translation initiation codon of the YE1 gene, a two-step strategy was employed to PCR the entire YE1 gene from Yarrowia. Specifically, using Yarrowia genomic DNA as template, the first half of YE1 was amplified by PCR using oligonucleotides YL567 and YL568 (SEQ ID NOs:320 and 321) as primers, while the second half of the YE1 gene was amplified similarly using oligonucleotides YL569 and YL570 (SEQ ID NOs:322 and 323) as primers. The PCR reactions were carried out in a 50 μl total volume, as described in the General Methods. The thermocycler conditions were set for 35 cycles at 95° C. for 1 min, 56° C. for 30 sec, 72° C. for 1 min, followed by a final extension at 72° C. for 10 min. The PCR products corresponding to the 5′ portion of YE1 were purified and then digested with NcoI and SacI to yield the YE1-1 fragment, while the PCR products of the 3′ portion of YE1 were purified and digested with SacI and NotI to yield the YE1-2 fragment. The YE1-1 and YE1-2 fragments were directly ligated with NcoI/NotI digested pZKUGPE1S (supra, Example 11) to generate pZKUGPYE1 (FIG. 19A, SEQ ID NO:150). The internal NcoI site of YE1 was then mutated by site-directed mutagenesis using pZKUGPYE1 as template and oligonucleotides YL571 and YL572 (SEQ ID NOs:324 and 325) as primers to generate pZKUGPYE1-N (SEQ ID NO:147). Sequence analysis showed that the mutation did not change the amino acid sequence of YE1. The addition of the NcoI site around the ATG translation initiation codon changed the second amino acid of YE1 from S to A.
The ClaI/NcoI fragment of pZF5T-PPC (containing the FBAIN promoter) and the NcoI/PacI fragment of pZKUGPYE1-N (containing the coding region of YE1 and the Aco terminator) were directionally ligated with ClaI/PacI-digested vector pZUF6S to produce pZUF6FYE1 (SEQ ID NO:151).
Analysis of Lipid Composition in Transformant Y. lipolytica Over-Expressing YE1
A chimeric gene comprising the Y. lipolytica YE1 ORF was cloned into plasmid pZUF6, such that the effect of the gene's overexpression could be determined by GC analysis of fatty acid composition in transformed Yarrowia strains. Specifically, the components of control plasmid pZUF6S (FIG. 18A; SEQ ID NO:145, comprising a FBAIN::D6S::Pex20 chimeric gene) are described in Example 19, while those components of pZUF6FYE1 (FIG. 19B; SEQ ID NO:151, comprising a FBAIN::D6S::Pex20 chimeric gene and the FBAIN::YE1::Aco chimeric gene) are described in Table 43 below.







TABLE 43







Description Of Plasmid pZUF6FYE1 (SEQ ID NO: 151)








RE Sites And



Nucleotides


Within SEQ
Description Of Fragment And Chimeric Gene


ID NO: 151
Components





EcoRI/ClaI
Yarrowia autonomous replicating sequence 18 (ARS18,


(7047-8445)
(GenBank Accession No. M91600)


SalI/PacI
Yarrowia Ura3 gene (GenBank Accession No.


(1493-1)
AJ306421)


EcoRI/BsiWI
FBAIN::Δ6S::Pex20: as described for pZUF6 (supra,


(1534-4251)
Example 19)


ClaI/PacI
FBAIN::YE1::Aco, comprising:


(8443-1)
FBAIN: FBAIN promoter (SEQ ID NO: 162)



YE1: Yarrowia YE1 gene (SEQ ID NO: 64; GenBank



Accession No. CAG83378)



Aco: Aco3 terminator sequence from Yarrowia Aco3



gene (Genbank Accession No. AJ001301)









Following transformation, transformants were grown for 2 days in synthetic MM supplemented with amino acids, followed by 4 days in HGM. The fatty acid profile of six clones containing pZUF6S and five clones containing pZUF6FYE1 are shown below in Table 44, based on GC analysis (as described in the General Methods). Fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid), 18:0, 18:1 (oleic acid), 18:2 (LA) and GLA; and the composition of each is presented as a % of the total fatty acids.







TABLE 44







Comparison Of Fatty Acid Composition In Yarrowia Strain


Y2031 Transformed With pZUF6S And pZUF6FYE1









Fatty Acid Composition



(% Of Total Fatty Acids)












Transformants
16:0
16:1
18:1
18:2
GLA















pZUF6S #1 (control)
12.9
18.2
29.6
23.5
10.7


pZUF6S #2 (control)
12.6
18.6
29.6
23.8
10.3


pZUF6S #3 (control)
13.0
17.8
29.8
23.9
10.6


pZUF6S #4 (control)
13.1
18.9
30.1
22.3
10.3


pZUF6S #5 (control)
13.0
17.8
29.6
23.4
10.9


pZUF6S #6 (control)
12.0
18.7
30.4
23.2
10.4


Average
12.8
18.3
29.9
23.4
10.5


pZUF6FYE1 #1
17.4
21.9
20.4
19.2
16.9


pZUF6FYE1 #2
16.7
22.8
21.1
19.1
16.1


pZUF6FYE1 #3
19.8
20.7
22.8
17.0
15.8


pZUF6FYE1 #4
16.8
22.4
23.7
16.1
16.8


pZUF6FYE1 #5
17.7
21.6
21.2
18.0
17.2


Average
17.7
21.9
21.9
17.9
16.5









GC analyses measured about 31.1% C16 (C16:0+C16:1) of total lipids produced in the Y2031 transformants with pZUF6S, while there was about 39.6% C16 produced in the Y2031 transformants with pZUF6FYE1. The total amount of C16 increased about 26.7% in the pZUF6FYE1 transformants, as compared to transformants with pZUF6S. Thus, these data demonstrated that YE1 functions as a C14/16 fatty acid elongase to produce C16 fatty acids in Yarrowia. Additionally, there was 57% more GLA produced in the pZUF6FYE1 transformants than in pZUF6S transformants, suggesting that the YE1 elongase could push carbon flux into the engineered pathway to produce more final product (i.e., GLA).
Example 21
Yarrowia lipolytica CPT1 Overexpression Increases Percent PUFAs
The present Example describes increased EPA biosynthesis and accumulation in Yarrowia lipolytica strain Y2067U (Example 10) that was transformed to overexpress the Y. lipolytica CPT1 cDNA (SEQ ID NO:109). PUFAs leading to the synthesis of EPA were also increased. It is contemplated that a Y. lipolytica host strain engineered to produce ARA via either the Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8 desaturase pathway could demonstrate increased ARA biosynthesis and accumulation, if the Y. lipolytica CPT1 was similarly co-expressed (e.g., in strains Y2034, Y2047 or Y2214).
Y. lipolytica strain ATCC #20326 cDNA was prepared using the following procedure. Cells were grown in 200 mL YPD medium (2% Bacto-yeast extract, 3% Bactor-peptone, 2% glucose) for 1 day at 30° C. and then pelleted by centrifugation at 3750 rpm in a Beckman GH3.8 rotor for 10 min and washed twice with HGM. Washed cells were resuspended in 200 mL of HGM and allowed to grow for an additional 4 hrs at 30° C. Cells were then harvested by centrifugation at 3750 rpm for 10 min in 4×50 mL tubes.
Total RNA was isolated using the Qiagen RNeasy total RNA Midi kit. To disrupt the cells, harvested cells were resuspended in 4×600 μl of kit buffer RLT (supplemented with β-mercaptoethanol, as specified by the manufacturer) and mixed with an equal volume of 0.5 mm glass beads in four 2 mL screwcap tubes. A Biospec Mini-beadbeater was used to break the cells for 2 min at the Homogenization setting. An additional 4×600 μl buffer RLT was added. Glass beads and cell debris were removed by centrifugation, and the supernatant was used to isolate total RNA according to manufacturer's protocol.
PolyA(+)RNA was isolated from the above total RNA sample using a Qiagen Oligotex mRNA purification kit according to the manufacturer's protocol. Isolated polyA(+) RNA was purified one additional round with the same kit to ensure the purity of mRNA sample. The final purified poly(A)+RNA had a concentration of 30.4 ng/μl.
cDNA was generated, using the LD-PCR method specified by BD-Clontech and 0.1 μg of polyA(+) RNA sample, as described in Example 13, with the exception that the PCR thermocycler conditions used for 1st strand cDNA synthesis were set for 95° C. for 20 sec, followed by 20 cycles of 95° C. for 5 sec and 68° C. for 6 min. The PCR product was quantitated by agarose gel electrophoresis and ethidium bromide staining.
The Y. lipolytica CPT1 cDNA was cloned as follows. Primers CPT1-5′-NcoI and CPT1-3′-NotI (SEQ ID NOs:326 and 327) were used to amplify the Y. lipolytica ORF from the cDNA of Y. lipolytica by PCR. The reaction mixture contained 0.5 μl of the cDNA, 0.5 μl each of the primers, 11 μl water and 12.5 μl ExTaq premix 2× Taq PCR solution (TaKaRa Bio Inc., Otsu, Shiga, 520-2193, Japan). Amplification was carried out as follows: initial denaturation at 94° C. for 300 sec, followed by 30 cycles of denaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec, and elongation at 72° C. for 60 sec. A final elongation cycle at 72° C. for 10 min was carried out, followed by reaction termination at 4° C. A  ̃1190 bp DNA fragment was obtained from the PCR reaction. It was purified using Qiagen's PCR purification kit according to the manufacturer's protocol. The purified PCR product was digested with NcoI and NotI, and cloned into Nco I-Not I cut pZUF17 vector (SEQ ID NO:118; FIG. 8B), such that the gene was under the control of the Y. lipolytica FBAIN promoter and the PEX20-3′ terminator region. Correct transformants were confirmed by miniprep analysis and the resultant plasmid was designated as “pYCPT1-17” (SEQ ID NO:152).
To integrate the chimeric FBAIN::CPT1::PEX20 gene into the genome of Yarrowia lipolytica, plasmid pYCPT1-ZP217 was created by digesting pYCPT1-17 with NcoI and NotI, and isolating the  ̃1190 bp fragment that contained the CPT1 ORF. This fragment was then cloned into pZP217+Ura (SEQ ID NO:153) digested with NcoI and NotI. As shown in FIG. 19C, plasmid pZP217+Ura is a Y. lipolytica integration plasmid comprising a chimeric TEF::synthetic Δ17 desaturase (codon-optimized for Y. lipolytica)::Pex20-3′ gene and a Ura3 gene, for use as a selectable marker. Correct transformants were confirmed by miniprep analysis and the resultant plasmid was designated as “pYCPT1-ZP217” (SEQ ID NO:154).
Y. lipolytica strain Y2067U (from Example 10) was transformed with BssHII/BbuI digested pYCPT1-ZP217 and pZUF-MOD-1 (supra, Example 14), respectively, according to the General Methods. Transformants were grown for 2 days in synthetic MM supplemented with amino acids, followed by 4 days in HGM. The fatty acid profile of two transformants containing pZUF-MOD-1 and four transformants having pYCPT1-ZP217 integrated into the genome are shown below in the Table, based on GC analysis (as described in the General Methods). Fatty acids are identified as 18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA and EPA; and the composition of each is presented as a % of the total fatty acids.







TABLE 45







Lipid Composition In Yarrowia Strain Y2067U


Engineered To Overexpress Y. lipolytica CPT1









Total Fatty Acids















Strain
18:0
18:1
18:2
GLA
DGLA
ARA
ETA
EPA


















Y2067U + pZUF-MOD-1 #1
1.3
6.9
12.0
23.1
5.7
1.1
3.8
13.2


Y2067U + pZUF-MOD-1 #2
1.4
6.8
12.1
22.0
5.8
1.1
3.8
13.5


Y2067U + pYCPT1-ZP2I7 #1
0.6
8.0
8.2
27.4
7.1
1.6
4.1
15.7


Y2067U + pYCPT1-ZP2I7 #2
0.6
8.1
8.2
27.2
7.0
1.6
4.0
15.7


Y2067U + pYCPT1-ZP2I7 #3
1.0
7.9
8.0
24.7
6.1
1.6
3.2
15.5


Y2067U + pYCPT1-ZP2I7 #4
0.6
7.1
8.6
25.5
6.9
1.8
4.0
16.0









As shown above, expression of the Y. lipolytica CPT1 under the control of the strong FBAIN promoter, by genome integration, increased the % EPA from 13.4% in the “control” strains to 15.7-16%. Furthermore, GLA, DGLA and ARA levels also were increased.
Example 22
Sacchromyces cerevisiae ISC1 Increases Percent PUFAs
The present Example describes increased EPA biosynthesis and accumulation in Yarrowia lipolytica strain M4 (Example 4) that was transformed to co-express the S. cerevisiae ISC1 gene (SEQ ID NO:111). It is contemplated that a Y. lipolytica host strain engineered to produce ARA via either the Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8 desaturase pathway could demonstrate increased ARA biosynthesis and accumulation, if the S. cerevisiae ISC1 was similarly co-expressed (e.g., in strains Y2034, Y2047 or Y2214).
The S. cerevisiae ISC1 ORF was cloned into plasmid pZP217+Ura as follows. First, the ORF was PCR-amplified using genomic DNA from S. cerevisiae strain S288C (Promega, Madison, Wis.) and primer pair Isc1F and Isc1R (SEQ ID NOs:328 and 329). Primer Isc1F modified the wildtype 5′ sequence of ISC1 from ‘ATGTACAA’ to ‘ATGGACM’ in the amplified ORF, as it was necessary to incorporate a NcoI site and thereby keep ISC1 in frame. Amplification was carried out as follows: initial denaturation at 94° C. for 120 sec, followed by 35 cycles of denaturation at 94° C. for 30 sec, annealing at 50° C. for 30 sec and elongation at 68° C. for 120 sec. A final elongation cycle at 68° C. for 10 min was carried out, followed by reaction termination at 4° C. A 1455 bp DNA fragment was obtained from the PCR reaction for ISC1 and the PCR product size was confirmed by electrophoresis, using a 1% agarose gel (120 V for 30 min) and a 1 kB DNA standard ladder from Invitrogen (Carlsbad, Calif.).
The DNA was purified using a DNA Clean & Concentrator-5 kit from Zymo Research Corporation (Orange, Calif.), per the manufacturer's instructions, and then digested with NcoI/NotI. The ISC1 fragment was then individually cloned into pZP217+Ura (SEQ ID NO:153; FIG. 19C) digested with NcoI and NotI. Correct transformants were confirmed by gel electrophoresis and the resultant plasmid was designated as “pTEF::ISC1” (SEQ ID NO:155). Thus, this plasmid contained a DNA cassette comprising the following: 3′-POX2, URA3, TEF::ISC1::Pex20 and a POX2 promoter region.
“Control” vector was prepared as follows. First, the S. cerevisiae pcl1 ORF (encoding a protein involved in entry into the mitotic cell cycle and regulation of morphogenesis) was PCR amplified using genomic DNA from S. cerevisiae strain S288C and primer pair Pcl1 F and Pcl1R (SEQ ID NOs:330 and 331). Amplification was carried out as described above. A 861 bp DNA fragment was obtained from the PCR reaction for pcl1 (confirmed by electrophoresis, supra). The DNA was purified using a DNA Clean & Concentrator-5 kit and then digested with NcoI/NotI. The fragment was then cloned into similarly digested pZP217+Ura. Correct transformants were confirmed by gel electrophoresis and the resultant plasmid was designated as “pTEF::pcl1”. Plasmid pTEF::plc1 was then digested with HincII to remove the pcl1 ORF. The remaining plasmid was religated, such that a linear DNA cassette comprising 3′-POX2, URA3, TEF::Pex20 and a POX2 promoter region resulted upon digestion with AscI/SphI.
Competent Y. lipolytica strain M4 cells (from Example 4) were transformed with Asc1/Sph1-digested pTEF::ISC1 and “control”, respectively (wherein 5 μg of each plasmid had been subject to digestion). Transformation was accomplished using the Frozen EZ Yeast Transformation II kit (Zymo Research) and transformants were selected on plates comprising YNB without Amino Acids (6.7 g/L; Becton, Dickinson and Co., Sparks, Md. [Catalog #291940]), glucose (20 g/L) and agar (20 g/L). Several hundred transformant colonies were obtained. Integration of each DNA cassette into the Yarrowia lipolytica POX2 locus was confirmed by PCR using the genomic DNA from 5 independent transformants for ISC1.
Transformants were grown in YNB without amino acids containing 2% glucose for 2 days. The cells were harvested by centrifugation and resuspended in media comprising 100 g/L dextrose, 2 g/L MgSO4 and 50 mM phosphate buffer at pH 6.5 for 5 additional days of growth. The cells from 0.75 mL of each culture were harvested by centrifugation and analyzed for their fatty acid composition. The fatty acid profile of 3 transformants comprising the “control” vector and 5 transformants comprising pTEF::ISC1 are shown below based on GC analysis (as described in the General Methods). Fatty acids are identified as 16:0, 16:1, 18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA and EPA; and the composition of each is presented as a % of the total fatty acids.







TABLE 46







Lipid Composition In Yarrowia Strain M4 Engineered


To Overexpress S. cerevisiae ISC1









Total Fatty Acids

















Strain
16:0
16:1
18:0
18:1
18:2
GLA
DGLA
ARA
ETA
EPA




















M4 + “control”
14.7
7.2
2.1
13.5
8.7
21.8
8.9
0.9
4.1
9.3


M4 + pTEF::ISC1
13.5
8.5
1.7
15.6
8.1
21.3
7.5
0.7
3.9
10.7









Expression of the S. cerevisiae ISC1 gene improved the percent EPA from 9.3% in the “control” strain to 10.7% (“M4+PTEF::ISC1”), representing a 14.5% increase.
Example 23
Generation of Yarrowia lipolytica Acyltransferase Knockouts
The present Example describes the creation of single, double and triple knockout strains of Yarrowia lipolytica that were disrupted in either PDAT, DGAT2, DGAT1, PDAT and DGAT2, PDAT and DGAT1, DGAT1 and DGAT2, or PDAT, DGAT1 and DGAT2 genes. Disruption of the gene(s) in each of the knock-out strains was confirmed and analysis of each of the disruptions on fatty acid content and composition was determined by GC analysis of total lipids in Example 24.
Targeted Disruption of the Yarrowia lipolytica DGAT2 Gene
Targeted disruption of the DGAT2 gene in Y. lipolytica ATCC #90812 was carried out by homologous recombination-mediated replacement of the endogenous DGAT2 gene with a targeting cassette designated as plasmid pY21 DGAT2. pY21 DGAT2 was derived from plasmid pY20 (FIG. 19D; SEQ ID NO:156). Specifically, pY21DGAT2 was created by inserting a 570 bp Hind III/Eco RI fragment into similarly linearized pY20. The 570 bp DNA fragment contained (in 5′ to 3′ orientation): 3′ homologous sequence from position +1090 to +1464 (of the coding sequence (ORF) in SEQ ID NO:89), a Bgl II restriction site and 5′ homologous sequence from position +906 to +1089 (of the coding sequence (ORF) shown in SEQ ID NO:89). The fragment was prepared by PCR amplification using two pairs of PCR primers, P95 and P96 (SEQ ID NOs:332 and 333), and P97 and P98 (SEQ ID NOs:334 and 335), respectively.
pY21 DGAT2 was linearized by Bgl II restriction digestion and transformed into mid-log phase Y. lipolytica ATCC #90812 cells, according to the General Methods. The cells were plated onto YPD hygromycin selection plates and maintained at 30° C. for 2 to 3 days.
Fourteen Y. lipolytica ATCC #90812 hygromycin-resistant colonies were isolated and screened for targeted disruption by PCR. One set of PCR primers (P115 and P116 [SEQ ID NOs:336 and 337]) was designed to amplify a specific junction fragment following homologous recombination. Another pair of PCR primers (P115 and P112 [SEQ ID NO:338]) was designed to detect the native gene.
Two of the 14 hygromycin-resistant colonies of ATCC #90812. strains were positive for the junction fragment and negative for the native fragment. Thus, targeted integration was confirmed in these 2 strains, one of which was designated as “S-D2”.
Targeted Disruption of the Yarrowia lipolytica PDAT Gene
Targeted disruption of the PDAT gene in Y. lipolytica ATCC #90812 was carried out by homologous recombination-mediated replacement of the endogenous PDAT gene with a targeting cassette designated as pLV13 (FIG. 19E; SEQ ID NO:157). pLV13 was derived from plasmid pY20 (FIG. 19D; SEQ ID NO:156). Specifically, the hygromycin resistant gene of pY20 was replaced with the Yarrowia Ura3 gene to create plasmid pLV5. Then, pLV13 was created by inserting a 992 bp Bam HI/Eco RI fragment into similarly linearized pLV5. The 992 bp DNA fragment contained (in 5′ to 3′ orientation): 3′ homologous sequence from position +877 to +1371 (of the coding sequence (ORF) in SEQ ID NO:76), a Bgl II restriction site and 5′ homologous sequence from position +390 to +876 (of the coding sequence (ORF) in SEQ ID NO:76). The fragment was prepared by PCR amplification using PCR primers P39 and P41 (SEQ ID NOs:339 and 340) and P40 and P42 (SEQ ID NOs:341 and 342), respectively.
pLV13 was linearized by Bgl II restriction digestion and was transformed into mid-log phase Y. lipolytica ATCC #90812 cells, according to the General Methods. The cells were plated onto Bio101 DOB/CSM-Ura selection plates and maintained at 30° C. for 2 to 3 days.
Ten Y. lipolytica ATCC #90812 colonies were isolated and screened for targeted disruption by PCR. One set of PCR primers (P51 and P52 [SEQ ID NOs:343 and 344]) was designed to amplify the targeting cassette. Another set of PCR primers (P37 and P38 [SEQ ID NOs:345 and 346]) was designed to detect the native gene. Ten of the ten strains were positive for the junction fragment and 3 of the 10 strains were negative for the native fragment, thus confirming successful targeted integration in these 3 strains. One of these strains was designated as “S—P”.
Targeted Disruption of the Yarrowia lipolytica DGAT1 Gene
The full-length YI DGAT1 ORF was cloned by PCR using degenerate PCR primers P201 and P203 (SEQ ID NOs:347 and 348, respectively) and Y. lipolytica ATCC #76982 genomic DNA as template. The degenerate primers were required, since the nucleotide sequence encoding YI DGAT1 was not known.
The PCR was carried out in a RoboCycler Gradient 40 PCR machine, with amplification carried out as follows: initial denaturation at 95° C. for 1 min, followed by 30 cycles of denaturation at 95° C. for 30 sec, annealing at 55° C. for 1 min, and elongation at 72° C. for 1 min. A final elongation cycle at 72° C. for 10 min was carried out, followed by reaction termination at 4° C. The expected PCR product (ca. 1.6 kB) was detected by agarose gel electrophoresis, isolated, purified, cloned into the TOPO® cloning vector (Invitrogen), and partially sequenced to confirm its identity.
Targeted disruption of the putative DGAT1 gene in Y. lipolytica ATCC #90812 was carried out by homologous recombination-mediated replacement of the endogenous DGAT1 gene with a targeting cassette (using the methodology described above for DGAT2). Specifically, the 1.6 kB isolated YI DGAT1 ORF (SEQ ID NO:81) was used as a PCR template molecule to construct a YI DGAT1 targeting cassette consisting of: 5′ homologous YI DGAT1 sequence (amplified with primers P214 and P215 (SEQ ID NOs:349 and 350)), the Yarrowia Leucine 2 (Leu2; GenBank Accession No. AAA35244) gene, and 3′ homologous YI DGAT1 sequence (amplified with primers P216 and P217 (SEQ ID NOs:351 and 352)). Following amplification of each individual portion of the targeting cassette with Pfu Ultra polymerase (Stratagene, Catalog #600630) and the thermocycler conditions described above, each fragment was purified. The three correct-sized, purified fragments were mixed together as template molecules for a second PCR reaction using PCR primers P214 and P219 (SEQ ID NO:353) to obtain the YI DGAT1 disruption cassette.
The targeting cassette was gel purified and used to transform mid-log phase wildtype Y. lipolytica (ATCC #90812). Transformation was performed as described in the General Methods. Transformants were plated onto Bio101 DOB/CSM-Leu selection plates and maintained at 30° C. for 2 to 3 days. Several leucine prototrophs were screened by PCR to confirm the targeted DGAT1 disruption. Specifically, one set of PCR primers (P226 and P227 [SEQ ID NOs:354 and 355]) was designed to amplify a junction between the disruption cassette and native target gene. Another set of PCR primers (P214 and P217 [SEQ ID NOs:349 and 352]) was designed to detect the native gene.
All of the leucine prototroph colonies were positive for the junction fragment and negative for the native fragment. Thus, targeted integration was confirmed in these strains, one of which was designated as “S-D1”.
Creation of Yarrowia lipolytica Double and Triple Knockout Strains Containing Disruptions in PDAT and/or DGAT2 and/or DGAT1 Genes
The Y. lipolytica ATCC #90812 hygromycin-resistant “S-D2” mutant (containing the DGAT2 disruption) was transformed with plasmid pLV13 (containing the PDAT disruption) and transformants were screened by PCR, as described for the single PDAT disruption. Two of twelve transformants were confirmed to be disrupted in both the DGAT2 and PDAT genes. One of these strains was designated as “S-D2-P”.
Similarly, strains with double knockouts in DGAT1 and PDAT (“S-D1-P”), in DGAT2 and DGAT1 (“S-D2-D1”), and triple knockouts in DGAT2, DGAT1 and PDAT (“S-D2-D1-P”) were made.
Example 24
Yarrowia lipolytica Acyltransferase Knockouts Decrease Lipid Content and Increase Percent PUFAs
The present Example analyzes the affect of single and/or double and/or triple acyltransferase knockouts in wildtype Yarrowia lipolytica and strains of Y. lipolytica that had been previously engineered to produce EPA, as measured by changes in fatty acid content and composition. It is contemplated that a Y. lipolytica host strain engineered to produce ARA via either the Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8 desaturase pathway could demonstrate increased ARA biosynthesis and accumulation, if similar manipulations to the host's native acyltransferases were created (e.g., within strains Y2034, Y2047 or Y2214).
TAG Content is Decreased in Y. lipolytica ATCC #90812 with Acyltransferase Disruptions
First, TAG content was compared in wildtype and mutant Y. lipolytica ATCC #90812 containing: (1) single disruptions in PDAT, DGAT2 and DGAT1; (2) double disruptions in PDAT and DGAT2, DGAT1 and PDAT, and DGAT1 and DGAT2; and (3) triple disruptions in PDAT, DGAT2 and DGAT1.
Specifically, one loopful of cells from plates containing wildtype and mutant Y. lipolytica ATCC #90812 (i.e., strains S-D1, S-D2, S-P, S-D1-D2, S-D1-P, S-D2-P, and S-D1-D2-P) were each individually inoculated into 3 mL YPD medium and grown overnight on a shaker (300 rpm) at 30° C. The cells were harvested and washed once in 0.9% NaCl and resuspended in 50 mL of HGM. Cells were then grown on a shaker for 48 hrs. Cells were washed in water and the cell pellet was lyophilized. Twenty (20) mg of dry cell weight was used for total fatty acid by GC analysis and the oil fraction following TLC (infra) and GC analysis.
The methodology used for TLC is described below in the following five steps: (1) The internal standard of 15:0 fatty acid (10 μl of 10 mg/mL) was added to 2 to 3 mg dry cell mass, followed by extraction of the total lipid using a methanol/chloroform method. (2) Extracted lipid (50 μl) was blotted across a light pencil line drawn approximately 1 inch from the bottom of a 5×20 cm silica gel 60 plate, using 25-50 μl micropipettes. (3) The TLC plate was then dried under N2 and was inserted into a tank containing about  ̃100 mL 80:20:1 hexane:ethyl ether:acetic acid solvent. (4) After separation of bands, a vapor of iodine was blown over one side of the plate to identify the bands. This permitted samples on the other side of the plate to be scraped using a razor blade for further analysis. (5) Basic transesterification of the scraped samples and GC analysis was performed, as described in the General Methods.
GC results are shown below in Table 47. Cultures are described as the “S” strain (wildtype), “S-P” (PDAT knockout), “S-D1” (DGAT1 knockout), “S-D2” (DGAT2 knockout), “S-D1-D2” (DGAT1 and DGAT2 knockout), “S-P-D1” (PDAT and DGAT1 knockout), “S-P-D2” (PDAT and DGAT2 knockout) and “S—P-D1-D2” (PDAT, DGAT1 and DGAT2 knockout). Abbreviations utilized are: “WT”=wildtype; “FAs”=fatty acids; “dcw”=dry cell weight; and, “FAs % dcw, % WT”=FAs % dcw relative to the % in wildtype, wherein the “S” strain is wildtype.







TABLE 47







Lipid Content In Yarrowia ATCC #90812 Strains With


Single, Double, Or Triple Disruptions In PDAT, DGAT2


And DGAT1










Total Fatty
TAG



Acids
Fraction




















FAs %


FAs %



Residual

FAs,
FAs %
dcw, %
FAs,
FAs %
dcw, %


Strain
DAG AT
dcw, mg
μg
dcw
WT
μg
dcw
WT


















S
D1, D2, P
32.0
797
15.9
100
697
13.9
100


S-D1
D2, P
78.8
723
13.6
86
617
11.6
83


S-D2
D1, P
37.5
329
6.4
40
227
4.4
32


S-P
D1, D2
28.8
318
6.0
38
212
4.0
29


S-D1-D2
P
64.6
219
4.1
26
113
2.1
15


S-D1-P
D2
76.2
778
13.4
84
662
11.4
82


S-D2-P
D1
31.2
228
4.3
27
122
2.3
17


S-D1-D2-P
None
52.2
139
2.4
15
25
0.4
3









The results in Table 47 indicate the relative contribution of the three DAG ATs to oil biosynthesis. DGAT2 contributes the most, while PDAT and DGAT1 contribute equally but less than DGAT2. The residual oil content ca. 3% in the triple knockout strain may be the contribution of Yarrowia lipolytica's acyl-CoA:sterol-acyltransferase enzyme, encoded by ARE2 (SEQ ID NOs:78 and 79).
TAG Content is Decreased and Percent EPA is Increased in Yarrowia lipolytica Strain EU with a Disrupted DGAT2 Gene
After examining the affect of various acyltransferase knockouts in wildtype Y. lipolytica ATCC #90812 (supra), TAG content and fatty acid composition was then studied in DGAT2 knockout strains of the EU strain (i.e., engineered to produce 10% EPA; see Example 10).
Specifically, the DGAT2 gene in strain EU was disrupted as described for the S strain (ATCC #90812) in Example 23. The DGAT2-disrupted strain was designated EU-D2. EU and EU-D2 strains were harvested and analyzed following growth according to two different conditions. In the condition referred to in the Table below as “3 mL”, cells were grown for 1 day in 3 mL MM medium, washed and then grown for 3 days in 3 mL HGM. Alternatively, in the condition referred to in the Table below as “51 mL”, cells were grown for 1 day in 51 mL MM medium, washed and then grown for 3 days in 51 mL HGM. The fatty acid compositions of phosphatidylcholine (PC), phosphatidyletanolamine (PE), and triacylglycerol (TAG or oil) were determined in the extracts of 51 mL cultures following TLC separation (“Fraction”).
GC results are shown below in Table 48. Cultures are described as the “EU” strain (wildtype) and the “EU-D2” strain (DGAT2 knockout). Fatty acids are identified as 16:0, 16:1, 18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA and EPA; and the composition of each is presented as a % of the total fatty acids.







TABLE 48







Lipid Content And Composition In Yarrowia Strain


EU With Disruption In DGAT2



















Strain &

TFAs %
%
%
%
%
%
%
%
%
%
%


Growth
Fraction
dcw
16:0
16:1
18:0
18:1
18:2
GLA
DGLA
ARA
ETA
EPA






















EU,
Total

19
10
2
16
12
19
6
0
3
10


3 mL


EU-
Total

17
10
1
6
7
24
5
0
6
19


D2, 3 mL


EU,
Total
37
18
11
3
19
31
5
1

1
4


51 mL
PC
2
12
9
1
8
43
7
3

5
4



PE
1
24
14
0
14
37
5
0

0
1



TAG
34
18
12
3
21
29
5
1

1
4


EU-
Total
18
18
8
1
5
7
25
5

5
20


D2, 51 mL
PC
1
18
6
1
2
4
26
5

11
22



PE
1
25
7
0
2
5
14
2

3
8



TAG
15
16
9
1
6
5
26
6

5
21









The results show that the DGAT2 knockout resulted in doubling of the % EPA (of total fatty acids) and halving of the lipid content (as % dcw). Furthermore, almost all of the changes observed in the lipid content are due to changes in the TAG fraction. The lower than expected % EPA in the 51 mL culture of strain EU is likely due to instability.
TAG Content is Decreased and Percent EPA is Increased in Yarrowia lipolytica Strain MU with Disrupted Acyltransferase Genes
Finally, based on the increased % EPA and reduced lipid content resulting from a single DGAT2 knockout in strain EU-D2, TAG content and fatty acid composition was then studied in various acyltransferase knockout strains of strain MU (engineered to produce 14% EPA; see Example 12).
Specifically, single disruptions in PDAT, DGAT2 and DGAT1 and double disruptions in PDAT and DGAT2 were created in strain MU. Lipid content and composition was compared in each of these strains, following growth in 4 different growth conditions.
More specifically, single disruptions in PDAT, DGAT2, DGAT1 were created in strain MU, using the methodology described in Example 23 (with the exception that selection for the DGAT1 disruption relied on the URA3 gene). This resulted in single knockout strains identified as “MU-D1” (disrupted in DGAT1), “MU-D2” (disrupted in DGAT2), and “MU-P” (disrupted in PDAT). Individual knockout strains were confirmed by PCR. Additionally, the MU-D2 strain was disrupted for the PDAT gene by the same method and the disruption confirmed by PCR. The resulting double knockout strain was designated “MU-D2-P”.
The MU-D1, MU-D2, MU-P, and M-D2-P knockout strains were analyzed to determine each knockout's effect on lipid content and composition, as described below. Furthermore, the growth conditions promoting oleaginy were also explored to determine their effect on total lipid content. Thus, in total, four different experiments were conducted, identified as “Experiment A”, “Experiment B”, “Experiment C” and “Experiment E”. Specifically, three loops of cells from plates containing each strain above was inoculated into MMU medium [3 mL for Experiments B and C; and 50 mL for Experiments A and E] and grown in a shaker at 30° C. for 24 hrs (for Experiments A, B and C) or 48 hrs (for Experiment E). Cells were harvested, washed once in HGM, resuspended in either HGM medium (50 mL for Experiments A and E; and 3 mL for Experiment B) or HGM medium with uracil (“HGMU”) (3 mL for Experiment C) and cultured as above for 4 days. One aliquot (1 mL) was used for lipid analysis by GC as described according to the General Methods, while a second aliquot was used for determining the culture OD at 600 nm. The remaining culture in Experiments A and E was harvested, washed once in water, and lyophilized for dry cell weight (dcw) determination. In contrast, the dcw in Experiments B and C were determined from their OD600 using the equation showing their relationship. The fatty acid compositions of each of the different strains in Experiments A, B, C and E was also determined.
The results are shown in Table 49 below. Cultures are described as the “MU” strain (the parent EPA producing strain), “MU-P” (PDAT knockout), “MU-D1” (DGAT1 knockout), “MU-D2” (DGAT2 knockout) and “MU-D2-P” (DGAT2 and PDAT knockouts). Abbreviations utilized are: “WT”=wildtype (i.e., MU); “OD”=optical density; “dcw”=dry cell weight; “TFAs”=total fatty acids; and, “TFAs % dcw, % WT”=TFAs % dcw relative to the wild type (“MU”) strain. Fatty acids are identified as 16:0, 16:1, 18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA and EPA; and the composition of each is presented as a % of the total fatty acids.







TABLE 49





Lipid Content And Composition In Yarrowia Strain MU


With Various Acyltransferase Disruptions



























1st Phase
2nd Phase



TFAs
TFAs %




Residual
Growth
Growth

dcw
TFAs
%
dcw,


Expt
Strain
DAG AT
Condition
Condition
OD
(mg)
(μg)
dcw
% WT



















A
MU
D1, D2, P
1 day,
4 days,
4.0
91
374
20.1
100


A
MU-D2
D1, P
50 mL
50 mL
3.1
75
160
10.4
52


A
MU-D1
D2, P
MMU
HGM
4.3
104
217
10.2
51


A
MU-P
D1, D2


4.4
100
238
11.7
58


B
MU
D1, D2, P
1 day,
4 days,
5.9
118
581
24.1
100


B
MU-D2
D1, P
3 mL
3 mL
4.6
102
248
11.9
50


B
MU-D1
D2, P
MMU
HGM
6.1
120
369
15.0
62


B
MU-P
D1, D2


6.4
124
443
17.5
72


C
MU
D1, D2, P
1 day,
4 days,
6.8
129
522
19.9
100


C
MU-D2
D1, P
3 mL
3 mL
5.6
115
239
10.2
51


C
MU-D1
D2, P
MMU
HGMU
6.9
129
395
15.0
75


C
MU-P
D1, D2


7.1
131
448
16.8
84


E
MU
D1, D2, P
2 days,
4 days,
4.6
89
314
17.3
100


E
MU-D2
D1, P
50 mL
50 mL
2.8
62
109
8.5
49


E
MU-P
D2, P
MM
HGM
5.0
99
232
11.5
66


E
MU-D2-P
D1


4.2
98
98
4.9
28























%
%
%
%
%
%
%
%
%
%



Expt
16:0
16:1
18:0
18:1
18:2
GLA
DGLA
ARA
ETA
EPA























A
17
10
2
18
10
22
7
1
3
9.7



A
16
12
0
8
9
23
7
0
8
17.4



A
15
10
2
11
10
22
7
0
7
17.4



A
16
9
2
11
7
24
7
1
6
17.5



B
17
9
3
18
10
22
8
1
3
9.1



B
16
10
0
7
10
24
7
1
7
17.8



B
18
9
3
14
11
20
7
1
5
12.0



B
15
8
3
16
10
25
6
1
4
11.9



C
16
10
2
13
11
21
10
1
4
12.6



C
17
9
1
6
11
21
8
1
7
18.9



C
15
9
2
12
12
20
10
1
5
13.5



C
17
8
3
14
11
20
10
1
4
11.3



E
16
12
2
18
9
22
7
1
4
11.2



E
14
12
1
6
8
25
6
0
7
20.0



E
16
10
2
14
7
24
7
1
5
15.8



E
18
10
0
7
12
20
5
0
6
22.5










The data showed that the lipid content within the transformed cells varied according to the growth conditions. Furthermore, the contribution of each acyltransferase on lipid content also varied. Specifically, in Experiments B, C and E, DGAT2 contributed more to oil biosynthesis than either PDAT or DGAT1. In contrast, as demonstrated in Experiment A, a single knockout in DGAT2, DGAT1 and PDAT resulted in approximately equivalent losses in lipid content (i.e., 48%, 49% and 42% loss, respectively [see “TFAs % dcw, % WT”]).
With respect to fatty acid composition, the data shows that knockout of each individual DAG AT gene resulted in lowered oil content and increased % EPA. For example, the DGAT2 knockout resulted in about half the lipid content and ca. double the % EPA in total fatty acids (similar to the results observed in strain EU-D2, supra). Knockout of both DAGAT2 and PDAT resulted in the least oil and the most % EPA.
On the basis of the results reported herein, it is contemplated that disruption of the native DGAT2 and/or DGAT1 and/or PDAT is a useful means to substantially increase the % PUFAs in a strain of Yarrowia lipolytica engineered to produce high concentrations of PUFAs, including ARA (e.g., strains Y2034, Y2047, Y2214). In fact, a disruption of the native DGAT2 gene in strain Y2214 resulted in a 1.7 fold increase in the percent ARA (data not shown).

## Claims

1. A recombinant oleaginous Yarrowia yeast cell for the production of arachidonic acid comprising at least one gene encoding a Δ8 desaturase wherein;
a) the at least one gene encoding Δ8 desaturase encodes a polypeptide having at least 95% identity to the amino acid sequence as set forth in SEQ ID NO:49; and
b) the oleaginous Yarrowia yeast cell accumulates in excess of about 25% of its dry cell weight as oil; and
c) the oleaginous Yarrowia yeast cell produces about 14% arachidonic acid of total lipid produced.

2. The recombinant oleaginous Yarrowia yeast cell according to claim 1 further comprising at least one gene encoding Δ12 desaturase having at least 95% identity to the amino acid sequence as set forth in SEQ ID NO:28.

3. The recombinant oleaginous Yarrowia yeast cell according to claim 2 wherein the Yarrowia cell is devoid of any native gene encoding a polypeptide having Δ12 desaturase activity.

4. The recombinant oleaginous Yarrowia yeast cell according to claim 1, wherein the at least one gene encoding Δ8 desaturase is under the control of a promoter sequence having the nucleic acid sequence selected from the group consisting of SEQ ID NOs:158-168 and 364.

5. The recombinant oleaginous Yarrowia yeast cell according to claim 1 further comprising at least one gene encoding C16/18 elongase having at least 95% identity to the amino acid sequence as set forth in SEQ ID NO:54.

6. The recombinant oleaginous Yarrowia yeast cell according to claim 1 wherein the yeast cell produces a microbial oil comprising arachidonic acid and wherein the microbial oil is devoid of any γ-linoleic acid.

7. A method for the production of a microbial oil comprising arachidonic acid comprising:
a) culturing the host of claim 1 wherein a microbial oil comprising arachidonic acid is produced; and
b) optionally recovering the microbial oil of step (a).