# Patent

## Title
Mortierella alpina diacylglycerol acyltransferase for alteration of polyunsaturated fatty acids and oil content in oleaginous organisms

## Abstract

An acyltransferase is provided, suitable for use in the manufacture of microbial oils enriched in omega fatty acids in oleaginous organisms. Specifically, the gene encoding diacylglycerol acyltransferase (DGAT2) has been isolated from Mortierella alpina. This gene encodes an enzyme that participates in the terminal step in oil biosynthesis in fungi and yeast and is expected to play a key role in altering the quantity of long-chain polyunsaturated fatty acids produced in oils of oleaginous organisms. Most desirably, the substrate specificity of the instant DGAT2 will be particularly useful to enable accumulation of long-chain PUFAs having chain lengths equal to or greater than C20 in oleaginous yeast, such as Yarrowia lipolytica.

## Background
This application claims the benefit of U.S. patent application Ser. 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 the identification of a nucleic acid fragment isolated from Mortierella alpina encoding a diacylglycerol acyltransferase (DGAT2). This enzyme is useful for altering the quantity of oil in oleaginous microorganisms, such as oleaginous yeast.
BACKGROUND OF THE INVENTION
The present invention is directed toward the development of an oleaginous yeast that accumulates oils enriched in long-chain ω-3 and/or ω-6 polyunsaturated fatty acids (“PUFAs”; e.g., 18:3, 18:4, 20:3, 20:4, 20:5, 22:6 fatty acids). Toward this end, the natural abilities of oleaginous yeast (mostly limited to 18:2 fatty acid production) have been enhanced by advances in genetic engineering, leading to the production of 20:4 (arachidonic acid or “ARA”), 20:5 (eicosapentaenoic acid or “EPA”) and 22:6 (docosahexaenoic acid or “DHA”) PUFAs in transformant Yarrowia lipolytica. These ω-3 and ω-6 fatty acids were produced by introducing and expressing heterologous genes encoding the ω-3/ω-6 biosynthetic pathway in the oleaginous host (see co-pending U.S. patent applications Ser. No. 10/840,579 and Ser. No. 60/624,812, each entirely incorporated herein by reference). However, in addition to developing techniques to introduce the appropriate fatty acid desaturases and elongases into these particular host organisms, it is also necessary to increase the transfer of PUFAs into storage lipid pools following their synthesis.
Most free fatty acids become esterified to coenzyme A (CoA) to yield acyl-CoAs. These molecules are then substrates for glycerolipid synthesis in the endoplasmic reticulum of the cell, where phosphatidic acid and diacylglycerol (DAG) are produced. Either of these metabolic intermediates may be directed to membrane phospholipids (e.g., phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine) or DAG may be directed to form triacylglycerols (TAGs), the primary storage reserve of lipids in eukaryotic cells.
In the yeast Saccharomyces cerevisiae, three pathways have been described for the synthesis of TAGs. First, TAGs are mainly synthesized from DAG and acyl-CoAs by the activity of diacylglycerol acyltransferases. More recently, however, a phospholipid:diacylglycerol acyltransferase has also been identified that is responsible for conversion of phospholipid and DAG to lysophospholipid and TAG, respectively, thus producing TAG via an acyl-CoA-independent mechanism (Dahlqvist et al., PNAS. 97(12): 6487–6492 (2000)). Finally, two acyl-CoA:sterol-acyltransferases 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)).
A comprehensive mini-review on TAG biosynthesis in yeast, including details concerning the genes involved and the metabolic intermediates that lead to TAG synthesis, is that of D. Sorger and G. Daum (Appl. Microbiol. Biotechnol. 61:289–299 (2003)). However, the authors acknowledge that most work performed thus far has focused on Saccharomyces cerevisiae and numerous questions regarding TAG formation and regulation remain. In this organism it has been conclusively demonstrated that only four genes are involved in storage lipid synthesis: ARE1 and ARE2 (encoding acyl-CoA:sterol-acyltransferases), LRO1 (encoding a phospholipid:diacylglycerol acyltransferase, or PDAT enzyme) and DGA1 (encoding a diacylglycerol acyltransferase, or DGAT2 enzyme) (Sandager, L. et al., J. Biol. Chem. 277(8):6478–6482 (2002)).
Although homologs of the acyltransferase genes described above have been identified in various other organisms and disclosed in the public literature, few genes are available from oleaginous organisms. Concerning diacylglycerol acyltransferases, a single DGAT2 enzyme from oleaginous yeast (i.e., Yarrowia lipolytica) has been isolated and characterized in co-pending U.S. patent application Ser. No. 10/882,760. Since the natural capabilities of this organism are limited to 18:2 fatty acid production, however, its native diacylglycerol acyltransferases (including DGAT2) are not likely to utilize longer chain PUFAs (i.e., C20 or greater) as efficiently as those from organisms that are naturally capable of producing longer chain PUFAs. The production of ARA, EPA and DHA PUFAs in transformant Yarrowia lipolytica therefore is likely to be improved by the use of heterologous acyltransferases (e.g., DGAT2) having altered substrate specificies, as compared to the native enzymes. Furthermore, techniques for modifying the transfer of fatty acids to the TAG pool in oleaginous yeast have not been developed.
Only three DGAT2 enzymes have been isolated from oleaginous fungi. Specifically, Lardizabal et al. isolated and characterized two DGAT2s (i.e., MrDGAT2A and MrDGAT2B) from Mortierella ramanniana (J. Biol. Chem. 276(42):38862–28869 (2001)); US 2003/0028923 A1, US 2003/0115632 A1) and one DGAT2 from Neurospora crassa (Nc DGAT2; see US 2004/0107459 A1). Upon expression of MrDGAT2A, MrDGAT2B, and NcDGAT2 in insect cells, high levels of DGAT activity were obtained on membranes isolated from those cells. Like Y. lipolytica, however, M. ramanniana and N. crassa are generally limited to production of 16:0, 18:0, 18:1, 18:2 and 18:3 fatty acids (although synthesis of 20:0 fatty acids is observed in M. ramanniana during lower temperature growth; see da Silva M., et al. Rev. Microbiol. 29:4 São Paulo (October/December 1998)). Thus, the DGAT2s from M. ramanniana and N. crassa are likely not preferred for the transfer of 20:4, 20:5 and 22:6 PUFAs to the TAG pool in a transformant oleaginous yeast.
A variety of microorganisms are known that naturally produce long-chain PUFAs (e.g., ARA and EPA, DHA). For example, microorganisms in the genera Mortierella (filamentous fungus), Entomophthora, Pythium and Porphyridium (red alga) can be used for commercial production of the ω-6 fatty acid, ARA. The fungus Mortierella alpina, for example, is used to produce an oil containing ARA, while U.S. Pat. No. 5,658,767 (Martek Corporation) teaches a method for the production of an oil containing ARA comprising cultivating Pythium insidiuosum in a culture medium containing a carbon and nitrogen source. Likewise, U.S. Pat. No. 5,244,921 (Martek Corporation) describes a process for producing an edible oil containing EPA, by cultivating the heterotrophic diatoms Cyclotella sp. and Nitzschia sp. in a fermentor. DHA can be obtained by cultivation of the heterotrophic microalgae Crypthecodinium cohnii (U.S. Pat. No. 5,492,938 and U.S. Pat. No. 5,407,957). Other long-chain PUFA-producing organisms include Thraustochytrium sp. and the green alga Parietochloris incisa. It is likely that many of these organisms possess genes encoding acyltransferases that would be preferred for the incorporation of long-chain PUFAs in a transformant oleaginous yeast, relative to the native acyltransferases that do not naturally produce long-chain PUFAs.
Thus, there is a need for the identification and isolation of a gene encoding an acyltransferase from an organism such as those above, to permit its use in the production and accumulation of long-chain PUFAs in the storage lipid pools (i.e., TAG fraction) of transformant oleaginous yeast.
Applicants have solved the stated problem by isolating the gene encoding DGAT2 from the oleaginous fungus, Mortierella alpina. This gene will be useful to enable one to modify the transfer of long-chain free fatty acids (e.g., ω-3 and/or ω-6 fatty acids) to the TAG pool in oleaginous yeast.
SUMMARY OF THE INVENTION
The invention relates to the discovery of a gene encoding a diacylglycerol acyltransferase enzyme from Mortierella. This gene and encoded enzyme are useful in manipulating the production of commercially useful oils in microorganisms, and particularly in oleaginous yeast. Accordingly the invention provides an isolated nucleic acid molecule encoding an diacylglycerol acyltransferase 2 enzyme, selected from the group consisting of:

    
    
        (a) an isolated nucleic acid molecule encoding the amino acid sequence as set forth in SEQ ID NO:2;
        (b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; or
        (c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).
    
    


Similarly the invention provides a polypeptide having diacylglycerol acyltransferase 2 activity encoded by the isolated nucleic acid molecule of the invention as well as genetic chimera of these molecules and host cells comprising the same.
In one preferred embodiment the invention provides a method of increasing triacylglycerol content in a transformed host cell comprising:

    (a) providing a transformed host cell comprising:
    
        (i) at least one gene encoding a diacylglycerol acyltransferase 2 enzyme having the amino acid sequence as set forth in SEQ ID NO:2 under the control of suitable regulatory sequences; and
        (ii) a source of fatty acids;
    
    
    (b) growing the cell of step (a) under conditions whereby the at least one gene encoding a diacylglycerol acyltransferase 2 enzyme is expressed, resulting in the transfer of the fatty acids to triacylglycerol; and
    (c) optionally recovering the triacylglycerol of step (b).


In an additional preferred embodiment the invention provides a method of increasing the ω-3 or ω-6 fatty acid content of triacylglycerols in a transformed host cell comprising:

    (a) providing a transformed host cell comprising:
    
        (i) at least one gene encoding at least one enzyme of the ω-3/ω-6 fatty acid biosynthetic pathway;
        (ii) at least one gene encoding a diacylglycerol acyltransferase 2 enzyme having the amino acid sequence as set forth in SEQ ID NO:2 under the control of suitable regulatory sequences;
    
    
    (b) growing the cell of step (a) under conditions whereby the genes of (i) and (ii) are expressed, resulting in the production of at least one ω-3 or ω-6 fatty acid and its transfer to triacylglycerol; and
    (c) optionally recovering the triacylglycerol of step (b).


Alternatively the invention provides a method of increasing triacylglycerol content in a transformed host cell comprising:

    (a) providing a transformed host cell comprising:
    
        (i) at least one gene encoding a heterologous diacylglycerol acyltransferase 2 enzyme having the amino acid sequence as set forth in SEQ ID NO:2 under the control of suitable regulatory sequences; and
        (ii) a source of fatty acids;
        wherein said transformed host has a disruption in the gene encoding the native diacylglycerol acyltransferase 2 enzyme;
    
    
    (b) growing the cell of step (a) under conditions whereby the at least one gene encoding a diacylglycerol acyltransferase 2 enzyme is expressed, resulting in the transfer of the fatty acids to triacylglycerol; and
    (c) optionally recovering the triacylglycerol of step (b).


In similar fashion the method provides a method of increasing the ω-3/ω-6 fatty acid content of triacylglycerols in a transformed host cell comprising:

    (a) providing a transformed host cell comprising:
    
        (i) at least one gene encoding at least one enzyme of the ω-3/ω-6 fatty acid biosynthetic pathway;
        wherein said transformed host has a disruption in the gene encoding the native diacylglycerol acyltransferase 2 enzyme;
        (ii) at least one gene encoding a diacylglycerol acyltransferase 2 enzyme having the amino acid sequence as set forth in SEQ ID NO:2 under the control of suitable regulatory sequences;
    
    
    (b) growing the cell of step (a) under conditions whereby the genes of (i) and (ii) are expressed, resulting in the production of at least one ω-3 or ω-6 fatty acid and its transfer to triacylglycerol; and
    (c) optionally recovering the triacylglycerol of step (b).





BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS
FIG. 1 shows a schematic illustration of the biochemical mechanism for lipid accumulation in oleaginous yeast.
FIG. 2 illustrates the ω-3 and ω-6 fatty acid biosynthetic pathways.
FIG. 3 graphically represents the relationship between SEQ ID NOs:1, 2, 18, 19, 20, 26, 31, 32, 33, 36 and 37, each of which relates to the diacylglycerol acyltransferase 2 (dgat2) gene in Mortierella alpina. 
FIG. 4A diagrams the development of Yarrowia lipolytica strain Y2067U, producing up to 14% EPA in the total lipid fraction. FIG. 4B provides a plasmid map for pKUNF12T6E; FIG. 4C provides a plasmid map for pDMW232; and FIG. 4D provides a plasmid map for pZP3L37.
FIG. 5 provides plasmid maps for the following: (A) pY37/F15; (B) pKO2UF2PE; (C) pZKUT16; (D) pZUF17; and (E) pZUF-Mod-1.



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:1–8, 18–20, 26, 31–33, 36, 37, 40–43, 45–48, 50, 51, 53, 54, 58, 59, 62 and 63 are ORFs encoding genes or proteins (or portions thereof) or protein motifs, as identified in Table 1.







TABLE 1







Summary of Gene and Protein SEQ ID Numbers










Nucleic acid
Protein


Description and Abbreviation
SEQ ID NO.
SEQ ID NO.














Mortierella alpina diacylglycerol
1
(996 bp)
2
(331 AA)


acyltransferase 2 (DGAT2) coding


region










Mortierella alpina DGAT2-partial
18
(370 bp)
—


cDNA sequence


Mortierella alpina DGAT2-internal
19
(654 bp)
—


genomic fragment


Mortierella alpina DGAT2-intron 1
32
(184 bp)
—


Mortierella alpina DGAT2-intron 2
20
(179 bp)
—


Mortierella alpina DGAT2-intron 3
33
(131 bp)
—


Mortierella alpina DGAT2-3′ fragment
26
(455 bp)
—


Mortierella alpina DGAT2-3′ fragment
31
(674 bp)
—


Mortierella alpina DGAT2-5′ fragment
36
(1261 bp)
—


Mortierella alpina DGAT2-genomic
37
(2299 bp)
—


fragment











Yarrowia lipolytica DGAT2
3
(2119 bp)
4
(514 AA)


(“YI DGAT2”)
5
(1380 bp)
6
(459 AA)



7
(1068 bp)
8
(355 AA)


Synthetic elongase gene derived from
40
(957 bp)
41
(318 AA)


Mortierella alpina, codon-optimized for


expression in Yarrowia lipolytica


Synthetic Δ6 desaturase, derived from
42
(1374 bp)
43
(457 AA)


Mortierella alpina, codon-optimized for


expression in Yarrowia lipolytica


Fusarium moniliforme Δ12 desaturase
45
(1434 bp)
46
(477 AA)


Synthetic elongase gene derived from
47
(819 bp)
48
(272 AA)


Thraustochytrium aureum, codon-


optimized for expression in Yarrowia


lipolytica


Mortierella alpina Δ5 desaturase
50
(1341 bp)
51
(446 AA)


Synthetic Δ17 desaturase gene derived
53
(1077 bp)
54
(358 AA)


from Saprolegnia diclina, codon-


optimized for expression in Yarrowia


lipolytica


Yarrowia lipolytica Δ12 desaturase
58
(1936 bp)
59
(419 AA)


Synthetic C16 elongase gene derived
62
(804 bp)
63
(267 AA)


from Rattus norvegicus, codon-


optimized for expression in Yarrowia


lipolytica









SEQ ID NOs:38, 49, 52, 56, 57, 61, 64, 67 and 68 are plasmids as identified in Table 2.







TABLE 2







Summary of Plasmid SEQ ID Numbers












Corresponding




Plasmid
Figure
SEQ ID NO
















pKUNF12T6E
4B
38
(12,649 bp)



pDMW232
4C
49
(10,945 bp)



pZP3L37
4D
52
(12,690 bp)



pY37/F15
5A
56
(8,194 bp)



pKO2UF2PE
5B
57
(10,838 bp)



pZKUT16
5C
61
(5,833 bp)



pZUF17
5D
64
(8,165 bp)



pMDGAT2-17
—
67
(8,084 bp)



pZUF-MOD-1
5E
68
(7,323 bp)










SEQ ID NOs:9, 10 and 11 correspond to BD-Clontech Creator Smart® cDNA library kit primers SMART IV oligonucleotide, CDSIII/3′ PCR primer and 5′-PCR primer.
SEQ ID NOs:12, 14 and 16 are the degenerate primers identified as MDGAT-FN1, MDGAT-RN1 and MDGAT-RN2, respectively, used for the amplification of a partial putative Mortierella alpina DGAT2.
SEQ ID NOs:13, 15 and 17 are the amino acid consensus sequences that correspond to the degenerate primers MDGAT-FN1, MDGAT-RN1 and MDGAT-RN2, respectively.
SEQ ID NOs:21–25 correspond to primers AP, MDGAT-3-1, UAP, MDGAT-3-2 and MDGAT-3-3, respectively, used for genome-walking to isolate the 3′-end region of the M. alpina DGAT2.
SEQ ID NOs:27 and 28 correspond to the Genome Walker adaptor from ClonTech's Universal GenomeWalkerTM Kit, used for genome-walking to isolate the 3′-end region of the M. alpina DGAT2.
SEQ ID NOs:29 and 30 correspond to primers AP1 and AP2, respectively, used for genome-walking to isolate the 3′-end region of the M. alpina DGAT2.
SEQ ID NOs:34 and 35 correspond to primers MDGAT-5-1 and MDGAT-5-2, respectively, used for genome-walking to isolate the 5′-end region of the M. alpina DGAT2.
SEQ ID NOs:39, 44, 55 and 60 correspond to the following Yarrowia lipolytica promoters, respectively: fructose-bisphosphate aldolase+intron (FBAIN; 973 bp), fructose-bisphosphate aldolase (FBA; 1001 bp), fructose-bisphosphate aldolase+modified intron (FBAINm; 924 bp), glycerol-3-phosphate acyltransferase (GPAT; 1130 bp).
SEQ ID NOs:65 and 66 correspond to primers MDGAT-F and MDGAT-R1, respectively, used for cloning of the M. alpina DGAT2 ORF.
SEQ ID NOs:69 and 70 correspond to primers pzuf-mod1 and pzuf-mod2, respectively, used for creating “control” plasmid pZUF-MOD-1.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the subject invention, Applicants have isolated and confirmed the identity of a Mortierella alpina gene encoding a diacylglycerol acyltransferase 2 (DGAT2) enzyme useful for transferring fatty acids into storage triacylglycerols (TAGs). This may be useful to alter the quantity of long chain polyunsaturated fatty acids (PUFAs) produced in oleaginous yeast.
The importance of PUFAs are undisputed. For example, certain PUFAs are important biological components of healthy cells and are recognized as: “essential” fatty acids that cannot be synthesized de novo in mammals and instead must be obtained either in the diet or derived by further desaturation and elongation of linoleic acid (LA) or α-linolenic acid (ALA); constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or TAGs; necessary for proper development (particularly in the developing infant brain) and for tissue formation and repair; and, precursors to several biologically active eicosanoids of importance in mammals (e.g., prostacyclins, eicosanoids, leukotrienes, prostaglandins). Additionally, a high intake of long-chain ω-3 PUFAs produces cardiovascular protective effects (Dyerberg, J. et al., Amer. J. Clin Nutr. 28:958–966 (1975); Dyerberg, J. et al., Lancet 2(8081):117–119 (Jul. 15, 1978); Shimokawa, H., World Rev Nutr Diet, 88:100–108 (2001); von Schacky, C., and Dyerberg, J., World Rev Nutr Diet, 88:90–99 (2001)). And, numerous other studies document wide-ranging health benefits conferred by administration of ω-3 and/or ω-6 fatty acids against a variety of symptoms and diseases (e.g., asthma, psoriasis, eczema, diabetes, cancer).
As such, the subject invention finds many applications. PUFAs, or derivatives thereof, accumulated 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 prostaglandins. 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.
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.
“Diacylglycerol” is abbreviated DAG.
“Triacylglycerols” are abbreviated TAGs.
“Co-enzyme A” is abbreviated CoA.
The term “fatty acids” refers to long chain aliphatic acids (alkanoic acids) of varying chain length, 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.
Generally, fatty acids are classified as saturated or unsaturated. The term “saturated fatty acids” refers to those fatty acids that have no “double bonds” between their carbon backbone. In contrast, “unsaturated fatty acids” have “double bonds” along their carbon backbones (which are most commonly in the cis-configuration). “Monounsaturated fatty acids” have only one “double bond” along the carbon backbone (e.g., usually between the 9th and 10th carbon atom as for palmitoleic acid (16:1) and oleic acid (18:1)), while “polyunsaturated fatty acids” (or “PUFAs”) have at least two double bonds along the carbon backbone (e.g., between the 9th and 10th, and 12th and 13th carbon atoms for linoleic acid (18:2); and between the 9th and 10th, 12th and 13th, and 15th and 16th for α-linolenic acid (18:3)).
“PUFAs” can be classified into two major families (depending on the position (n) of the first double bond nearest the methyl end of the fatty acid carbon chain). Thus, the “omega-6 fatty acids” (ω-6 or n-6) have the first unsaturated double bond six carbon atoms from the omega (methyl) end of the molecule and additionally have a total of two or more double bonds, with each subsequent unsaturation occurring 3 additional carbon atoms toward the carboxyl end of the molecule. In contrast, the “omega-3 fatty acids” (ω-3 or n-3) have the first unsaturated double bond three carbon atoms away from the omega end of the molecule and additionally have a total of three or more double bonds, with each subsequent unsaturation occurring 3 additional carbon atoms toward the carboxyl end of the molecule.
For the purposes of the present disclosure, the omega-reference system will be 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). This nomenclature is shown below in Table 3, in the column titled “Shorthand Notation”. The remainder of the Table summarizes the common names of ω-3 and ω-6 fatty acids, the abbreviations that will be used throughout the specification and each compounds' chemical name.







TABLE 3







Nomenclature Of Polyunsaturated Fatty Acids













Shorthand


Common Name
Abbreviation
Chemical Name
Notation





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









“Microbial oils” or “single cell oils” are those oils naturally produced by microorganisms (e.g., algae, oleaginous yeast 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 “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):38862–28869 (2001)). A representative DGAT2 enzyme is encoded by the DGA1 gene of Saccharomyces cerevisiae (locus NP—014888 of Genbank Accession No. NC—001147; Oelkers et. al. J. Biol. Chem. 277:8877 (2002)); a gene encoding DGAT2 isolated from Mortierella alpina is provided as SEQ ID NO:1.
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 “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 and/or an elongase(s).
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 ω-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, elongase, Δ5 desaturase, Δ17 desaturase, Δ15 desaturase, Δ9 desaturase, Δ8 desaturase and Δ4 desaturase. A representative pathway is illustrated in FIG. 2, 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 “desaturase” refers to a polypeptide that can desaturate, i.e., introduce a double bond, in one or more fatty acids to produce a mono- or polyunsaturated fatty acid. Despite use of the omega-reference system throughout the specification in reference 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: Δ12 desaturases that desaturate a fatty acid between the 12th and 13th carbon atoms numbered from the carboxyl-terminal end of the molecule and that catalyze the conversion of oleic acid to LA; Δ15 desaturases that catalyze the conversion of LA to ALA; Δ17 desaturases that catalyze the conversion of ARA to EPA and/or DGLA to ETA; Δ6 desaturases that catalyze the conversion of LA to GLA and/or ALA to STA; Δ5 desaturases that catalyze the conversion of DGLA to ARA and/or ETA to EPA; Δ4 desaturases that catalyze the conversion of DPA to DHA; Δ8 desaturases that catalyze the conversion of eicosadienoic acid (EDA; C20:2) to DGLA and/or eicosatrienoic acid (ETrA; C20:3) to ETA; and Δ9 desaturases that catalyze the conversion of palmitate to palmitoleic acid (16:1) and/or stearate to oleic acid (18:1).
The term “elongase” refers to a polypeptide that can elongate a fatty acid carbon chain to produce an acid that is 2 carbons longer than the fatty acid substrate that the elongase acts upon. This process of elongation occurs in a multi-step mechanism in association with fatty acid synthase, whereby CoA is the acyl carrier (Lassner et al., The Plant Cell 8:281–292 (1996)). Briefly, 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 elongases are the conversion of GLA to DGLA, STA to ETA, and EPA to DPA. In general, the substrate selectivity of elongases is somewhat broad but segregated by both chain length and the degree of unsaturation. Accordingly, elongases can have different specificities. For example, a C16/18 elongase will prefer a C16 substrate, a C18/20 elongase will prefer a C18 substrate and a C20/22 elongase will prefer a C20 substrate. In like manner, a Δ9 elongase is able to catalyze the conversion of LA and ALA to eicosadienoic acid (EDA; C20:2) and eicosatrienoic acid (ETrA; C20:3), respectively.
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 substrate” 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, the terms “isolated nucleic acid fragment” or “isolated nucleic acid molecule” will be used interchangeably and will mean 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 nucleic acid molecule is “hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringent conditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.
Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50–9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7–11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.
A “substantial portion” of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403–410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify 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 instant specification teaches partial or complete amino acid and nucleotide sequences encoding one or more particular yeast proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the instant invention also includes isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing, as well as those substantially similar nucleic acid sequences.
The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is 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 are: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
Suitable nucleic acid fragments (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% identical, preferably at least about 75% identical, and more preferably at least about 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragments encode amino acid sequences that are about 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.
The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403–410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111–20. Suhai, Sandor, Ed. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters that originally load with the software when first initialized.
“Codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting 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.
The term “codon-optimized”, as it refers to genes or coding regions of nucleic acid molecules, refers to modification of codons such that the altered codons reflect the typical codon usage of the host organism without altering the polypeptide for which the DNA codes.
“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 “3′ non-coding sequences” or “transcription terminator” refers to DNA sequences located downstream of a coding sequence. This includes polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The 3′ region can influence the transcription, RNA processing or stability, or translation of the associated coding sequence.
“RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA” or “mRNA” refers to the RNA that is without introns and that can be translated into protein by the cell. “cDNA” refers to a double-stranded DNA that is complementary to, and derived from, mRNA. “Sense” RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell. “Antisense RNA” refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065; 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 fragment(s) of the invention. Expression may also refer to translation of mRNA into a polypeptide.
“Transformation” refers to the transfer of a nucleic acid molecule into a host organism, resulting in genetically stable inheritance. The nucleic acid molecule may be a plasmid that replicates autonomously, for example; or, it may integrate into the genome of the host organism. Host organisms containing the transformed nucleic acid 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. “Transformation cassette” refers to a specific vector containing a foreign gene(s) and having elements in addition to the foreign gene(s) that facilitate transformation of a particular host cell. “Expression cassette” refers to a specific vector containing a foreign gene(s) and having elements in addition to the foreign gene(s) 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.
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).
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 (FIG. 1). When cells have exhausted available nitrogen supplies (e.g., when the carbon to nitrogen ratio is greater than about 40), the depletion of cellular adenosine monophosphate (AMP) leads to the cessation of AMP-dependent isocitrate dehydrogenase activity in the mitochondria and the accumulation of citrate, transport of citrate into the cytosol, and subsequent cleavage of the citrate by ATP-citrate lyase to yield acetyl-CoA and oxaloacetate. Acetyl-CoA is the principle building block for de novo biosynthesis of fatty acids. Although any compound that can effectively be metabolized to produce acetyl-CoA can serve as a precursor of fatty acids, glucose is the primary source of carbon in this type of reaction (FIG. 1). Glucose is converted to pyruvate via glycolysis and pyruvate is then transported into the mitochondria where it can be converted to acetyl-CoA by pyruvate dehydrogenase (“PD”). Since acetyl-CoA can not be transported directly across the mitochondrial membrane into the cytoplasm, the two carbons from acetyl-CoA condense with oxaloacetate to yield citrate (catalyzed by citrate synthase). Citrate is transported directly into the cytoplasm, where it is cleaved by ATP-citrate lyase to regenerate acetyl-CoA and oxaloacetate. The oxaloacetate reenters the tricarboxylic acid cycle, via conversion to malate.
The synthesis of malonyl-CoA is the first committed step of fatty acid biosynthesis, which takes place in the cytoplasm. Malonyl-CoA is produced via carboxylation of acetyl-CoA by acetyl-CoA carboxylase (“ACC”). Fatty acid synthesis is catalyzed by a multi-enzyme fatty acid synthase complex (“FAS”) and occurs by the condensation of eight two-carbon fragments (acetyl groups from acetyl-CoA) to form a 16-carbon saturated fatty acid, palmitate. More specifically, FAS catalyzes a series of 7 reactions, which involve the following (Smith, S. FASEB J, 8(15):1248–59 (1994)). First, acetyl-CoA and malonyl-CoA are transferred to the acyl carrier peptide (ACP) of FAS. The acetyl group is then transferred to the malonyl group, forming β-ketobutyryl-ACP and releasing CO2. Secondly, the β-ketobutyryl-ACP undergoes reduction (via β-ketoacyl reductase) and dehydration (via β-hydroxyacyl dehydratase) to form a trans-monounsaturated fatty acyl group. The third reaction occurs when the double bond is reduced by NADPH, yielding a saturated fatty-acyl group two carbons longer than the initial one. The butyryl-group's ability to condense with a new malonyl group and repeat the elongation process is then regenerated. Finally, when the fatty acyl group becomes 16 carbons long, a thioesterase activity hydrolyses it, releasing free palmitate.
Whereas palmitate synthesis occurs in the cytosol, formation of longer chain saturated and unsaturated fatty acid derivates occur in both the mitochondria and endoplasmic reticulum (ER), wherein the ER is the dominant system. Specifically, palmitate (16:0) is the precursor of stearic (18:0), palmitoleic (16:1) and oleic (18:1) acids through the action of elongases and desaturases. For example, palmitate and stearate are converted to their unsaturated derivatives, palmitoleic (16:1) and oleic (18:1) acids, respectively, by the action of a Δ9 desaturase.
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 a DAG acyltransferase (e.g., PDAT, DGAT1 or DGAT2) to form TAG (FIG. 1).
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 (e.g., DGAT2) 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), linoleic (18:2), eleostearic (18:3), γ-linolenic (18:3), α-linolenic (18:3), stearidonic (18:4), arachidic (20:0), eicosadienoic (20:2), dihomo-γ-linoleic (20:3), eicosatrienoic (20:3), arachidonic (20:4), eicosa-tetraenoic (20:4), eicosa-pentaenoic (20:5), behenic (22:0), docosa-pentaenoic (22:5), docosa-hexaenoic (22:6), lignoceric (24:0), nervonic (24:1), cerotic (26:0), and montanic (28:0) fatty acids. In preferred embodiments of the present invention, incorporation of PUFAs into TAG is most desirable.
Genes Encoding DGAT2
Historically, DGAT1 (responsible for the third acyl transferase reaction, wherein an acyl-CoA group is transferred from acyl-CoA to the sn-3 position of DAG to form TAG) was thought to be the only enzyme specifically involved in TAG synthesis. This enzyme was known to be homologous to acyl-CoA:cholesterol acyltransferases (ACATs); however, recent studies have identified a new family of DAG acyltransferase enzymes that are unrelated to the ACAT gene family. Thus, nomenclature now distinguishes between the DAG acyltransferase 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):38862–28869 (2001)). Members of the DGAT2 family appear to be present in all major phyla of eukaryotes (fungi, plants, animals and basal eukaryotes).
Many genes encoding DGAT2 enzymes have been identified through genetic means and the DNA sequences of some of these genes are publicly available. For example, some non-limiting examples include the following GenBank Accession Numbers: NC—001147 (locus NP—014888; Saccharomyces cerevisiae); NM—012079 (human); NM—127503, AF051849 and AJ238008 (Arabidopsis thaliana); NM—026384, NM—010046 and AB057816 (mouse); AY093657 (pig); AB062762 (rat); AF221132 (Caenorhabditis elegans); AF391089 and AF391090 (Mortierella ramanniana); AF129003 (Nicotiana tabacum); and, AF251794 and AF164434 (Brassica napus). Additionally, the patent literature provides many additional DNA sequences of DGAT2 genes (and/or details concerning several of the genes above and their methods of isolation). See, for example: US 2003/124126 (Cases et al.); WO 2001/034814 (Banas et al.); and US 2003/115632, US2003/0028923 and US 2004/0107459 (Lardizabal et al.). The work of Lardizabal et al. includes DNA sequences of DGAT2s from, e.g., Mortierella ramanniana, Neurospora crassa (GenBank Accession No. CAE76475), Saccharomyces cerevisiae (see also Sorger and Daum, J. Bacteriol. 184(2):519–524 (2002)), Hordeum vulgare, Zea mays, Glycine max, Triticum aestivum, Drosophilia, Homo sapiens, Schizosaccharomyces pombe (see also Zhang et al., J. Biol. Chem. 278(47):47145–47155 (2003)), Candida albicans and Arabidopsis thaliana. Despite disclosure of several complete and incomplete sequences encoding DGAT2 (supra), very few of these sequences have been shown to have DGAT2 activity. The exceptions include the work of: 1.) Bouvier-Nave, P. et al. (Biochem. Soc. Trans. 28(6):692–695 (2000)), wherein the DGAT2 of the nematode worm Caenorhabditis elegans was expressed in Saccharomyces cerevisiae, leading to an increase in TAG content and in microsomal oleyl-CoA:DAG acyltransferase activity; and, 2.) Lardizabal et al. (supra), wherein two DGAT2s of the fungus Mortierella ramanniana and one DGAT2 from the fungus Neurospora crassa were expressed in insect cells, leading to high levels of DGAT activity on membranes isolated from those cells. In addition to these demonstrations where oil biosynthesis was increased by over-expression of DGAT2, disruption of the genes encoding DGAT2 have also been shown to result in a decrease in the cellular TAG content (Oelkers et al., J Biol. Chem. 277(11):8877–81 (2002); Sandager et al., J Biol. Chem. 277:6478–6482 (2002); Sorger and Daum, J. Bacteriol. 184:519–524 (2002)).
Most recently, a single DGAT2 enzyme from the oleaginous yeast Yarrowia lipolytica has been isolated and characterized in co-pending U.S. patent application Ser. No. 10/882,760. Briefly, following cloning of a partial putative DGAT2 DNA fragment from Y. lipolytica, targeted disruption of the endogenous Y. lipolytica gene was carried out to test the identity of the fragment. Lower oil content in the disrupted strain confirmed that the native DGAT2 activity was disrupted. Subsequently, a full-length Y. lipolytica DGAT2 gene (2119 bp; SEQ ID NO:3) was assembled, which included three nested open reading frames [ORF 1: nucleotides +291 to +1835 of SEQ ID NO:3, corresponding to a deduced encoded amino acid sequence of 514 residues (SEQ ID NO:4); ORF 2: nucleotides +456 to +1835 of SEQ ID NO:3 (i.e., SEQ ID NO:5), corresponding to a deduced encoded amino acid sequence of 459 residues (SEQ ID NO:6); and ORF 3: nucleotides +768 to +1835 of SEQ ID NO:3 (i.e., SEQ ID NO:7), corresponding to a deduced encoded amino acid sequence of 355 residues (SEQ ID NO:8)].
Biosynthesis of Omega-3 and Omega-6 Polyunsaturated Fatty Acids
The metabolic process that converts LA to GLA, DGLA and ARA (the ω-6 pathway) and ALA to STA, ETA, EPA, DPA and DHA (the ω-3 pathway) involves elongation of the carbon chain through the addition of two-carbon units and desaturation of the molecule through the addition of double bonds (FIG. 2). This requires a series of desaturation and elongation enzymes. Specifically, oleic acid is converted to LA (18:2), the first of the ω-6 fatty acids, by the action of a Δ12 desaturase. Subsequent ω-6 fatty acids are produced 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 an elongase; and 3.) DGLA is converted to ARA by the action of a Δ5 desaturase. In like manner, linoleic acid (LA) is converted to ALA, the first of the ω-3 fatty acids, by the action of a Δ15 desaturase. Subsequent ω-3 fatty acids are produced in a series of steps similar to that for the ω-6 fatty acids. Specifically, 1.) ALA is converted to STA by the activity of a Δ6 desaturase; 2.) STA is converted to ETA by the activity of an elongase; and 3.) ETA is converted to EPA by the activity of a Δ5 desaturase. Alternatively, ETA and EPA can be produced from DGLA and ARA, respectively, by the activity of a Δ17 desaturase. EPA can be further converted to DHA by the activity of an elongase and a Δ4 desaturase.
In alternate embodiments, a Δ9 elongase is able to catalyze the conversion of LA and ALA to eicosadienoic acid (EDA; C20:2) and eicosatrienoic acid (ETrA; C20:3), respectively. A Δ8 desaturase then converts these products to DGLA and ETA, respectively.
Many microorganisms, including algae, bacteria, molds, fungi and yeast, can synthesize PUFAs and omega fatty acids in the ordinary course of cellular metabolism. Particularly well-studied are fungi including Schizochytrium aggregatm, species of the genus Thraustochytrium and Mortierella alpina. Additionally, many dinoflagellates (Dinophyceaae) naturally produce high concentrations of PUFAs. As such, a variety of desaturase and elongase genes involved in PUFA production have been identified through genetic means and the DNA sequences of some of these genes are publicly available. For instance, the GenBank Accession Numbers of some non-limiting examples are: AY131238, Y055118, AY055117, AF296076, AF007561, L11421, NM—031344, AF465283, AF465281, AF110510, AF465282, AF419296, AB052086, AJ250735, AF126799, AF126798 (Δ6 desaturases); AF199596, AF226273, AF320509, AB072976, AF489588, AJ510244, AF419297, AF07879, AF067654, AB022097 (Δ5 desaturases); AF489589.1, AY332747 (Δ4 fatty acid desaturases); AAG36933, AF110509, AB020033, AAL13300, AF417244, AF161219, AY332747, AAG36933, AF110509, AB020033, AAL13300, AF417244, AF161219, X86736, AF240777, AB007640, AB075526, AP002063 (Δ12 desaturases); NP—441622, BAA18302, BAA02924, AAL36934 (Δ15 desaturases); AF338466, AF438199, E11368, E11367, D83185, U90417, AF085500, AY504633, NM—069854, AF230693 (Δ9 desaturases); and AX464731, NM—119617, NM—134255, NM—134383, NM—134382, NM—068396, NM—068392, NM—070713, NM—068746, NM—064685 (elongases).
Additionally, 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. See, for example: 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 91/13972 and U.S. Pat. No. 5,057,419 (Δ9 desaturases); WO 93/11245 (Δ15 desaturases); WO 94/11516, U.S. Pat. No. 5,443,974 and WO 03/099216 (Δ12 desaturases); WO 00/12720 and U.S. 2002/0139974A1 (elongases); U.S. 2003/0196217 A1 (Δ17 desaturase); WO 00/34439 (Δ8 desaturases); and, WO 02/090493 (Δ4 desaturases). Each of these patents and applications are herein incorporated by reference in their entirety.
Depending upon the host cell, the availability of substrate, and the desired end product(s), several desaturases and elongases are of interest for use in production of PUFAs. 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 are therefore 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 fatty acid substrate.
Sequence Identification of Mortierella alpina DGAT2 Acyltransferase
Despite the availability of several genes encoding DGAT2 (supra) that could be used for heterologous expression in oleaginous yeast (e.g., Yarrowia lipolytica), only three DGAT2 genes have been isolated and characterized from oleaginous organisms (e.g., from Mortierella ramanniana and Yarrowia lipolytica). And, none of the three DGAT2 genes from oleaginous organisms are expected to favor longer chain PUFAs (i.e., those PUFAs having a chain length equal to or greater than C20). In the present invention, a DGAT2 gene has been isolated from Mortierella alpina. M. alpina is an organism that naturally accumulates fatty acids having chain-lengths equal to or greater than C20 in its TAG fraction, thus indicating that the DGAT2 is likely to have the desired substrate specificity.
Comparison of the DGAT2 nucleotide base and deduced amino acid sequences to public databases, using a BLAST algorithm (Altschul, S. F., et al., Nucleic Acids Res. 25:3389–3402 (1997)), reveals that the most similar known sequences are about 47% identical to the amino acid sequence of DGAT2 reported herein over a length of 331 amino acids. Preferred amino acid fragments are at least about 70%–80% identical to the sequences herein, where those sequences that are 85%–90% identical are particularly suitable and those sequences that are about 95% identical are most preferred. Similarly, preferred DGAT2 encoding nucleic acid sequences corresponding to the instant ORF are those encoding active proteins and which are at least about 70%–80% identical to the nucleic acid sequences encoding DGAT2 reported herein, where those sequences that are 85%–90% identical are particularly suitable and those sequences that are about 95% identical are most preferred.
Isolation of Homologs
DGAT2 nucleic acid fragments of the instant invention may be used to isolate genes encoding homologous proteins from the same or other microbial species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to: 1.) methods of nucleic acid hybridization; 2.) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and 3.) methods of library construction and screening by complementation.
For example, genes encoding similar proteins or polypeptides to the DGAT2 described herein could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired yeast or fungus using methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan (e.g., random primers DNA labeling, nick translation or end-labeling techniques), or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of (or full-length of) the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length DNA fragments under conditions of appropriate stringency.
Typically, in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PCR primer design are common and well known in the art (Thein and Wallace, “The use of oligonucleotides as specific hybridization probes in the Diagnosis of Genetic Disorders”, in Human Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33–50, IRL: Herndon, V A; and Rychlik, W., In Methods in Molecular Biology, White, B. A. Ed., (1993) Vol. 15, pp 31–39, PCR Protocols: Current Methods and Applications. Humania: Totowa, N.J.).
Generally two short segments of the instant sequences may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding microbial genes.
Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (BRL, Gaithersburg, Md.), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217 (1989)).
Alternatively, the instant DGAT2 sequences may be employed as hybridization reagents for the identification of homologs. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the gene or gene fragment of interest, and a specific hybridization method. Probes of the present invention are typically single-stranded nucleic acid sequences that are complementary to the nucleic acid sequences to be detected. Probes are “hybridizable” to the nucleic acid sequence to be detected. The probe length can vary from 5 bases to tens of thousands of bases, and will depend upon the specific test to be done. Typically a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.
Hybridization methods are well defined. Typically the probe and sample must be mixed under conditions that will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed. Optionally, a chaotropic agent may be added. The chaotropic agent stabilizes nucleic acids by inhibiting nuclease activity. Furthermore, the chaotropic agent allows sensitive and stringent hybridization of short oligonucleotide probes at room temperature (Van Ness and Chen, Nucl. Acids Res. 19:5143–5151 (1991)). Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide and cesium trifluoroacetate, among others. Typically, the chaotropic agent will be present at a final concentration of about 3 M. If desired, one can add formamide to the hybridization mixture, typically 30–50% (v/v).
Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent. A common hybridization solution employs about 30–50% v/v formamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6–9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), or between 0.5–20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300–500 kdal), polyvinylpyrrolidone (about 250–500 kdal) and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to 2% wt/vol glycine. Other additives may also be included, such as volume exclusion agents that include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate) and anionic saccharidic polymers (e.g., dextran sulfate).
Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions. A primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the sequence.
Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of DNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can then be used to screen DNA-expression libraries to isolate full-length DNA clones of interest (Lerner, R. A. Adv. Immunol. 36:1 (1984); Maniatis, supra).
Gene Optimization for Improved Heterologous Expression
It may be desirable to modify the expression of the instant DGAT2 and/or PUFA biosynthetic pathway enzymes to achieve optimal conversion efficiency of each, according to the specific TAG composition of interest. As such, a variety of techniques can be utilized to improve/optimize the expression of a polypeptide of interest in an alternative host. Two such techniques include codon-optimization and mutagenesis of the gene.
Codon Optimization
As will be appreciated by one skilled in the art, 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. 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 codons are used with highest frequency. Thus, the coding sequence for a polypeptide having acyltransferase activity 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. All (or portions) of the DNA also can be synthesized to alter the base composition to one more preferable in the desired host cell.
Thus, for example, it may be desirable to modify a portion of the codons encoding the polypeptide having DGAT2 activity, to enhance the expression of the gene in Yarrowia lipolytica. The codon usage profile and the consensus sequence around the ‘ATG’ translation initiation codon for this particular organism are taught in co-pending U.S. patent application Ser. No. 10/840,478 (herein incorporated entirely by reference); likewise, a method for rapid synthesis of genes optimized for expression in Yarrowia lipolytica is also provided.
Mutagenesis
Methods for synthesizing sequences and bringing sequences together are well established in the literature. For example, in vitro mutagenesis and selection, site-directed mutagenesis, error prone PCR (Melnikov et al., Nucleic Acids Research, 27(4):1056–1062 (Feb. 15, 1999)), “gene shuffling” or other means can be employed to obtain mutations of naturally occurring DGAT2 genes. This would permit production of a polypeptide having acyltransferase activity in vivo with more desirable physical and kinetic parameters for function in the host cell (e.g., a longer half-life or a higher rate of synthesis of TAGs from fatty acids).
If desired, the regions of a DGAT2 polypeptide important for enzymatic activity can be determined through routine mutagenesis, expression of the resulting mutant polypeptides and determination of their activities. Mutants may include deletions, insertions and point mutations, or combinations thereof. A typical functional analysis begins with deletion mutagenesis to determine the N- and C-terminal limits of the protein necessary for function, and then internal deletions, insertions or point mutants are made to further determine regions necessary for function. Other techniques such as cassette mutagenesis or total synthesis also can be used. Deletion mutagenesis is accomplished, for example, by using exonucleases to sequentially remove the 5′ or 3′ coding regions. Kits are available for such techniques. After deletion, the coding region is completed by ligating oligonucleotides containing start or stop codons to the deleted coding region after the 5′ or 3′ deletion, respectively. Alternatively, oligonucleotides encoding start or stop codons are inserted into the coding region by a variety of methods including site-directed mutagenesis, mutagenic PCR or by ligation onto DNA digested at existing restriction sites. Internal deletions can similarly be made through a variety of methods including the use of existing restriction sites in the DNA, by use of mutagenic primers via site-directed mutagenesis or mutagenic PCR. Insertions are made through methods such as linker-scanning mutagenesis, site-directed mutagenesis or mutagenic PCR. Point mutations are made through techniques such as site-directed mutagenesis or mutagenic PCR.
Chemical mutagenesis also can be used for identifying regions of a DGAT2 polypeptide important for activity. A mutated construct is expressed, and the ability of the resulting altered protein to function as an acyltransferase is assayed. Such structure-function analysis can determine which regions may be deleted, which regions tolerate insertions, and which point mutations allow the mutant protein to function in substantially the same way as the native acyltransferase.
All such mutant proteins and nucleotide sequences encoding them that are derived from the DGAT2 described herein are within the scope of the present invention.
Microbial Production of Fatty Acids and Triacylglycerols
Microbial production of fatty acids and TAGs has several advantages over purification from natural sources such as fish or plants. For example: (1) many microbes are known with greatly simplified oil compositions compared with those of higher organisms, making purification of desired components easier; (2) microbial production is not subject to fluctuations caused by external variables, such as weather and food supply; (3) microbially produced oil is substantially free of contamination by environmental pollutants; and, (4) microbial oil production can be manipulated by controlling culture conditions, notably by providing particular substrates for microbially expressed enzymes, or by addition of compounds or genetic engineering approaches to suppress undesired biochemical pathways. With respect to the production of ω-3 and/or ω-6 fatty acids in particular, and TAGs containing those PUFAs, additional advantages are incurred since microbes can provide fatty acids in particular forms that may have specific uses; and, recombinant microbes provide the ability to alter the naturally occurring microbial fatty acid profile by providing new biosynthetic pathways in the host or by suppressing undesired pathways, thereby increasing levels of desired PUFAs, or conjugated forms thereof, and decreasing levels of undesired PUFAs.
Thus, knowledge of the sequence of the present DGAT2 will be useful for manipulating fatty acid biosynthesis and accumulation in oleaginous yeast, and particularly, in Yarrowia lipolytica. This may require metabolic engineering directly within the fatty acid or TAG biosynthetic pathways or additional manipulation of pathways that contribute carbon to the fatty acid biosynthetic pathway. Methods useful for manipulating biochemical pathways are well known to those skilled in the art.
Metabolic Engineering to Up-Regulate Genes and Biosynthetic Pathways Affecting Fatty Acid Synthesis and Oil Accumulation in Oleaginous Yeast
It is expected that introduction of chimeric genes encoding the DGAT2 described herein, under the control of the appropriate promoters, will result in increased transfer of fatty acids to storage TAGs. As such, the present invention encompasses a method for increasing the TAG content in an oleaginous yeast comprising expressing the DGAT2 enzyme of the present invention in a transformed oleaginous yeast host cell producing a fatty acid, such that the fatty acid is transferred to the TAG pool.
Additional copies of DGAT2 genes may be introduced into the host to increase the transfer of fatty acids to the TAG fraction. Expression of the genes also 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). Yet another approach to increase expression of heterologous genes is to increase the translational efficiency of the encoded mRNAs by replacement of codons in the native gene with those for optimal gene expression in the selected host microorganism.
In one specific embodiment, the present invention encompasses a method of increasing the ω-3 and ω-6 fatty acid content of TAGs in an oleaginous yeast, since it is possible to introduce an expression cassette encoding each of the enzymes necessary for ω-3 and ω-6 fatty acid biosynthesis into the organism (since naturally produced PUFAs in these organisms are limited to 18:2 (i.e., LA), and less commonly 18:3 (i.e., ALA) fatty acids). Thus, the method comprises:

    
    
        a) providing a transformed oleaginous yeast host cell possessing at least one gene encoding at least one enzyme of the ω-3/ω-6 fatty acid biosynthetic pathway and the DGAT2 enzyme of the present invention;
        b) growing the yeast cells of step (a) in the presence of a fermentable carbon substrate, whereby the gene(s) of the ω-3/ω-6 fatty acid biosynthetic pathway and the DGAT2 are expressed, whereby a ω-3 and/or ω-6 fatty acid is produced, and whereby the ω-3 and/or ω-6 fatty acid is transferred to TAGs.
    
    


A variety of PUFA products can be produced (prior to their transfer to TAGs), depending on the fatty acid substrate and the particular genes of the ω-3/ω-6 fatty acid biosynthetic pathway that are transformed into the host cell. As such, production of the desired fatty acid product can occur directly (wherein the fatty acid substrate is converted directly into the desired fatty acid product without any intermediate steps or pathway intermediates) or indirectly (wherein multiple genes encoding the PUFA biosynthetic pathway may be used in combination, such that a series of reactions occur to produce a desired PUFA). Specifically, for example, it may be desirable to transform an oleaginous yeast with an expression cassette comprising a Δ12 desaturase, Δ6 desaturase, an elongase, a Δ5 desaturase and a Δ17 desaturase for the overproduction of EPA. As is well known to one skilled in the art, various other combinations of the following enzymatic activities may be useful to express in a host in conjunction with the DGAT2 described herein: a Δ15 desaturase, a Δ4 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ17 desaturase, a Δ9 desaturase, a Δ8 desaturase and/or an elongase(s) (see FIG. 2). The particular genes included within a particular expression cassette will depend on the host cell (and its PUFA profile and/or desaturase profile), the availability of substrate and the desired end product(s).
Thus, within the context of the present invention, it may be useful to modulate the expression of the TAG biosynthetic pathway by any one of the methods described above. For example, the present invention provides a gene encoding a key enzyme in the fatty acid biosynthetic pathway leading to the storage of TAGs. This gene encodes the DGAT2 enzyme. It will be particularly useful to express this gene in oleaginous yeast to maximize production and accumulation of TAGs using various means for metabolic engineering of the host organism. In preferred embodiments, modification of the expression levels of this gene in combination with expression of ω-3/ω-6 biosynthetic genes can be utilized to maximize production and accumulation of preferred PUFAs in the TAG pool.
Metabolic Engineering to Down-Regulate Undesirable Genes and Biosynthetic Pathways Affecting Fatty Acid Synthesis and Oil Accumulation in Oleaginous Yeast
In some embodiments, it may be useful to disrupt or inactivate a host organism's native DGAT2, based on the complete sequences described herein, the complement of those complete sequences, substantial portions of those sequences, codon-optimized acyltransferases derived therefrom, and those sequences that are substantially homologous thereto. In an alternate embodiment, a transformant host organism comprising a disruption or inactivation of its native DGAT2 may then be advantageously transformed to express a heterologous DGAT2 (e.g., if the heterologous DGAT2 has different substrate specificity than the native DGAT2).
For gene disruption, a foreign DNA fragment (typically a selectable marker gene) 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. For example, cells may be exposed to UV radiation and then screened for the desired phenotype. Mutagenesis with chemical agents is also effective for generating mutants and commonly used substances include chemicals that affect nonreplicating DNA (e.g., HNO2 and NH2OH), as well as agents that affect replicating DNA (e.g., acridine dyes, notable for causing frameshift mutations). Specific methods for creating mutants using radiation or chemical agents are well documented in the art. See, for example: Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd ed. (1989) Sinauer Associates: Sunderland, Mass.; or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227 (1992).
Another non-specific method of gene disruption is the use of transposable elements or transposons. Transposons are genetic elements that insert randomly into DNA but can be later retrieved on the basis of sequence to determine where the insertion has occurred. Both in vivo and in vitro transposition methods are known. Both methods involve the use of a transposable element in combination with a transposase enzyme. When the transposable element or transposon is contacted with a nucleic acid fragment in the presence of the transposase, the transposable element will randomly insert into the nucleic acid fragment. The technique is useful for random mutagenesis and for gene isolation, since the disrupted gene may be identified on the basis of the sequence of the transposable element. Kits for in vitro transposition are commercially available [see, for example: 1.) The Primer Island Transposition Kit, available from Perkin Elmer Applied Biosystems, Branchburg, N.J., based upon the yeast Ty1 element; 2.) The Genome Priming System, available from New England Biolabs, Beverly, Mass., based upon the bacterial transposon Tn7; and 3.) the EZ::TN Transposon Insertion Systems, available from Epicentre Technologies, Madison, Wis., based upon the Tn5 bacterial transposable element].
As described in U.S. Patent Application No. 60/624,812, the Applicants have discovered that expression of heterologous acyltransferases in conjunction with knockouts of the corresponding native Yarrowia lipolytica acyltransferase can significantly increase the overall long-chain w-3 PUFAs that are produced in transformant Yarrowia lipolytica host cells engineered for PUFA biosynthesis. This manipulation is thought to reduce substrate competition between the native and heterologous acyltransferase; and, when the heterologous acyltransferase has specificity for those fatty acids that are 18:3 and greater (in comparison to the native enzymes that may not efficiently catalyze reactions with longer-chain fatty acids since naturally produced PUFAs in Yarrowia lipolytica are limited to 18:2 fatty acids) likely enables more efficient acyltransferase reactions to occur within the transformant host. Thus, within the context of the present invention, it may be useful to disrupt or inactivate a host organism's native DGAT2 (e.g., the Yarrowia lipolytica DGAT2 encoded by SEQ ID NO:4, 6 or 8) that does not have specificity for long-chain PUFAs (e.g., 20:0, 22:0) or that has difficulty efficiently synthesizing TAGs comprising fatty acids that are 18:3 and greater in length (e.g., EPA). Then, the heterologous (or “foreign”) DGAT2 of the present invention (i.e. SEQ ID NO:2) could be expressed to enable increased accumulation of long chain PUFAs in the organism's TAG fraction, since substrate competition between the native and heterologous acyltransferase would be reduced. One skilled in the art would readily be able to apply the teachings herein toward the advantageous manipulation of DGAT2 acyltransferases in other oleaginous organisms.
In conjunction with this approach, or alternatively, it may be necessary to disrupt genes and pathways that diminish the existing fatty acid pool and/or that hydrolyze TAGs to regulate (and/or maximize) TAG accumulation.
Expression Systems, Cassettes and Vectors
The gene and gene product of the instant sequences described herein may be produced in microbial host cells, particularly in the cells of oleaginous yeast (e.g., Yarrowia lipolytica). Expression in recombinant microbial hosts may be useful for the transfer of various fatty acids to TAGs.
Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes for production of the gene products of the instant DGAT2 sequences. These chimeric genes could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded enzymes.
Vectors or DNA cassettes useful for the transformation of suitable host cells are well known in the art. The specific choice of sequences present in the construct is dependent upon the desired expression products (supra), 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 and a region 3′ of the DNA fragment that controls transcriptional termination. 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.
Initiation control regions or promoters which are useful to drive expression of the instant ORFs 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 the selected host cell is suitable for the present invention. Expression in a 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. 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 (see U.S. patent application Ser. No. 10/869,630, incorporated herein by reference), phosphoglycerate mutase (see U.S. patent application Ser. No. 10/869,630, incorporated herein by reference), fructose-bisphosphate aldolase (see U.S. patent application Ser. No. 10/987,548, incorporated herein by reference), phosphoglucose-isomerase, phosphoglycerate kinase, glycerol-3-phosphate O-acyltransferase (see U.S. Patent Application No. 60/610,060), 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), 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.
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, 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 (see, e.g., U.S. patent application Ser. No. 10/840,478 for specific teachings applicable for Yarrowia lipolytica).
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.
As one of skill in the art is aware, merely inserting a gene into a cloning vector does not ensure that it will be successfully expressed at the level needed. In response to the need for a high expression rate, many specialized expression vectors have been created by manipulating a number of different genetic elements that control aspects of transcription, translation, protein stability, oxygen limitation and secretion from the host cell. More specifically, some of the molecular features that have been manipulated to control gene expression include: 1.) the nature of the relevant transcriptional promoter and terminator sequences; 2.) the number of copies of the cloned gene and whether the gene is plasmid-borne or integrated into the genome of the host cell; 3.) the final cellular location of the synthesized foreign protein; 4.) the efficiency of translation in the host organism; 5.) the intrinsic stability of the cloned gene protein within the host cell; and 6.) the codon usage within the cloned gene, such that its frequency approaches the frequency of preferred codon usage of the host cell. Each of these types of modifications are encompassed in the present invention, as means to further optimize expression of the DGAT2 enzyme described herein.
Preferred Microbial Hosts for Recombinant Expression of DGAT2
Host cells for expression of the instant DGAT2 gene and nucleic acid fragments may include microbial hosts that grow on a variety of feedstocks, including simple or complex carbohydrates, organic acids and alcohols and/or hydrocarbons over a wide range of temperature and pH values. Although the gene described in the instant invention has been isolated for expression in an oleaginous yeast, and in particular Yarrowia lipolytica, it is contemplated that because transcription, translation and the protein biosynthetic apparatus is highly conserved, any bacteria, yeast, algae and/or filamentous fungus will be a suitable host for expression of the present nucleic acid fragments.
Preferred microbial hosts are oleaginous organisms, such as oleaginous yeast. These oleaginous organisms are naturally capable of oil synthesis and accumulation, wherein the total oil content 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. Additionally, there is basis for the use of these organisms for the production of PUFA's, as seen in co-pending U.S. patent applications Ser. No. 10/840,579 and No. 60/624,812, each incorporated entirely by reference herein.
Genera typically identified as oleaginous yeast include, but are not limited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. More specifically, illustrative oil-synthesizing yeast include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R. graminis, and Yarrowia lipolytica (formerly classified as Candida lipolytica).
Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in a further embodiment, most preferred are the Y. lipolytica strains designated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982, ATCC #90812 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol. 82(1):43–9 (2002)).
Transformation of Microbial Hosts
Once the DNA encoding a polypeptide suitable for expression in an oleaginous yeast has been obtained, 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 within the host locus. Where constructs are targeted to an endogenous locus, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus.
Where two or more genes are expressed from separate replicating vectors, it is desirable that each vector has a different means of selection and should lack homology to the other construct(s) to maintain stable expression and prevent reassortment of elements among constructs. Judicious choice of regulatory regions, selection means and method of propagation of the introduced construct(s) can be experimentally determined so that all introduced genes are expressed at the necessary levels to provide for synthesis of the desired products.
Constructs comprising the gene 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, biolistic impact, electroporation, microinjection, or any other method that introduces the gene of interest into the host cell. More specific teachings applicable for oleaginous yeast (i.e., Yarrowia lipolytica) include U.S. Pat. Nos. 4,880,741 and 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 selection for a marker contained on the introduced construct. Alternatively, a separate marker construct may be co-transformed with the desired construct, as many transformation techniques introduce many DNA molecules into host cells. Typically, transformed hosts are selected for their ability to grow on selective media. Selective media may incorporate an antibiotic or lack a factor necessary for growth of the untransformed host, such as a nutrient or growth factor. An introduced marker gene may confer antibiotic resistance, or encode an essential growth factor or enzyme, thereby permitting growth on selective media when expressed in the transformed host. Selection of a transformed host can also occur when the expressed marker protein can be detected, either directly or indirectly. The marker protein may be expressed alone or as a fusion to another protein. The marker protein can be detected by: 1.) its enzymatic activity (e.g., β-galactosidase can convert the substrate X-gal [5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside] to a colored product; luciferase can convert luciferin to a light-emitting product); or 2.) its light-producing or modifying characteristics (e.g., the green fluorescent protein of Aequorea victoria fluoresces when illuminated with blue light). Alternatively, antibodies can be used to detect the marker protein or a molecular tag on, for example, a protein of interest. Cells expressing the marker protein or tag can be selected, for example, visually, or by techniques such as FACS or panning using antibodies. For selection of yeast transformants, any marker that functions in yeast may be used. Desirably, resistance to kanamycin, hygromycin and the amino glycoside G418 are of interest, as well as ability to grow on media lacking uracil or leucine.
Following transformation, substrates suitable for the gene products of the instant sequence (and optionally other PUFA enzymes that are expressed within the host cell), may be produced by the host either naturally or transgenically, or they may be provided exogenously.
Fermentation Processes for Triacylglycerol Biosynthesis and Accumulation
The transformed microbial host cell is grown under conditions that optimize activity of fatty acid biosynthetic genes and acyltransferase genes. This leads to production of the greatest and the most economical yield of fatty acids, which can in turn be transferred to TAGs for storage. 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. Microorganisms of interest, such as oleaginous yeast, are 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 substrate may include one-carbon substrates (e.g., carbon dioxide, methanol, formaldehyde, formate, 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 substrates and will only be limited by the choice of the host organism. Although all of the above mentioned carbon substrates and mixtures thereof are expected to be suitable in the present invention, preferred carbon substrates 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 source (e.g., urea, glutamate). In addition to appropriate carbon and nitrogen sources, the fermentation media must also contain suitable minerals, salts, cofactors, buffers, vitamins and other components known to those skilled in the art suitable for the growth of the microorganism and promotion of the enzymatic pathways necessary for fatty acid 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 the particular microorganism will be known by one skilled in the art of microbiology or fermentation science. A suitable pH range for the fermentation is typically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.0 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 fatty acids and TAGs 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 oils in oleaginous yeast. In this approach, the first stage of the fermentation is dedicated to the generation and accumulation of cell mass and is characterized by rapid cell growth and cell division. In the second stage of the fermentation, it is preferable to establish conditions of nitrogen deprivation in the culture to promote high levels of lipid accumulation. The effect of this nitrogen deprivation is to reduce the effective concentration of AMP in the cells, thereby reducing the activity of the NAD-dependent isocitrate dehydrogenase of mitochondria. When this occurs, citric acid will accumulate, thus forming abundant pools of acetyl-CoA in the cytoplasm and priming fatty acid synthesis. Thus, this phase is characterized by the cessation of cell division followed by the synthesis of fatty acids and accumulation of TAGs.
Although cells are typically grown at about 30° C., some studies have shown increased synthesis of unsaturated fatty acids at lower temperatures (Yongmanitchai and Ward, Appl. Environ. Microbiol. 57:419–25 (1991)). Based on process economics, this temperature shift should likely occur after the first phase of the two-stage fermentation, when the bulk of the organisms' growth has occurred.
It is contemplated that a variety of fermentation process designs may be applied, where commercial production of fatty acids and TAGs using the instant DGAT2 is desired. For example, commercial production of TAGs containing PUFAs from a recombinant microbial host may be produced by a batch, fed-batch or continuous fermentation process.
A batch fermentation process is a closed system wherein the media composition is set at the beginning of the process and not subject to further additions beyond those required for maintenance of pH and oxygen level during the process. Thus, at the beginning of the culturing process the media is inoculated with the desired organism and growth or metabolic activity is permitted to occur without adding additional substrates (i.e., carbon and nitrogen sources) to the medium. In batch processes the metabolite and biomass compositions of the system change constantly up to the time the culture is terminated. In a typical batch process, cells moderate through a static lag phase to a high-growth log phase and finally to a stationary phase, wherein the growth rate is diminished or halted. Left untreated, cells in the stationary phase will eventually die. A variation of the standard batch process is the fed-batch process, wherein the substrate is continually added to the fermentor over the course of the fermentation process. A fed-batch process is also suitable in the present invention. Fed-batch processes are useful when catabolite repression is apt to inhibit the metabolism of the cells or where it is desirable to have limited amounts of substrate in the media at any one time. Measurement of the substrate concentration in fed-batch systems is difficult and therefore may be estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases (e.g., CO2). Batch and fed-batch culturing methods are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd ed., (1989) Sinauer Associates: Sunderland, Mass.; or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227 (1992), herein incorporated by reference.
Commercial production of fatty acids using the instant DGAT2 may also be accomplished by a continuous fermentation process wherein a defined media is continuously added to a bioreactor while an equal amount of culture volume is removed simultaneously for product recovery. Continuous cultures generally maintain the cells in the log phase of growth at a constant cell density. Continuous or semi-continuous culture methods permit the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one approach may limit the carbon source and allow all other parameters to moderate metabolism. In other systems, a number of factors affecting growth may be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth and thus the cell growth rate must be balanced against cell loss due to media being drawn off the culture. Methods of modulating nutrients and growth factors for continuous culture processes, as well as techniques for maximizing the rate of product formation, are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.
Purification of Fatty Acids
Fatty acids, including PUFAs, 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 fatty acids (including 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. Of particular interest is extraction with methanol and chloroform in the presence of water (E. G. Bligh & W. J. Dyer, Can. J. Biochem. Physiol. 37:911–917 (1959)). Where desirable, the aqueous layer can be acidified to protonate negatively-charged moieties and thereby increase partitioning of desired products into the organic layer. After extraction, the organic solvents can be removed by evaporation under a stream of nitrogen. When isolated in conjugated forms, the products may be enzymatically or chemically cleaved to release the free fatty acid or a less complex conjugate of interest, and can then be subject to further manipulations to produce a desired end product. Desirably, conjugated forms of fatty acids are cleaved with potassium hydroxide.
If further purification is necessary, standard methods can be employed. Such methods may include extraction, treatment with urea, fractional crystallization, HPLC, fractional distillation, silica gel chromatography, high-speed centrifugation or distillation, or combinations of these techniques. Protection of reactive groups, such as the acid or alkenyl groups, may be done at any step through known techniques (e.g., alkylation, iodination). Methods used include methylation of the fatty acids to produce methyl esters. Similarly, protecting groups may be removed at any step. Desirably, purification of fractions containing GLA, STA, ARA, DHA and EPA may be accomplished by treatment with urea and/or fractional distillation.
DESCRIPTION OF PREFERRED EMBODIMENTS
The ultimate goal of the work described herein is the development of an oleaginous yeast that accumulates TAGs enriched in ω-3 and/or ω-6 PUFAs. Toward this end, acyltransferases must be identified that function efficiently in oleaginous yeast to enable synthesis and high accumulation of preferred TAGs in these hosts. Specifically, modification of the expression levels of these acyltransferases will enable increased transfer of fatty acids (and particularly, PUFAs having chain-lengths equal to or greater than C20) to TAGs. Thus, identification of efficient acyltransferases is necessary for the manipulation of the amount of ω-3/ω-6 PUFAs incorporated into the TAG fraction produced in transformant host cells.
In the present invention, Applicants have isolated and cloned the gene from Mortierella alpina that encodes DGAT2. Based on the ability of the native organism to synthesize ARA at concentrations greater than 50% of the total fatty acids (TFAs), it was expected that the DGAT2 would have excellent efficient synthesizing TAGs comprising long chain fatty acids. Confirmation of this gene's activity was provided based upon sequence homology with DGAT2 from other species.
The Applicants hypothesized that the M. alpina acyltransferase gene encoding DGAT2 would be useful for expression in various microbial hosts, and particularly for over-expression in oleaginous yeast whose native DGAT2 may not have the substrate specificity necessary to enable efficient incorporation of PUFAs having chain-lengths equal to or greater than C20 into the TAG fraction. To test this, the M. alpina DGAT2 was over-expressed in an engineered strain of Yarrowia lipolytica producing about 14% EPA. Transformant strains possessed increased oil content (total fatty acids as a % of dry cell weight) relative to the parental strains. Additional benefits may result, since expression of the DGAT2 of the instant invention can also be put under the control of strong constitutive or regulated promoters that do not have the regulatory constraints of the native gene.
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 TOP10 cells and E. coli Electromax DH10B cells were obtained from Invitrogen (Carlsbad, Calif.). Max Efficiency competent cells of E. coli DH5α were obtained from GIBCO/BRL (Gaithersburg, Md.). 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.). 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 (DNASTAR, Inc., (Madison, Wis.).
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).
Transformation of Yarrowia 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, 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 leucine and/or uracil were added as appropriate to a final concentration of 0.01% (thereby producing “MMLe” 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, Detroit, Mich.) 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).
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
Preparation of Mortierella alpina Genomic DNA and cDNA
The present Example describes the preparation of genomic DNA and cDNA from Mortierella alpina (ATCC #16266).
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% Bactor-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 (Bartlesville, Okla.) 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 a 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 Pharmacia's kit protocol. 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:9) and 1 μl of CDSIII/3′ PCR primer (SEQ ID NO:10). 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:11), 2 μl CDSIII/3′-PCR primer (SEQ ID NO:10), 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 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.
Example 2
Cloning of a Partial Putative DGAT2 Sequence from Mortierella alpina By PCR Using Degenerate PCR Primers 
PCR amplifications were performed using either M. alpina genomic DNA or cDNA as template and several sets of degenerate primers (see Table 4 below) designed to encode conserved amino acid sequences among different known DGAT2s (i.e., GenBank Accession NOs. NC—001147 [Saccharomyces cerevisiae] and AF391089 and AF391090 [Mortierella ramanniana]). The best results were obtained with degenerate primers MDGAT-FN1 and MDGAT-RN1.








TABLE 4







Degenerate Primers Used For



Amplification Of A Partial Putative DGAT2













Degenerate
Corresponding



Primer

Nucleotide
Amino Acid


Set
Description
Sequence
Sequence





MDGAT-
(16) 20-mers
5′-GAACTACATCTTY
NYIFGYH



FN1

GGNTAYCA-3′
(SEQ ID NO:13)




(SEQ ID NO:12)


 


MDGAT-
(32) 20-mers
5′-TACAGCTCRTTYT
Complement of


RN1

CNCCRAA-3′
FGENELY




(SEQ ID NO:14)
(SEQ ID NO:15)


 


MDGAT-
(32) 20-mers
5′-CCAAAGTCRTART
Complement of


RN2

TRAANAC-3′
VFNYDFG




(SEQ ID NO:16)
(SEQ ID NO:17)





[Note: Abbreviations are standard for nucleotides and proteins. The nucleic acid degeneracy code used is as follows: Y = C/T; R = A/G, and N = A/C/G/T.]






The PCR 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.
Using cDNA as template, and MDGAT-FN1 and MDGAT-RN1 as primers, a fragment with a size of ca. 370 bp was obtained (SEQ ID NO:18). 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 DGAT2s, based on BLAST program analysis (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403–410 (1993)).
Using genomic DNA as template, and MDGAT-FN1 and MDGAT-RN2 as primers, a fragment of ca 670 bp was obtained. The fragment was purified, cloned into pCR2.1-TOPO and sequenced, as above. 654 bp of sequence was obtained (SEQ ID NO:19). Again, when translated, the sequence had homology to known DGAT2s, based on the BLAST program analysis. Comparison of the genomic sequence and the cDNA sequence revealed the presence of an intron of 179 bp (SEQ ID NO:20).
Example 3
Isolation of the Full-Length DGAT2 Sequence from Mortierella alpina 
Based on the sequence of the 370 bp fragment, the 5′ and 3′ end regions of the M. alpina DGAT2 were cloned using genome walking techniques. This enabled assembly of a contig, corresponding to the −747 bp to +1552 bp region of the M. alpina DGAT2 (SEQ ID NO:37). This contig included the entire coding region of DGAT2 and three introns.
InVitrogen's 3′-End RACE Protocol
3′-end RACE was carried out using the InVitrogen 3′-end RACE kit, following the manufacturer's protocol. Briefly, 90 ng of M. alpina polyA(+)RNA in 11 μl of water was mixed with 1 μl of 10 μM Adaptor primer (“AP”; SEQ ID NO:21) solution. The mixture was heated at 70° C. for 10 min and cooled on ice for 2 min. To this, 2 μl 10×PCR buffer, 2 μl 25 mM MgCl2, 2 μl 0.1 M DTT, and 1 μl of 10 mM dNTP mix were added (from the kit). The reaction mixture was heated to 42° C. for 3 min and then 1 μl of Superscript II reverse transcriptase was added. The reaction was allowed to proceed for 50 min at 42° C., then was heated to 70° C. for 15 min and cooled on ice for 2 min. 1 μl of RNaseH from the kit was added. The entire mixture was then incubated at 37° C. for 20 min.
The above reaction mixture (2 μl) was used directly as a PCR template, while the remainder of the PCR reaction mixture contained 1 μl of 20 μM primer MDGAT-3-1 (SEQ ID NO:22, nested at the 3′ end), 2 μl of 10 μM kit primer UAP (SEQ ID NO:23), 25 μl of ExTaq premix Taq 2×PCR solution (TaKaRa Bio Inc., Otsu, Shiga, Japan) and 20 μl of water. PCR amplification was carried out for 30 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 90 sec. A final elongation cycle at 72° C. for 7 min was carried out, followed by reaction termination at 4° C.
A second round of PCR was carried out using the same conditions described above, with the exception that the template used was 1 μl of 1:10 diluted PCR reaction mixture and primer MDGAT-3-2 (SEQ ID NO:24, nested at the 3′ end) was used in place of primer MDGAT-3-1. This was followed by a third round of PCR using primers MDGAT-3-3 (SEQ ID NO:25, nested at the 3′ end) and UAP.
A ca 455 bp fragment was obtained from the PCR. After purification with a Qiagen QiaQuick PCR purification kit, the fragment was cloned into pCR2.1-TOPO and sequenced. Sequence analysis showed that the fragment was the 3′-end of the M. alpina DGAT2 cDNA and it included the polyA tail (SEQ ID NO:26).
Genome Walking to Isolate the 3′-End Region of the M. alpine DGAT2
A Clontech Universal GenomeWalkerTM kit was used to obtain a piece of genomic DNA corresponding to the 3′-end region of the M. alpina DGAT2. 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:27 [top strand] and 28 [bottom strand]), as shown below:








5′-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT-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, 1 μl of 20 μM MDGAT-3-1 (SEQ ID NO:22), 2 μl of 10 μM kit primer AP1 (SEQ ID NO:29), 21 μl water, and 25 μl ExTaq premix Taq 2×PCR solution (TaKaRa). The PCR reactions were carried out for 30 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 90 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, 1 μl of 20 μM MDGAT-3-2 (SEQ ID NO:24), 2 μl of 10 μM kit primer AP2 (SEQ ID NO:30), 21 μl water and 25 μl of ExTaq premix Taq 2×PCR solution (TaKaRa). PCR reactions were carried out for 30 cycles using the same thermocycler conditions described above.
A 674 bp 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 fragment was the 3′-end of the M. alpina DGAT2 gene (SEQ ID NO:31). Additionally, this fragment was identical to the cDNA fragment (SEQ ID NO:26), with two exceptions: (1) the 3′-end did not extend as far as the cDNA fragment; and (2) two additional regions were present, representing introns that had been spliced off from the cDNA (wherein one intron [SEQ ID NO:20] was located between bases 35–213 of SEQ ID NO:31 and the second intron [SEQ ID NO:33] was located between bases 369–499 of SEQ ID NO:31).
Genome Walking to Isolate the 5′-End Region of the M. alpine DGAT2
The same set of four ligation mixtures used in the Clontech 3′-end RACE protocol were also used to obtain the 5′-end region of the M. alpina DGAT2. Specifically, a first round of PCR using the same components and conditions as described above was conducted, with the exception that MDGAT-5-1 (SEQ ID NO:34, nested at the 5′ end) and AP1 were used as primers. The second round of PCR used MDGAT-5-2 (SEQ ID NO:35, nested at the 5′ end) and AP2 as primers. A DNA fragment with 1261 bp was obtained. It was purified and cloned into pCR2.1-TOPO and sequenced. Analysis of the sequence showed that it was the 5′-region of the DGAT2 gene (SEQ ID NO:36).
Assembly of the Full-Length DGAT2 Sequence from Mortierella alpina 
A 2299 bp sequence (SEQ ID NO:37) containing the complete DGAT2 gene (comprising a region extending 747 bases upstream of the DGAT2 translation initiation ‘ATG’ codon and extending 62 bases beyond the DGAT2 termination codon) was assembled from the original partial cDNA fragment (SEQ ID NO:18), the partial genomic fragment (SEQ ID NO:19), the 3′ cDNA fragment (SEQ ID NO:26) and 3′ and 5′ genomic sequences (SEQ ID NOs:31 and 36) described above (graphically illustrated in FIG. 3). The complete nucleotide sequence of the M. alpina DGAT2 cDNA from ‘ATG’ to the stop codon ‘TAG’ is provided as SEQ ID NO:1 (corresponding to bases 748 to 2237 of SEQ ID NO:37, excluding the three introns (i.e., intron 1 [SEQ ID NO:32], corresponding to bases 1018 to 1201 of SEQ ID NO:37; intron 2 [SEQ ID NO:20], corresponding to bases 1570 to 1748 of SEQ ID NO:37; and intron 3 [SEQ ID NO:32], corresponding to bases 1903 to 2034 of SEQ ID NO:37). The translated amino acid sequence (SEQ ID NO:2) showed homology with a number of fungal, plant and animal DGAT2s.
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 for 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). The sequence was analyzed for similarity to all publicly available DNA sequences contained in the “nr” database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequence was 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 the BLAST comparison summarizing the sequence to which SEQ ID NO:2 has 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. Thus, the amino acid fragment described herein as SEQ ID NO:2 had 47% identity and 66% similarity with the protein sequence of Mortierella ramanniana DGAT2A (GenBank Accession No. MK84179.1), with an expectation value of 1e-87; additionally, SEQ ID NO:2 had 47% identity and 65% similarity with hypothetical protein CNBF4150 from Cryptococcus neoformans var. neoformans B-3501A (GenBank Accession No. EAL20089), with an expectation value of 6e-89.
Example 4
Generation Of EPA-Producing Yarrowia lipolytica ATCC #20362 Strain Y2067U
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. 4A). The affect of M. alpina DGAT2 gene overexpression was examined in this EPA producing strain based on analysis of TAG content, as described in Example 5 (infra).
The development of strain Y2067U herein required the construction of strain M4 (producing 8% DGLA), strain Y2034 (producing 10% ARA), strain E (producing 10% EPA), strain EU (producing 10% EPA) and strain Y2067 (producing 15% EPA).
Construction of Strain M4 Producing 8% DGLA
Construct pKUNF12T6E (FIG. 4B; SEQ ID NO:38) was generated to integrate four chimeric genes (comprising a Δ12 desaturase, a Δ6 desaturase and 2 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 5







Description of Plasmid pKUNF12T6E (SEQ ID NO: 38)








RE Sites And



Nucleotides


Within SEQ ID
Description


NO: 38
Of Fragment And Chimeric Gene Components





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


(9420-8629)
Accession No. AJ306421)


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


(12128-1)
Accession No. AJ306421)


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


(6380-8629)
FBAIN: FBAIN promoter (SEQ ID NO: 39; see also



U.S. Patent Application No. 10/987548)



EL1S: codon-optimized elongase 1 gene (SEQ ID



NO: 40), 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: 42), 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: 44; see also U.S.



Patent Application No. 10/987548)



F.Δ12: Fusarium moniliforme Δ12 desaturase gene



(SEQ ID NO: 45)



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: 47), derived from Thraustochytrium aureum (U.S.



6,677,145)



XPR: XPR terminator sequence of Yarrowia Xpr gene



(GenBank Accession No. M17741)









The pKUNF12T6E plasmid was digested with Ascl/Sphl, 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 (FIG. 4B), 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.
Construction of Strain Y2034 Producing about 10% ARA
Constructs pDMW232 (FIG. 4C; SEQ ID NO:49) was generated to integrate two Δ5 chimeric genes into the Leu2 gene of Yarrowia strain M4. The plasmid pDMW232 contained the following components:







TABLE 6







Description of Plasmid pDMW232 (SEQ ID NO: 49)








RE Sites And



Nucleotides


Within SEQ ID
Description


NO: 49
Of Fragment And Chimeric Gene Components





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


(5550-4755)
Accession No. AF260230)


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


(8258-8967)
Accession No. AF260230)


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


(2114-4755)
FBAIN: FBAIN Promoter (SEQ ID NO: 39; see also



U.S. Patent Application No. 10/987548)



MAΔ5: Mortierella alpina Δ5 desaturase gene (SEQ ID



NO: 50) (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: as described for FBAIN::MAΔ5::Pex20 (supra)



Lip1: Lip1 terminator sequence of Yarrowia Lip1 gene



(GenBank Accession No. Z50020)


PmeI/ClaI
Yarrowia Ura3 gene (GenBank Accession No.


(5550-4755)
AJ306421)









Plasmid pDMW232 was digested with Ascl/Sphl, and then used to transform strain M4 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 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”.
Construction of Strain E, Producing about 10% EPA
Construct pZP3L37 (FIG. 4D; SEQ ID NO:52) was created to integrate three synthetic Δ17 desaturase chimeric genes into the acyl-CoA oxidase 3 (i.e., POX3) gene of the Y2034 strain. The plasmid pZP3L37 contained the following components:







TABLE 7







Description of Plasmid pZP3L37 (SEQ ID NO: 52)








RE Sites And



Nucleotides


Within SEQ ID
Description


NO: 52
Of 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: 53), 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: 39; see also



U.S. Patent Application No. 10/987548)



Δ17S: SEQ ID NO: 53 (supra)



Lip2: Lip2 terminator sequence of Yarrowia Lip2 gene



(GenBank Accession No. AJ012632)


PmeI/SwaI
Yarrowia Leu2 gene (GenBank Accession No.


(1811-1)
AF260230)


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


(10345-1)
FBAINm: FBAINm promoter (SEQ ID NO: 55; see also



U.S. Patent Application No. 10/987548)



Δ17S: SEQ ID NO: 53 (supra)



Pex16: Pex16 terminator sequence of Yarrowia Pex16



gene (GenBank Accession No. U75433)









Plasmid pZP3L37 was digested with Ascl/Sphl, 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 as follows (“two-stage growth conditions”). 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 72 hrs at 30° C. with shaking at 250 rpm/min. The cells were again collected by centrifugation and the liquid supernatant was extracted.
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.
Construction of Strain EU Producing about 10% EPA
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 with 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 FOA selection 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 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. 5A; SEQ ID NO:56). 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 5-FOA resistant 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.
Construction of Strain Y2067 Producing about 15% EPA
Plasmid pKO2UF2PE (FIG. 5B; SEQ ID NO:57) was created to integrate a cluster containing two chimeric genes (comprising a heterologous A12 desaturase and an elongase) and a Ura3 gene into the native Yarrowia Δ12 desaturase gene of strain EU (supra). Plasmid pKO2UF2PE contained the following components:







TABLE 8







Description of Plasmid pKO2UF2PE (SEQ ID NO: 57)








RE Sites And



Nucleotides


Within SEQ ID
Description


NO: 57
Of Fragment And Chimeric Gene Components





AscI/BsiWI
730 bp 5′ part of Yarrowia Δ12 desaturase gene (SEQ ID


(3382-2645)
NO: 58)


SphI/EcoRI
556 bp 3′ part of Yarrowia Δ12 desaturase gene (SEQ ID


(6090-6646)
NO: 58)


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


(1-2645)
FBAINm: FBAINm promoter (SEQ ID NO: 55; see also



U.S. Patent Application No. 10/987548)



F.Δ12: Fusarium moniliforme Δ12 desaturase gene



(SEQ ID NO: 45)



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: 60; see also U.S.



Patent Application No. 60/610060)



EL1S: codon-optimized elongase 1 gene (SEQ ID



NO: 40), 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 No.


(6646-8163)
AJ306421)









Plasmid pKO2UF2PE was digested with Ascl/Sphl 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 two-stage growth conditions. 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”.
Construction of Strain Y2067U Producing about 14% EPA with Ura-Phenotype
In order to disrupt the Ura3 gene in Y2067 strain, construct pZKUT16 (FIG. 5C; SEQ ID NO:61) 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. The plasmid pZKUT16 contained the following components:







TABLE 9







Description of Plasmid pZKUT16 (SEQ ID NO: 61)








RE Sites And



Nucleotides


Within SEQ ID
Description


NO: 61
Of Fragment 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: 62), derived from rat (GenBank Accession No.



AB071986)



Pex 20: Pex20 terminator sequence of Yarrowia Pex20



gene (GenBank Accession No. AF054613)









The plasmid pZKUT16 was digested with Sall/Pacl, and then used to transform Y2067 strain according to the General Methods. Following transformation, cells were plated onto FOA selection plates and maintained at 30° C. for 2 to 3 days.
A total of 24 transformants grown on FOA plates were picked and re-streaked onto MM plates and FOA plates, separately. The strains that could grow on 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 two-stage growth conditions (48 hrs MM+96 hrs in 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::EIS:: Oct, TEF::E2S::Xpr; FBAIN::MAΔ5::Pex20, TEF::MAΔ5::Lip1, FBAIN::Δ17S::Lip2, FBAINm::Δ17S::Pex16, TEF::Δ17S::Pex20 and TEF::rELO2S::Pex20.
Example 5
Heterologous Expression of the Mortierella alpina DGAT2 ORF Under the Control of a Yarrowia Promoter in Yarrowia lipolytica 
The present Example describes the over-expression of the M. alpina DGAT2 ORF in a chimeric gene under the control of a Yarrowia lipolytica promoter in Y. lipolytica strain Y2067U, and the affect of the overexpression as determined by an analysis of TAG content.
The M. alpina DGAT2 ORF was cloned into plasmid pZUF17 (SEQ ID NO:64; FIG. 5D) 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. First, the ORF was PCR-amplified using upper primer MDGAT-F (SEQ ID NO:65) and lower primer MDGAT-R1 (SEQ ID NO:66) from the M. alpina cDNA (supra, Example 1). 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. Correct transformants were confirmed by miniprep analysis and the resultant plasmid was designated as “pMDGAT2-17” (SEQ ID NO:67).
“Control” vector pZUF-MOD-1 (SEQ ID NO:68; FIG. 5E) was prepared as follows. First, primers pzuf-mod1 (SEQ ID NO:69) and pzuf-mod2 (SEQ ID NO:70) 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 at room temperature overnight into similarly digested NcoI-/NotI-cut pZUF17 vector (wherein the ligation consisted of 0.5 μg of the digested PCR fragment and 0.3 μg of  ̃7079 bp digested pZUF17 vector fragment with 2 μl of 10×T4 ligase buffer and 3 units of T4 DNA ligase (Promega) in a total volume of 20 μl) and the resulting ligation mixture was used to transform E. coli Top10 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”.
Y. lipolytica strain Y2067U (from Example 4, producing 14% EPA of total lipids) was transformed with pMDGAT2-17 and pZUF-MOD-1, 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 10







Lipid Content 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 concentration 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 DAG ATs [i.e., DGAT2 (SEQ ID NOs:3–8), DGAT1 and/or PDAT] were knocked-out in strain Y2067U+pMDGAT2-17 using means well known to one of skill in the art.

## Claims

1. An isolated nucleic acid molecule selected from the group consisting of:
(a) an isolated nucleic acid molecule encoding a diacylglycerol acyltransferase 2 enzyme, having the amino acid sequence as set forth in SEQ ID NO:2;
(b) an isolated nucleic acid molecule encoding an diacylglycerol acyltransferase 2 enzyme, that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; or
(c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).

2. The isolated nucleic acid molecule of claim 1 as set forth in SEQ ID NO:1.

3. A chimeric gene comprising the isolated nucleic acid molecule of claim 1 operably linked to suitable regulatory sequences.

4. An isolated host cell comprising the chimeric gene of claim 3.

5. An isolated host cell of claim 4, selected from the group consisting of algae, bacteria, molds, fungi and yeast.

6. An isolated host cell of claim 5, wherein the yeast is an oleaginous yeast.

7. An isolated host cell of claim 6, wherein the oleaginous yeast cell is selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidlum, Cryptococcus, Trichosporon and Lipomyces.

8. An isolated host cell of claim 7, wherein the host cell is Yarrowia lipolyzica.