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 and 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 application Ser. No. 10/840579, 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.
Two comprehensive mini-reviews on TAG biosynthesis in yeast, including details concerning the genes involved and the metabolic intermediates that lead to TAG synthesis are: D. Sorger and G. Daum, Appl. Microbiol. Biotechnol. 61:289-299 (2003); and H. Müllner and G. Daum, Acta Biochimica Polonica, 51(2):323-347 (2004). However, the authors acknowledge that most work performed thus far has focused on the yeast Saccharomyces cerevisiae and numerous questions regarding TAG formation and regulation remain.
Briefly, three pathways have been described for the synthesis of TAGs in S. cerevisiae (Sandager, L. et al., J. Biol. Chem. 277(8):6478-6482 (2002)). First, TAGs are mainly synthesized from DAG and acyl-CoAs by the activity of a diacylglycerol acyltransferase (i.e., DGAT2, encoded by the DGA1 gene). More recently, however, a phospholipid:diacylglycerol acyltransferase (i.e., PDAT, encoded by the LRO1 gene) 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 (encoded by the ARE1 and ARE2 genes) are known that utilize acyl-CoAs and sterols to produce sterol esters (and TAGs in low quantities; see Sandager, L. et al., Biochem. Soc. Trans. 28(6):700-702 (2000)). Together, PDAT and DGAT2 are responsible for approximately 95% of oil biosynthesis in S. cerevisiae. 
Although homologs of each of the acyltransferase genes described above have been identified in various other organisms and disclosed in the public literature, few genes are available from organisms classified as oleaginous. With respect to yeast, those species included within the genera of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces and that can accumulate at least 25% of their dry cell weight as oil are classified as oleaginous. Within this unique family of yeast, however, only two acyltransferases have been isolated and characterized. These include a DGAT2 and PDAT from Yarrowia lipolytica (see co-pending U.S. patent application Ser. No. 10/882,760, entirely incorporated herein by reference). However, in contrast to the findings in Saccharomyces cerevisiae, the Y. lipolytica DGAT2 and PDAT were discovered to only be partially responsible for the organism's total oil biosynthesis.
There remains a need there for to identify genes encoding diacylglycerol acyltransferases (DGAT1) and acyl-CoA:sterol-acyltransferases useful for expression in oleaginous yeast for the production of PFUA's. The present work was conducted to identify and characterize the additional gene(s) involved in oil biosynthesis in the oleaginous yeast, Yarrowia lipolytica. An understanding of the native mechanisms of oil biosynthesis in this organism is useful, prior to the development of techniques that modify the transfer of recombinantly produced fatty acids (e.g., long-chain PUFAs, such as ARA, EPA and DHA) to the storage lipid pools (i.e., TAG fraction) within transformant oleaginous yeast.
Applicants have solved the stated problem by isolating the genes encoding a diacylglycerol acyltransferase (DGAT1) and an acyl-CoA:sterol-acyltransferase (ARE2) from the oleaginous yeast, Yarrowia lipolytica. Together, the PDAT, DGAT2 and DGAT1 of Yarrowia lipolytica are responsible for up to ˜95% of oil biosynthesis (while ARE2 may additionally be a minor contributor to oil biosynthesis). Additionally, an orthologous DGAT1 gene was cloned from Mortierella alpina (an oleaginous fungus) and four other fungal DGAT1 orthologs from public sequence databases (i.e., Neurospora crassa, Gibberella zeae PH-1, Magnaporthe grisea and Aspergillus nidulans) were identified. With these fungal DGAT1 protein sequences, the Applicants have discovered diagnostic features that will be useful to identify subsequent genes within this family of proteins. These DGAT1 genes 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.
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