Identification And Functional Analysis Of Acyl-CoA:Diacylglycerol Acyltransferase From Oleaginous Fungus Mucor Circinelloides

Acyl-CoA:diacylglycerol acyltransferases(DGAT) catalyzes the final and committed step of triacylglycerol(TAG) biosynthesis. DGAT plays a key role in determining the carbon flux into TAG, has been used as targets to increase overall oil production or to acccumulate desired fatty acids(FAs) in oilseed and oleaginous microorganisms engineering, and also as therapeutic targets for human diseases, such as obesity and diabetes mellitus, all of those diseases are associated with excessive accumulation of TAG. Oleaginous fungus Mucor circinelloides has the capability to synthesize and accumulate high amounts of TAG andγ-linolenic acid(GLA). So far, most of the research has been focused on the mechanism of fatty acid biosynthesis in this fungus, not much work has been done with TAG biosynthesis.The key gene for TAG biosynthesis, ie, DGAT gene in M. circinelloides has not been studied,which may be a promising target to increase TAG accumulation via metabolic engineering. So it is essential to identify and characterize the DGAT genes from M. circinelloides. In this research, the DGAT from M. circinelloides(designed Mc DGAT1/2/3/4, respectively) were studied, mainly on its characterization and function.Firstly, fermentation conditions of M. circinelloides were carried out and the relative m RNA expression patterns of the four putative DGATs in relation to TAG biosynthesis in M.circinelloides were investigated. The dry cell weight(DCW) of M. circinelloides was 12 g/L,the proportions of TAG and total lipids in DCW were 6% and 10%, respectively, after fermentation for 80 h. And overexpression Mc DGAT may be linked with TAG accumulation.Secondly, the bioinformatics of the four DGAT proteins were analyzed, including sequence alignment, evolutionary tree, physiochemical properties, secondary structures and transmembrane domains. Mc DGAT1 and Mc DGAT4 were clearly within the DGAT2 subfamily with 68% similarity. Mc DGAT2 and Mc DGAT3 were clustered into the DGAT1 subfamily with 32% similarity. The four Mc DGAT proteins had different biochemical properties, secondary structures and transmembrane domains.Thirdly, the four DGAT homologue genes from M. circinelloides were heterologously expressed in mutant TAG-deficient Saccharomyces cerevisiae mutant strain H1246,respectively. The four genes were cloned, the corresponding expression vectors p YES2-Mc DGAT1/2/3/4 were successfully constructed and transformed into mutant strain H1246. And the m RNA reletive levels of the four Mc DGATs were determined. The empty vector p YES2 and the yeast endogenous DGAT gene Sc DGA1 were used as the negative and positive controls, respectively.Finally, the characterization of growth and lipids accumulation in recombinant strains were studied and the effect of linoleic acid(LA) or GLA on recombinant strains with DGAT activity were also investigated. The growth was inhibited with 1 m M LA expect the recombinant strains expressing Mc DGAT4 or Sc DGA1. None of the recombinant strains were inhibited with GLA. Only the recombinant strain expressing Mc DGAT4 produced TAG and restored the oil body formation. Without exogenous fatty acids, the TAG and total lipids contents of the recombinant strains expressing Mc DGAT4 were 1.8 fold and 1.1 fold that of the positive carrying Sc DGA1, respectively. The substrate specificity of C18:0 in this strain wassignificantly higher than that of the positive. When 1 m M exogenous LA was added, the contents of TAG and total lipids in the recombinant strain carrying Mc DGAT4 were 2.5 fold and 1.9 fold that of the positive, respectively. The acyl-Co A preference of Mc DGAT4 had no significant difference compared with the positive control Sc DGA1. While with 1 m M exogenous GLA, the contents of TAG and total lipids in the recombinant strain carrying Mc DGAT4 were 2.2 fold and 1.3 fold that of the positive, respectively. The substrate specificity of C18:1 in Mc DGAT4 was significantly decreased compared with Sc DGA1. The Mc DGAT4 tend to use more LA than GLA.