Overexpression of the active diacylglycerol acyltransferase variant transforms Saccharomyces cerevisiae into an oleaginous yeast

• Published in: Applied microbiology and biotechnology 2013-08, Vol.97 (16), p.7345-7355
• Main Authors: Kamisaka, Yasushi, Kimura, Kazuyoshi, Uemura, Hiroshi, Yamaoka, Masakazu
• Format: Article
• Language: English
• Subjects: Accumulation; Amino acids; Analysis; Applied Genetics and Molecular Biotechnology; Biomedical and Life Sciences; Biosynthesis; Biotechnology; Biotechnology - methods; Culture Media - chemistry; Diacylglycerol O-Acyltransferase - biosynthesis; Diacylglycerol O-Acyltransferase - genetics; Enzymes; Fluorides; Gene Expression; Gene Expression Profiling; Genetic engineering; Glucose; Glucose - metabolism; Life Sciences; Lipid Metabolism; Lipids; Metabolic Engineering - methods; Microbial Genetics and Genomics; Microbiology; Mutants; Nitrogen; Nitrogen - metabolism; Reagents; Real-Time Polymerase Chain Reaction; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - biosynthesis; Saccharomyces cerevisiae Proteins - genetics; Sequence Deletion; Studies; Yeast; Yeasts
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-013-4915-9

Lipid production by Saccharomyces cerevisiae was improved by overexpression of the yeast diacylglycerol acyltransferase Dga1p lacking the N-terminal 29 amino acids (Dga1∆Np), which was previously found to be an active form in the ∆ snf2 mutant. Overexpression of Dga1∆Np in the ∆ snf2 mutant, however, did not increase lipid content as expected, which prompted us to search for a more suitable strain in which to study the role of Dga1∆Np in lipid accumulation. We found that the overexpression of Dga1∆Np in the ∆ dga1 mutant effectively increased the lipid content up to about 45 % in the medium containing 10 % glucose. The high lipid content of the transformant was dependent on glucose concentration, nitrogen limitation, and active leucine biosynthesis. To better understand the effect of dga1 disruption on the ability of Dga1∆Np to stimulate lipid accumulation, the ∆ dga1 - 1 mutant, in which the 3′-terminal 36 bp of the dga1 open reading frame (ORF) remained, and the ∆ dga1 - 2 mutant, in which the 3′-terminal 36 bp were also deleted, were prepared with URA3 disruption cassettes. Surprisingly, the overexpression of Dga1∆Np in the ∆ dga1 - 1 mutant had a lower lipid content than the original ∆ dga1 mutant, whereas overexpression in the ∆ dga1 - 2 mutant led to a high lipid content of about 45 %. These results indicated that deletion of the 3′ terminal region of the dga1 ORF, rather than abrogation of genomic Dga1p expression, was crucial for the effect of Dga1∆Np on lipid accumulation. To investigate whether dga1 disruption affected gene expression adjacent to DGA1 , we found that the overexpression of Esa1p together with Dga1∆Np in the ∆ dga1 mutant reverted the lipid content to the level of the wild-type strain overexpressing Dga1∆Np. In addition, RT-qPCR analysis revealed that ESA1 mRNA expression in the ∆ dga1 mutant was decreased compared to the wild-type strain at the early stages of culture, suggesting that lowered Esa1p expression is involved in the effect of dga1 disruption on Dga1∆Np-dependent lipid accumulation. These results provide a new strategy to engineer S. cerevisiae for optimal lipid production.