Identification of Six Thiolases and their Effects on Fatty Acid and Ergosterol Biosynthesis in Aspergillus oryzae

Abstract

Thiolase plays important roles in lipid metabolism. It can be divided into degradative thiolases (Thioase I) and biosynthetic thiolases (thiolases II), which are involved in fatty acid β-oxidation and acetoacetyl-CoA biosynthesis, respectively. The Saccharomyces cerevisiae (S. cerevisiae) genome harbors only one gene each for thioase I and thiolase II, namely, Pot1 and Erg10, respectively. In this study, six thiolases (named AoErg10A−AoErg10F) were identified in Aspergillus oryzae (A. oryzae) genome using bioinformatics analysis. Quantitative reverse transcription–PCR (qRT-PCR) indicated that the expression of these six thiolases varied at different growth stages and under different forms of abiotic stress. Subcellular localization analysis showed that AoErg10A was located in the cytoplasm, AoErg10B and AoErg10C in the mitochondria, and AoErg10D-AoErg10F in the peroxisome. Yeast heterologous complementation assays revealed that AoErg10A, AoErg10D, AoErg10E, AoErg10F and cytoplasmic AoErg10B (AoErg10BΔMTS) recovered the phenotypes of S. cerevisiae erg10 weak and lethal mutants, and that only AoErg10D-F recovered the phenotype of the pot1 mutant that cannot use oleic acid as the carbon source. Overexpression of AoErg10s either affected the growth speed or sporulation of the transgenic strains. In addition, the fatty acid and ergosterol content changed in all the AoErg10-overexpressing strains. This study revealed the function of six thiolases in A. oryzae and their effect on growth, and fatty acid and ergosterol biosynthesis, which may lay the foundation for genetic engineering for lipid metabolism in A. oryzae or other fungi.ImportanceThiolase including thioase I and thiolase II, plays important roles in lipid metabolism. A. oryzae, one of the most industrially important filamentous fungi, has been widely used for manufacturing oriental fermented food such as sauce, miso, and sake for a long time. Besides, A. oryzae has a high capability in production of high lipid content and has been used for lipid production. Thus, it is very important to investiagte the function of thiolases in A. oryzae. In this study, six thiolase (named AoErg10A-AoErg10F) were identified by bioinformatics analysis. Unlike other reported thiolases in fungi, three of the six thiolases showed dual function of thioase I and thiolase II in S. cerevisiae, indicating the lipid metabolism is more complex in A. oryzae. The reveal of founction of these thiolases in A. oryzae can lay the foundation for genetic engineering for lipid metabolism in A. oryzae or other fungi.