[Objective] Ergosterol is a fungal metabolite with economic importance.For ergosterol biosynthesis,to identify the bottleneck enzymes in the metabolic pathway is of crucial importance.[Methods] Sterol C-8 isomerase en...[Objective] Ergosterol is a fungal metabolite with economic importance.For ergosterol biosynthesis,to identify the bottleneck enzymes in the metabolic pathway is of crucial importance.[Methods] Sterol C-8 isomerase encoding gene ERG2 was cloned from Saccharomyces cerevisiae by PCR.To evaluate the effect of ERG2 overexpression on the sterol content in the yeast,the expression plasmid pPERG2 was constructed and transformed into S.cerevisiae strain YS58 to generate recombinant strain YS58(pPERG2).In addition,the regulation role of sterol C-24 methyltransferase,encoded by ERG6,in ergosterol biosynthesis was further verified by analysis of sterol components and levels in yeast strains overexpressing ERG6,ERG2 respectively,or overexpressing ERG6 and ERG2 simultaneously.[Results] Ergosterol content and all sterol intermediates increased largely by overexpressing ERG6 in S.cerevisiae.Although the overexpression of sterol C-8 isomerase encoded by ERG2 alone had negative effect on ergosterol biosynthesis,overexpression of ERG6 and ERG2 simultaneously led to an increased ergosterol level,which was 1.41-fold of that in empty vector strain,1.92-fold of that in ERG2 only overexpressing strain and 1.12-fold of that in ERG6 only overexpressing strain.[Conclusion] These results demonstrated that sterol C-24 methyltransferase is an important bottleneck enzyme for ergosterol biosynthesis in S.cerevisiae.展开更多
文摘[Objective] Ergosterol is a fungal metabolite with economic importance.For ergosterol biosynthesis,to identify the bottleneck enzymes in the metabolic pathway is of crucial importance.[Methods] Sterol C-8 isomerase encoding gene ERG2 was cloned from Saccharomyces cerevisiae by PCR.To evaluate the effect of ERG2 overexpression on the sterol content in the yeast,the expression plasmid pPERG2 was constructed and transformed into S.cerevisiae strain YS58 to generate recombinant strain YS58(pPERG2).In addition,the regulation role of sterol C-24 methyltransferase,encoded by ERG6,in ergosterol biosynthesis was further verified by analysis of sterol components and levels in yeast strains overexpressing ERG6,ERG2 respectively,or overexpressing ERG6 and ERG2 simultaneously.[Results] Ergosterol content and all sterol intermediates increased largely by overexpressing ERG6 in S.cerevisiae.Although the overexpression of sterol C-8 isomerase encoded by ERG2 alone had negative effect on ergosterol biosynthesis,overexpression of ERG6 and ERG2 simultaneously led to an increased ergosterol level,which was 1.41-fold of that in empty vector strain,1.92-fold of that in ERG2 only overexpressing strain and 1.12-fold of that in ERG6 only overexpressing strain.[Conclusion] These results demonstrated that sterol C-24 methyltransferase is an important bottleneck enzyme for ergosterol biosynthesis in S.cerevisiae.
