摘要
麦角甾醇是真菌细胞膜的重要组成成分,也是重要的医药化工原料,具有广阔的应用前景,如何提高麦角甾醇产量并降低生物生产成本,是当今国内外研究的重点。本研究采用PCR克隆得到含有酿酒酵母角鲨烯环氧酶基因Erg1编码序列、C-24甾醇还原酶基因Erg4编码序列、GAL10启动子序列和PGK1终止子序列,并以大肠-酵母穿梭质粒pAUR112为载体,GAL10启动子和PGK1终止子序列为调控元件构建了酵母菌单基因表达质粒p112-g1和p112-g4,并利用两个表达质粒分别转化野生型酿酒酵母菌株S288C从而获得NJ-g1与NJ-g4两个重组菌株;通过Aba抗性筛选到转化子实现质粒上Erg1和Erg4基因在NJ-g1与NJ-g4中酶活性的过量表达,使得细胞内麦角甾醇的含量有大幅度提高。结果表明NJ-g1与NJ-g4的麦角甾醇含量分别是受体菌NJ的3.47倍和2.3倍,从而证明了角鲨烯环氧酶基因和C-24甾醇还原酶基因的过量表达可以有效提高酿酒酵母体内的麦角甾醇含量。本文研究可为确定工业麦角甾醇的最优生物合成条件具有重要理论意义和应用价值。
Ergosterol is an important component of fungal cell membranes and a key raw material in the pharmaceutical and chemical industries,with broad application prospects.Enhancing ergosterol production while reducing bioproduction costs is a major focus of current research worldwide.In this study,PCR cloning was employed to obtain the coding sequences of the Erg1(squalene epoxidase)and Erg4(C-24 sterol reductase)genes from Saccharomyces cerevisiae,along with the GAL10 promoter and PGK1 terminator sequences.Using the E.coli-yeast shuttle plasmid pAUR112 as the vector and the GAL10 promoter and PGK1 terminator as regulatory elements,two single-gene expression plasmids,p112-g1 and p112-g4,were constructed.These plasmids were then transformed into the wild-type S.cerevisiae strain S288C,yielding the recombinant strains NJ-g1 and NJ-g4.Transformants were selected via Aba resistance,enabling the overexpression of Erg1 and Erg4 genes in NJ-g1 and NJ-g4,respectively,which significantly increased intracellular ergosterol levels.The results demonstrated that the ergosterol content in NJ-g1 and NJ-g4 was 3.47 times and 2.3 times higher,respectively,than that in the host strain NJ.This confirms that the overexpression of squalene epoxidase and C-24 sterol reductase genes can effectively enhance ergosterol production in S.cerevisiae.This study provides important theoretical insights and practical value for determining the optimal biosynthesis conditions for industrial ergosterol production.
作者
鹿永华
李伟
牛浩
未雅楠
LU Yonghua;LI Wei;NIU Hao;WEI Yanan(Shandong Xinhua Pharmaceutical Co.,Ltd.,Zibo 255005,China)
出处
《山东化工》
2025年第16期29-32,共4页
Shandong Chemical Industry
