甾醇C-22去饱和酶高表达对酵母细胞麦角甾醇合成的影响

Effect of over-expression of sterol C-22 desaturase on ergosterol production in yeast strains

摘要:

通过PCR扩增克隆到酵母菌甾醇C-22去饱和酶基因(ERG5)的编码序列及其终止子序列,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以磷酸甘油酸激酶基因PGK1启动子为上游调控元件构建了酵母菌表达质粒pYPE5。以铜离子螯合蛋白基因CUP1替换ERG5基因内部序列获得ERG5破坏菌株YSE5,其中麦角甾醇的合成被阻断,而积累了甾醇中间体Ergosta-5,7-dien-3β-ol。表达质粒pYPE5转化破坏菌株后使细胞恢复了合成麦角甾醇的能力。说明表达质粒上的ERG5基因得到了功能性的表达。将表达质粒pYPE5转化酿酒酵母单倍体菌株YS58,通过营养缺陷互补筛选到重组菌株YS58(pYPE5)。对重组菌株、破坏菌株和互补菌株细胞甾醇组分和含量进行测定,发现重组菌株和互补菌株的麦角甾醇和总甾醇含量明显低于对照菌YS58(YEp352)。测定不同培养时间细胞的麦角甾醇含量,发现重组菌株的麦角甾醇含量始终低于对照菌YS58(YEp352)。可见,ERG5在酵母中的高表达导致细胞麦角甾醇含量降低。

Abstract:

Ergosterol, the main sterol in yeast, is responsible for structural membrane features such as fluidity and permeability. Additionally, ergosterol is economically important as a precursor of vitamin D₂. The biosynthesis of sterols in yeast is complex. As an enzyme of the later ergosterol biosynthesis, the sterol C-22 desaturase encoded by ERG5 gene is required to form the C-22 (23) double bond in the sterol side chain. In order to know the regulation of C-22 sterol desaturase in the ergosterol biosynthesis, ERG5 gene was cloned and over-expressed in the Saccharomyces cerevisiae. Primer 1 (5′-GTCGGTACCTCCAATGACAATAAATACC-3′, KpnⅠ) and primer 2 (5′-AAGGATCCTAGCAGA TCATTAGCTGTAG-3′, BamHⅠ) were designed according to the ERG5 sequence in GenBank. A 1.8kb DNA fragment containing the open reading frame and terminator of ERG5 gene was amplified from Saccharomyces cerevisiae YSF-20 by PCR and inserted into YEp352 to generate recombinant plasmid pYE5. To express ERG5 gene properly in S. cerevisiae, the recombinant expression plasmid pYPE5 containing ERG5 from pYE5 under the control of PGK1 promoter, the URA3 gene as the selection marker and the plasmid YEp352 as the vector was constructed, and then they were introduced into Saccharomyces cerevisiae YS58. To make sure the plasmid pYPE5 in the YS58 acted properly, the disruptant (YSE5) was created by deleting a 0.4kb fragment of ERG5 gene and inserting the CUP1 gene into the ERG5 and transforming the YS58. And then the disruptant (YSE5) was transformed with the plasmid pYPE5 carrying the corresponding complementing ERG5 gene to control the activity of the over-expressed ERG5 gene and restauration of the wild-type sterol pattern. The sterol profile of the disruptant (YSE5) demonstrated that ergosta-5, 7-dien-3β-ol was accumulated which was very similar to ergosterol but with a saturated side chain. In contrast, the YSE5 (pYPE5) strain contains predominantly ergosterol. The sterol content of the transformant was analyzed using gas chromatography (GC) analysis. The result shows that ergosterol production in recombinant strains was reduced. And the experiment of the effect of culturing time shows that ergosterol productions in recombinant strains were always lower than YS58 (pYPE5) from 24～48h culturing time. Under the optimal culture condition, ergosterol content in recombinant strain YS58 (pYPE5) was about 0.70-fold of that in the referring strain.