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

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.