关键词: 7-脱氢胆固醇还原酶, 酿酒酵母, 菜油甾醇, 合成生物学, 补料分批发酵

Abstract:

Campesterol, as an important synthetic precursor of steroid drugs (progesterone, androstenedione, hydrocortisone, etc.), has received extensive attention from domestic and foreign researchers. First, through bioinformatics analysis, 10 different sources of 7-dehydrocholesterol reductase DHCR7 encoding gene DHCR7 were isolated, and the endogenous ERG5 gene of Saccharomyces cerevisiae was inactivated by using the CRISPR/Cas9 gene editing technology to construct the recombinant strain for producing campesterol. The results showed that the Zw507, integrated Pangasianodon hypophthalmus DHCR7, obtains the yield of campesterol 216.93 mg/L. Then, 10 yeast endogenous strong promoters were selected to combine with PhDHCR7. The results showed that when TEF1p was used as the promoter of PhDHCR7, the production of campesterol was up to 253.35 mg/L. In order to further increase the output of campesterol, the copy number of the PhDHCR7 expression cassette in the yeast genome was increased. When the copy number is 3, the output of campesterol reaches the highest 302.27 mg/L. Finally, fed-batch fermentation was carried out in a 5 L fermentor, and the yield of campesterol was 916.88 mg/L. Thus this strain can be used as an excellent chassis cell for subsequent biosynthesis of steroidal drugs.

Key words: 7-dehydrocholesterol reductase, Saccharomyces cerevisiae, campesterol, synthetic biology, fed-batch fermentation