酿酒酵母稳定整合位点鉴定及其在橘烯生物合成中的应用

Stable integration sites in Saccharomyces cerevisiae: identification and application in the biosynthesis of valencene

摘要:

橘烯(valencene)是一种带有柑橘香气的高值倍半萜类化合物，被广泛应用于食品、化妆品等行业，以及工业合成圆柚酮。本研究在酿酒酵母基因组中鉴定到了16个位于基因间区(intergenic region, IGR)的基因组位点，利用CRISPR-Cas9技术，将Ypet表达盒整合到不同的基因组位点，整合成功率高达87.50%，不同插入位点之间的表达差异达1.91倍。研究显示，位置效应在基因表达中相对稳定，基本不受启动子和报告基因变更的影响。之后筛选高效表达的元件组合P_TDH3-T_PRC1，在优选的整合位点迭代整合来自阿拉斯加黄扁柏(Callitropsis nootkatensis)橘烯合成酶(VSm)，橘烯产量提升至254.67 mg/L。过表达多个拷贝的关键基因tHMG1-ERG20，橘烯的产量提高了93.49%。所得工程菌株L-13在3 L发酵罐中进行2阶段补料分批发酵能够生产橘烯9 530.18 mg/L，相较出发菌株提高了近100倍，展现了筛选出的基因组位点在橘烯生产优化过程中的潜力。

Abstract:

Valencene, a high-value sesquiterpene with a citrus aroma, is widely employed in the food and cosmetic fields and the industrial synthesis of nootkatone. In this study, 16 genomic loci in the intergenic regions (IGRs) of Saccharomyces cerevisiae were identified. A Ypet expression cassette was successfully integrated into various genomic loci by CRISPR-Cas9, with an impressive integration success rate of 87.50% and exhibiting expression variations of up to 1.91-fold depending on the insertion site. The study demonstrates that the positional effect exhibits relative stability in gene expression, and is essentially unaffected by changes in promoters and reporter genes. Furthermore, a high-expression element combination, P_TDH3-T_PRC1, was selected. The iterative integration of the valencene synthase gene VSm from Callitropsis nootkatensis at the selected loci increased the valencene yield to 254.67 mg/L. Overexpression of key genes tHMG1-ERG20 with multiple copies increased the valencene yield by 93.49%. The engineered strain L-13 achieved the valencene yield of 9 530.18 mg/L by two-stage fed-batch fermentation in a 3 L fermenter. This yield represents a nearly 100-fold increase compared with that of the starting strain, highlighting the significant potential of the screened genomic loci in optimizing valencene production.