Chen Dongying, Zhu Chaoyi, Chen Hefeng, Zhou Jingtao, Li Shuang
School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, Guangdong, China.
Sheng Wu Gong Cheng Xue Bao. 2024 Jun 25;40(6):1924-1934. doi: 10.13345/j.cjb.230792.
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 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-T, was selected. The iterative integration of the valencene synthase gene from at the selected loci increased the valencene yield to 254.67 mg/L. Overexpression of key genes 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.
瓦伦烯是一种具有柑橘香气的高价值倍半萜烯，广泛应用于食品、化妆品领域以及诺卡酮的工业合成。在本研究中，在[具体物种名称]的基因间区域（IGRs）中鉴定出了16个基因组位点。通过CRISPR-Cas9技术成功地将一个Ypet表达盒整合到了不同的基因组位点，整合成功率高达87.50%，并且根据插入位点的不同，表达变化高达1.91倍。该研究表明，位置效应在基因表达中表现出相对稳定性，并且基本上不受启动子和报告基因变化的影响。此外，还筛选出了一个高表达元件组合P-T。在选定的位点上反复整合来自[具体物种名称]的瓦伦烯合酶基因，使瓦伦烯产量提高到了254.67 mg/L。通过多拷贝过表达关键基因使瓦伦烯产量提高了93.49%。工程菌株L-13在3 L发酵罐中通过两阶段补料分批发酵实现了9530.18 mg/L的瓦伦烯产量。与出发菌株相比，该产量提高了近100倍,突出了所筛选的基因组位点在优化瓦伦烯生产方面的巨大潜力。
