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目的:通过遗传改造和发酵工艺优化等方法进一步提高齐墩果酸酿酒酵母细胞工厂的生产能力。方法:利用多片段基因同源重组法,增加齐墩果酸酿酒酵母工程菌BY-OA中甘草β-香树脂合酶(GgbAS),蒺藜苜蓿齐墩果酸合成酶(MtOAS)和拟南芥烟酰胺腺嘌呤二核苷磷酸-细胞色素P450还原酶1(AtCPR1)等基因的拷贝数;并通过优化YPD发酵液中初糖浓度的方式提高齐墩果酸的产量。结果:增加工程菌BY-OA中GgbAS,MtOAS和AtCPR1基因的拷贝数能显著提高工程菌中目标产物的产量,获得的工程菌BY-2OA在初始葡萄糖浓度为20 g·L-1的YPD培养基中发酵7 d后β-香树脂和齐墩果酸分别达到136.5 mg·L-1(提高54%)和92.5 mg·L-1(提高30%),当初糖浓度提高到40 g·L-1时,齐墩果酸产量能达到165.7 mg·L-1。结论:新获得的工程菌BY-2OA生产齐墩果酸的能力显著提高,为发酵法生产齐墩果酸奠定了基础。
OBJECTIVE: To further improve the production capacity of oleanolic acid yeast cell factory through genetic modification and fermentation process optimization. Methods: The homologous recombination method of multi-segment gene was used to increase the content of Glycoside-β-Arylanin synthase (GgbAS), Medicago truncatula Oleanutase (MtOAS) and Arabidopsis thaliana Nicotinamide adenine dinucleotide phosphate - cytochrome P450 reductase 1 (AtCPR1) and other genes copy number; and by optimizing the YPD fermentation broth initial concentration of oleanolic acid to improve the yield. Results: Increasing the copy number of GgbAS, MtOAS and AtCPR1 in BY-OA could significantly increase the yield of the target product in the engineered bacteria. The obtained engineered BY-2OA strain was cultured in YPD with an initial glucose concentration of 20 g · L -1 After 7 days of fermentation, β-aromatase and oleanolic acid reached 136.5 mg · L-1 (increased 54%) and 92.5 mg · L-1 (increased 30%), respectively. -1, oleanolic acid yield reached 165.7 mg · L-1. CONCLUSION: The newly obtained engineering bacterium BY-2OA has significantly improved the ability of producing oleanolic acid and laid the foundation for the production of oleanolic acid by fermentation.