Sun Meng-Chu, Chao Er-Kun, Su Xin-Yao, Zhu Min, Su Yong, Qian Guang-Tao, Chen Shi-Lin, Wang Cai-Xia, Xue Jian-Ping
College of Life Sciences, Huaibei Normal University Huaibei 235000, China Institute of Chinese Medicine,China Academy of Chinese Medical Sciences Beijing 100700, China.
Institute of Chinese Medicine,China Academy of Chinese Medical Sciences Beijing 100700, China.
Zhongguo Zhong Yao Za Zhi. 2019 Apr;44(7):1341-1349. doi: 10.19540/j.cnki.cjcmm.20190129.011.
In this study, the synthetic pathway of β-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing β-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced β-amyrin up to 5.97 mg·L(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of β-amyrin synthesis for further improving β-amyrin production, resulting the strain Y2-C2-4 which produced β-amyrin of 10.3 mg·L(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of β-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of β-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of β-amyrin-based triterpenoids.
在本研究中，通过引入甘草的β-香树脂醇合酶，在预先构建的酿酒酵母底盘菌株Y0中构建了β-香树脂醇的合成途径，得到菌株Y1-C20-6，其成功产生了高达5.97 mg·L⁻¹的β-香树脂醇。然后，过表达甲羟戊酸焦磷酸脱羧酶基因（ERG19）、甲羟戊酸激酶基因（ERG12）、3-羟基-3-甲基戊二酰辅酶A合酶基因（ERG13）、磷酸甲羟戊酸激酶基因（ERG8）和异戊烯基焦磷酸异构酶基因（IDI1），以促进代谢流朝着β-香树脂醇合成的方向流动，从而进一步提高β-香树脂醇的产量，得到菌株Y2-C2-4，其在摇瓶发酵条件下产生了10.3 mg·L⁻¹的β-香树脂醇。这比菌株Y1-C20-6的产量高出100%，说明了本研究中应用的代谢工程策略的积极效果。在补料分批发酵中，β-香树脂醇的产量进一步提高到157.4 mg·L⁻¹，几乎是菌株Y1-C20-6产量的26倍。本研究不仅为β-香树脂醇的生物合成奠定了基础，也为阐明基于β-香树脂醇的三萜类化合物的细胞色素氧化酶和糖基转移酶提供了优良的底盘菌株。
