Zhang Yu, Yuan Mengdi, Wu Xinxin, Zhang Qiuhui, Wang Yuzhu, Zheng Liming, Chiu Tsan-Yu, Zhang Huiming, Lan Lei, Wang Feng, Liao Ying, Gong Xuemei, Yan Shirui, Wang Yun, Shen Yue, Fu Xian
BGI Research Shenzhen China.
BGI Research Hangzhou China.
mLife. 2023 Dec 26;2(4):438-449. doi: 10.1002/mlf2.12099. eCollection 2023 Dec.
Microbial production of monoterpenoid indole alkaloids (MIAs) provides a sustainable and eco-friendly means to obtain compounds with high pharmaceutical values. However, efficient biosynthesis of MIAs in heterologous microorganisms is hindered due to low supply of key precursors such as geraniol and its derivative 8-hydroxygeraniol catalyzed by geraniol 8-hydroxylase (G8H). In this study, we developed a facile evolution platform to screen strains with improved yield of geraniol by using the SCRaMbLE system embedded in the Sc2.0 synthetic yeast and confirmed the causal role of relevant genomic targets. Through genome mining, we identified several G8H enzymes that perform much better than the commonly used CrG8H for 8-hydroxygeraniol production in vivo. We further showed that the N-terminus of these G8H enzymes plays an important role in cellular activity by swapping experiments. Finally, the combination of the engineered chassis, optimized biosynthesis pathway, and utilization of G8H led to the final strain with more than 30-fold improvement in producing 8-hydroxygeraniol compared with the starting strain. Overall, this study will provide insights into the construction and optimization of yeast cells for efficient biosynthesis of 8-hydroxygeraniol and its derivatives.
微生物生产单萜吲哚生物碱（MIAs）为获得具有高药用价值的化合物提供了一种可持续且环保的方法。然而，由于关键前体如香叶醇及其衍生物8-羟基香叶醇的供应不足，由香叶醇8-羟化酶（G8H）催化，异源微生物中MIAs的高效生物合成受到阻碍。在本研究中，我们开发了一个简便的进化平台，通过使用嵌入Sc2.0合成酵母中的SCRaMbLE系统筛选香叶醇产量提高的菌株，并证实了相关基因组靶点的因果作用。通过基因组挖掘，我们鉴定了几种G8H酶，它们在体内生产8-羟基香叶醇方面比常用的CrG8H表现要好得多。我们通过交换实验进一步表明，这些G8H酶的N端在细胞活性中起重要作用。最后，工程底盘、优化的生物合成途径和G8H的利用相结合，导致最终菌株在生产8-羟基香叶醇方面比起始菌株提高了30多倍。总体而言，本研究将为构建和优化酵母细胞以高效生物合成8-羟基香叶醇及其衍生物提供见解。
