大肠杆菌
生物化学
代谢工程
异源表达
发酵
生物合成
异源的
生物
跨膜蛋白
酵母
蛋白质工程
酶
化学
重组DNA
计算生物学
基因
受体
作者
Yuwei Sun,Zhuo Chen,Guangyi Wang,Huajun Lv,Yuxin Mao,Ke Ma,Yong Wang
标识
DOI:10.1016/j.ymben.2022.08.001
摘要
The oxidized kaurene (Ox-Kau) compounds are the core structures of many important diterpenoids with biological activities and economical values. However, easy access to diverse Ox-Kau products is still limited by low natural abundance, and large-scale manufacture remain challenging due to lack of proper heterologous production. To achieve an abundant source alternative to natural extracts, we here report a highly effective Escherichia coli-based platform for the de novo production of multiple Ox-Kau molecules from simple carbon source. Pathway optimization in prokaryotic cells through modification of transmembrane CYP450 oxidases, cytochrome b5 co-expression and AlphaFold-based protein engineering improved a 50-fold yield of steviol (1.07 g L−1), a key intermediate in the kaurenoid biosynthesis. Combinatorial biosynthetic strategy further led to a series of oxidized derivatives (20–600 mg L−1) with rich oxygenated functional groups on C3, C7, C16 and C19 previously hard to be introduced. Our engineered strains not only laid a foundation for realizing the industrial fermentation of gram-scale ent-kaurene diterpenoids, but also provided a reliable platform for characterization and utilization of kaurene-modifying oxidases, which may generate naturally rare or unnatural ent-kaurenoids with potential bioactivity.
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