Metabolic engineering of Saccharomyces cerevisiae for high-level production of (+)-ambrein from glucose

文献类型： 外文期刊

第一作者： Lin, Chumin

作者： Lin, Chumin;Chen, Yaman;Wang, Feng;Zhang, Xiaopeng;Ji, Zhongju;Fan, Baolian;Wu, Yuhong;Gan, Yuhong;Li, Zhengping;Duan, Lixin;Shang, Yi

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关键词： Saccharomyces cerevisiae; (+)-Ambrein; Tetraprenyl-beta-curcumene cyclase; Metabolic engineering

期刊名称：BIOTECHNOLOGY LETTERS （ 影响因子：2.0； 五年影响因子：2.4 ）

ISSN： 0141-5492

年卷期： 2024 年 46 卷 4 期

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收录情况： SCI

摘要： (+)-Ambrein is the primary component of ambergris, a rare product found in sperm whales (Physeter microcephalus). Microbial production using sustainable resources is a promising way to replace animal extraction and chemical synthesis. We constructed an engineered yeast strain to produce (+)-ambrein de novo. Squalene is a substrate for the biosynthesis of (+)-ambrein. Firstly, strain LQ2, with a squalene yield of 384.4 mg/L was obtained by optimizing the mevalonate pathway. Then we engineered a method for the de novo production of (+)-ambrein using glucose as a carbon source by overexpressing codon-optimized tetraprenyl-beta-curcumene cyclase (BmeTC) and its double mutant enzyme (BmeTC(Y167A/D373C)), evaluating different promoters, knocking out GAL80, and fusing the protein with BmeTC and squalene synthase (AtSQS2). Nevertheless, the synthesis of (+)-ambrein is still limited, causing low catalytic activity in BmeTC. We carried out a protein surface amino acid modification of BmeTC. The dominant mutant BmeTC(K6A/Q9E/N454A) for the first step was obtained to improve its catalytic activity. The yield of (+)-ambrein increased from 35.2 to 59.0 mg/L in the shake flask and finally reached 457.4 mg/L in the 2 L fermenter, the highest titer currently available for yeast. Efficiently engineered strains and inexpensive fermentation conditions for the industrial production of (+)-ambrein. The metabolic engineering tools provide directions for optimizing the biosynthesis of other high-value triterpenes.

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