Modular Pathway Compartmentalization for Agroclavine Overproduction in Saccharomyces cerevisiae

酿酒酵母 分区(防火) 内质网 生物化学 生产过剩 生物 酵母 细胞生物学 化学
作者
Nan Wu,Mingdong Yao,Wenhai Xiao,Tianyu Dong,Haidi Ma,Xianli Du,Ying Wang,Ying‐Jin Yuan
出处
期刊:ACS Synthetic Biology [American Chemical Society]
卷期号:12 (4): 1133-1145 被引量:8
标识
DOI:10.1021/acssynbio.2c00626
摘要

Agroclavine, which has anti-depressant activity and anti-Alzheimer effects, is the raw material used to synthesize ergo-based drugs. Although the production of agroclavine from Saccharomyces cerevisiae is possible, its yield is exceptionally low. The current study proposes a modular compartmentalization strategy for identifying and modifying the bottleneck step in agroclavine overproduction. The agroclavine synthetic pathway was reconstituted in yeast, and the best combination of Claviceps fusiformis EasA with Claviceps purpurea EasD/EasG was identified. According to the data on the expression and subcellular localization of agroclavine pathway proteins, the whole pathway was divided into two modules by chanoclavine-I. Separate enzyme distribution within the downstream module and low expression of DmaW and EasE in the upstream module were identified as the bottleneck steps in the pathway. The pathway efficiency was enhanced 2.06-fold when the downstream module was entirely anchored to the endoplasmic reticulum compartment. Increasing NADPH supply by overexpressing POS5 further improved the agroclavine yield by 27.4%. Altering the intracellular localization of DmaW from the peroxisome to the endoplasmic reticulum (ER) not only improved protein expression but also accelerated the accumulation of agroclavine by 59.9%. Integration of all modified modules into the host chromosome resulted in an improved yield of agroclavine at 101.6 mg/L with flask fermentation (a 241-fold improvement over the initial strain) and ultimately produced 152.8 mg/L of agroclavine on fed-batch fermentation. The current study unlocked the potential of S. cerevisiae in the advanced biosynthesis of ergot alkaloids. It also provides a promising strategy to reconstitute compartmentalized pathways.
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