Discovery of a new gene target enabling carotenoids improvement of recombinant Saccharomyces cerevisiae using ethanol-induced adaptive laboratory evolution

Adaptive laboratory evolution (ALE) is an increasingly popular technique, which allows screening strains with excellent phenotype through accumulative positive changes. However, little strategy based on ALE has been previously reported about improving carotenoids yield in S. cerevisiae. Herein, a new ethanol-induced ALE was successfully applied to boost carotenoids accumulation in a carotenoid producer BL03-D-4. Following the novel ALE strategy, an evolved strain M3 was obtained through adaptive evolution with a 5.1-fold increase in carotenoids yield from 3.9 mg/g CDW (cell dry weight) to 19.7 mg/g CDW in the YPD medium. Through whole-genome resequencing and reverse engineering, loss-of-function mutation of PFK1 was revealed as the cause of increased carotenoids yield. Subsequent transcriptome analysis was conducted to reveal the potential mechanisms for improved carotenoids yield, and strengthening of gluconeogenesis and downregulation of cell wall-related genes were observed in M3. This study demonstrates that ethanol is a powerful stress to obtain high carotenoids accumulation strains and provides PFK1 as a new target for improving carotenoids yield in S. cerevisiae.自适应实验室进化（Adaptive laboratory evolution，ALE）是一种日益流行的技术，可通过累积正向遗传改变筛选获得具有优良表型的菌株。然而，此前基于ALE提升酿酒酵母（S. cerevisiae）类胡萝卜素产量的策略鲜有报道。本研究成功将一种新型乙醇诱导型自适应实验室进化策略应用于类胡萝卜素生产菌株BL03-D-4，以提升其类胡萝卜素积累水平。通过该新型ALE策略，经适应性进化获得了进化菌株M3；在YPD培养基中，该菌株的类胡萝卜素产量较初始菌株提升5.1倍，从3.9 mg/g细胞干重（cell dry weight，CDW）增至19.7 mg/g CDW。通过全基因组重测序与反向工程分析，研究揭示PFK1的功能丧失性突变是类胡萝卜素产量提升的关键原因。后续转录组分析阐明了类胡萝卜素产量提升的潜在机制：在M3菌株中，糖异生通路得到增强，且与细胞壁相关的基因表达出现下调。本研究表明，乙醇可作为一种有效的胁迫因子，用于筛选获得高类胡萝卜素积累菌株，并为酿酒酵母提升类胡萝卜素产量提供了PFK1这一新的靶标。