Identification of a novel metabolic engineering target for carotenoid production in Saccharomyces cerevisiae via ethanol-induced adaptive laboratory evolution

Carotenoids are a large family of health-beneficial compounds that have been widely used in the food and nutraceutical industries. There have been extensive studies to engineer Saccharomyces cerevisiae for the production of carotenoids, which already gained high level. However, it was difficult to discover new targets that were relevant to the accumulation of carotenoids. Herein, a new, ethanol-induced adaptive laboratory evolution was applied to boost carotenoid accumulation in a carotenoid producer BL03-D-4, subsequently, an evolved strain M3 was obtained with a 5.1-fold increase in carotenoid yield. Through whole-genome resequencing and reverse engineering, loss-of-function mutation of phosphofructokinase 1 (PFK1) was revealed as the major cause of increased carotenoid yield. Transcriptome analysis was conducted to reveal the potential mechanisms for improved yield, and strengthening of gluconeogenesis and downregulation of cell wall-related genes were observed in M3. This study provided a classic case where the appropriate selective pressure could be employed to improve carotenoid yield using adaptive evolution and elucidated the causal mutation of evolved strain. Overall design: RNA-seq of strain BL03-D-4 and M3.

类胡萝卜素（Carotenoids）是一类具有健康益处的大型化合物家族，已被广泛应用于食品及营养保健品行业。已有大量研究通过基因工程改造酿酒酵母（Saccharomyces cerevisiae）以生产类胡萝卜素，且已实现较高产量。然而，此前难以发现与类胡萝卜素积累相关的新靶点。本研究采用一种新型乙醇诱导的自适应实验室进化（adaptive laboratory evolution）策略，以提升类胡萝卜素生产菌株BL03-D-4的类胡萝卜素积累水平，最终获得进化菌株M3，其类胡萝卜素产量提升了5.1倍。通过全基因组重测序与反向工程分析，研究揭示磷酸果糖激酶1（PFK1）的功能丧失突变是类胡萝卜素产量提升的主要原因。为解析产量提升的潜在机制，本研究对菌株M3开展转录组分析，结果显示其糖异生通路被增强，且细胞壁相关基因的表达被下调。本研究提供了一个经典案例：通过自适应进化施加适宜的选择压力可有效提升类胡萝卜素产量，同时阐明了进化菌株的因果突变位点。实验整体设计：对菌株BL03-D-4与M3进行RNA测序（RNA-seq）。