Mining novel gene targets for improving tolerance to furfural and acetic acid in Yarrowia lipolytica using whole-genome clustered regularly interspaced short palindromic repeats interference library [RNA-seq]

Lignocellulosic biomass is an abundant renewable resource with tremendous potential to alleviate climate crisis. Yarrowia lipolytica is an attractive biochemical production host, while the presence of inhibitors furfural and acetic acid in lignocellulosic hydrolysate restricts the efficient utilization of this resource. Given a deficient understanding of the inherent interactions between these inhibitors and cellular metabolism, sufficiently mining relevant genes is necessary. Herein, 14 novel gene targets were discovered using CRISPR interference library in Y. lipolytica, achieving tolerance to 0.35% (v/v) acetic acid (the highest concentration reported in Y. lipolytica), 4.8 mM furfural, or a combination of 2.4 mM furfural and 0.15% (v/v) acetic acid. The tolerance mechanism might involve improvements of signal transduction, PP pathway, and TCA cycle. Transcriptional repression of effective gene targets still enabled tolerance when xylose was a carbon source. This work forms a robust foundation for significantly improving microbial tolerance to inhibitors in lignocellulosic hydrolysate and profoundly revealing underlying mechanism. In this study, a whole-genome CRISPRi library was developed in Y. lipolytica and applied for enforcing the tolerance to furfural and acetic acid. Several novel gene targets were discovered. And to analyze the tolerance mechanism caused by transcriptional repression of the most prominent gene targets, the transcriptome and proteomics were performed.

木质纤维素生物质（Lignocellulosic biomass）是储量丰富的可再生资源，在缓解气候危机方面具备巨大应用潜力。解脂耶氏酵母（Yarrowia lipolytica）是极具吸引力的生化生产宿主，但木质纤维素水解液（Lignocellulosic hydrolysate）中存在的糠醛（furfural）与乙酸（acetic acid）两类抑制剂，限制了该资源的高效利用。鉴于当前对这类抑制剂与细胞代谢间的内在互作机制认知不足，充分挖掘相关基因靶点显得尤为必要。本研究通过CRISPR干扰文库（CRISPR interference library）在解脂耶氏酵母中筛选得到14个全新基因靶点，使菌株可耐受0.35%（体积比）乙酸（为解脂耶氏酵母中已报道的最高耐受浓度）、4.8 mM糠醛，或2.4 mM糠醛与0.15%（体积比）乙酸的混合体系。该耐受机制可能涉及信号转导通路、磷酸戊糖途径（PP pathway）以及三羧酸循环（TCA cycle）的功能优化。当以木糖作为碳源时，对有效基因靶点进行转录抑制仍可赋予菌株耐受性能。本研究为大幅提升微生物对木质纤维素水解液中抑制剂的耐受能力，以及深入解析其内在耐受机制奠定了坚实基础。本研究构建了解脂耶氏酵母的全基因组CRISPR干扰文库，用于筛选可提升菌株对糠醛与乙酸耐受能力的靶点，最终获得多个全新基因靶点。为解析核心靶点转录抑制所介导的耐受机制，本研究开展了转录组与蛋白质组学分析。