Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
Microb Cell Fact. 2022 Oct 15;21(1):214. doi: 10.1186/s12934-022-01938-7.
Acetic acid tolerance is crucial for the development of robust cell factories for conversion of lignocellulosic hydrolysates that typically contain high levels of acetic acid. Screening mutants for growth in medium with acetic acid is an attractive way to identify sensitive variants and can provide novel insights into the complex mechanisms regulating the acetic acid stress response.
An acetic acid biosensor based on the Saccharomyces cerevisiae transcription factor Haa1, was used to screen a CRISPRi yeast strain library where dCas9-Mxi was set to individually repress each essential or respiratory growth essential gene. Fluorescence-activated cell sorting led to the enrichment of a population of cells with higher acetic acid retention. These cells with higher biosensor signal were demonstrated to be more sensitive to acetic acid. Biosensor-based screening of the CRISPRi library strains enabled identification of strains with increased acetic acid sensitivity: strains with gRNAs targeting TIF34, MSN5, PAP1, COX10 or TRA1.
This study demonstrated that biosensors are valuable tools for screening and monitoring acetic acid tolerance in yeast. Fine-tuning the expression of essential genes can lead to altered acetic acid tolerance.
对于木质纤维素水解物转化的稳健细胞工厂的开发，乙酸耐受性至关重要，因为木质纤维素水解物通常含有高水平的乙酸。在含有乙酸的培养基中筛选生长的突变体是一种识别敏感变体的有吸引力的方法，可以为调节乙酸应激反应的复杂机制提供新的见解。
利用基于酿酒酵母转录因子 Haa1 的乙酸生物传感器，筛选了 CRISPRi 酵母菌株文库，其中 dCas9-Mxi 被设定为分别抑制每个必需或呼吸生长必需基因。荧光激活细胞分选导致具有更高乙酸保留的细胞群体富集。具有更高生物传感器信号的这些细胞被证明对乙酸更敏感。基于生物传感器的 CRISPRi 文库菌株筛选可鉴定出对乙酸敏感性增加的菌株：靶向 TIF34、MSN5、PAP1、COX10 或 TRA1 的 gRNA 菌株。
这项研究表明，生物传感器是筛选和监测酵母乙酸耐受性的有价值工具。精细调节必需基因的表达可能导致乙酸耐受性的改变。
