Mukherjee Vaskar, Lenitz Ibai, Lind Ulrika, Blomberg Anders, Nygård Yvonne
Department of Life Sciences, Chalmers University of Technology, Gothenburg, Sweden.
Department of Biorefinery and Energy, RISE Research Institutes of Sweden, Örnsköldsvik, Sweden.
Eng Microbiol. 2023 Feb 3;3(2):100076. doi: 10.1016/j.engmic.2023.100076. eCollection 2023 Jun.
Formic acid is one of the main weak acids in lignocellulosic hydrolysates that is known to be inhibitory to yeast growth even at low concentrations. In this study, we employed a CRISPR interference (CRISPRi) strain library comprising >9000 strains encompassing >98% of all essential and respiratory growth-essential genes, to study formic acid tolerance in . To provide quantitative growth estimates on formic acid tolerance, the strains were screened individually on solid medium supplemented with 140 mM formic acid using the Scan-o-Matic platform. Selected resistant and sensitive strains were characterized in liquid medium supplemented with formic acid and in synthetic hydrolysate medium containing a combination of inhibitors. Strains with gRNAs targeting genes associated with chromatin remodeling were significantly enriched for strains showing formic acid tolerance. In line with earlier findings on acetic acid tolerance, we found genes encoding proteins involved in intracellular vesicle transport enriched among formic acid sensitive strains. The growth of the strains in synthetic hydrolysate medium followed the same trend as when screened in medium supplemented with formic acid. Strains sensitive to formic acid had decreased growth in the synthetic hydrolysate and all strains that had improved growth in the presence of formic acid also grew better in the hydrolysate medium. Systematic analysis of CRISPRi strains allowed identification of genes involved in tolerance mechanisms and provided novel engineering targets for bioengineering strains with increased resistance to inhibitors in lignocellulosic hydrolysates.
甲酸是木质纤维素水解产物中的主要弱酸之一，已知即使在低浓度下也会抑制酵母生长。在本研究中，我们使用了一个包含9000多个菌株的CRISPR干扰（CRISPRi）菌株库，这些菌株涵盖了所有必需基因和呼吸生长必需基因的98%以上，以研究对甲酸的耐受性。为了提供对甲酸耐受性的定量生长估计，使用Scan-o-Matic平台在补充有140 mM甲酸的固体培养基上对菌株进行单独筛选。在补充有甲酸的液体培养基和含有抑制剂组合的合成水解产物培养基中对选定的抗性和敏感菌株进行表征。靶向与染色质重塑相关基因的gRNA菌株中，显示出对甲酸耐受性的菌株显著富集。与早期关于乙酸耐受性的研究结果一致，我们发现在对甲酸敏感的菌株中，编码参与细胞内囊泡运输的蛋白质的基因富集。菌株在合成水解产物培养基中的生长趋势与在补充有甲酸的培养基中筛选时相同。对甲酸敏感的菌株在合成水解产物中的生长减少，并且在甲酸存在下生长得到改善的所有菌株在水解产物培养基中也生长得更好。对CRISPRi菌株的系统分析有助于鉴定参与耐受机制的基因，并为对木质纤维素水解产物中抑制剂具有更高抗性的生物工程菌株提供新的工程靶点。
