College of Food and Biological Engineering, Anhui Key Laboratory of Intensive Processing of Agricultural Products, Hefei University of Technology, 420 Feicui Road, Shushan District, Hefei, 230601, Anhui, China.
Department of Biological, Food and Environment Engineering, Hefei University, 158 Jinxiu Avenue, Hefei, 230601, China.
Microb Cell Fact. 2022 Aug 13;21(1):160. doi: 10.1186/s12934-022-01885-3.
Saccharomyces cerevisiae generally consumes glucose to produce ethanol accompanied by the main by-products of glycerol, acetic acid, and lactic acid. The minimization of the formation of by-products in S. cerevisiae was an effective way to improve the economic viability of the bioethanol industry. In this study, S. cerevisiae GPD2, FPS1, ADH2, and DLD3 genes were knocked out by the Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR-Cas9) approach. The mechanism of gene deletion affecting ethanol metabolism was further elucidated based on metabolic flux and transcriptomics approaches.
The engineered S. cerevisiae with gene deletion of GPD2, FPS1, ADH2, and DLD3 was constructed by the CRISPR-Cas9 approach. The ethanol content of engineered S. cerevisiae GPD2 Delta FPS1 Delta ADH2 Delta DLD3 Delta increased by 18.58% with the decrease of glycerol, acetic acid, and lactic acid contents by 22.32, 8.87, and 16.82%, respectively. The metabolic flux analysis indicated that the carbon flux r in engineered strain increased from 60.969 to 63.379. The sequencing-based RNA-Seq transcriptomics represented 472 differential expression genes (DEGs) were identified in engineered S. cerevisiae, in which 195 and 277 genes were significantly up-regulated and down-regulated, respectively. The enriched pathways of up-regulated genes were mainly involved in the energy metabolism of carbohydrates, while the down-regulated genes were mainly enriched in acid metabolic pathways.
The yield of ethanol in engineered S. cerevisiae increased with the decrease of the by-products including glycerol, acetic acid, and lactic acid. The deletion of genes GPD2, FPS1, ADH2, and DLD3 resulted in the redirection of carbon flux.
酿酒酵母通常消耗葡萄糖来生产乙醇，同时产生甘油、乙酸和乳酸等主要副产物。减少酿酒酵母中副产物的形成是提高生物乙醇工业经济可行性的有效方法。在本研究中，通过 Clustered Regularly Interspaced Short Palindromic Repeats Cas9（CRISPR-Cas9）方法敲除了酿酒酵母的 GPD2、FPS1、ADH2 和 DLD3 基因。进一步通过代谢通量和转录组学方法阐明了基因缺失影响乙醇代谢的机制。
通过 CRISPR-Cas9 方法构建了 GPD2、FPS1、ADH2 和 DLD3 基因缺失的工程酿酒酵母。工程酿酒酵母 GPD2ΔFPS1ΔADH2ΔDLD3Δ的乙醇含量增加了 18.58%，甘油、乙酸和乳酸的含量分别降低了 22.32%、8.87%和 16.82%。代谢通量分析表明，工程菌株的碳通量 r 从 60.969 增加到 63.379。基于测序的 RNA-Seq 转录组学共鉴定出工程酿酒酵母中 472 个差异表达基因（DEGs），其中 195 个和 277 个基因分别显著上调和下调。上调基因的富集途径主要涉及碳水化合物的能量代谢，而下调基因主要富集在酸代谢途径中。
工程酿酒酵母中乙醇的产量随着甘油、乙酸和乳酸等副产物的减少而增加。GPD2、FPS1、ADH2 和 DLD3 基因的缺失导致碳通量重新定向。
