Yang Peizhou, Feng Jiaqi, Chen Jianchao
School of Food and Biological Engineering, Anhui Province Key Laboratory of Agricultural Products Modern Processing, Hefei University of Technology, Feicui Road 420, Shushan District, Hefei, 230601, China.
Appl Microbiol Biotechnol. 2025 Mar 19;109(1):67. doi: 10.1007/s00253-025-13446-w.
Strong sugar tolerance and high bioethanol yield of yeast under high-gravity fermentation have caused great attention in the bioethanol industry. In this study, Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR-Cas9) technology was used to knock out S. cerevisiae GPD2, FPS1, ADH2, DLD3, ERG5, NTH1, and AMS1 to construct engineering strain S. cerevisiae GFADENA. Under high-gravity fermentation with 400 g/L of sucrose, S. cerevisiae GFADENA produced 135 g/L ethanol, which increased 17% compared with the wild-type strain. In addition, S. cerevisiae GFADENA produced 145 g/L of ethanol by simultaneous saccharification and fermentation (SSF) using 400 g/L of corn syrup with a sugar-ethanol conversion rate of 41.1%. Further, the targeted metabolomics involving energy, amino acid, and free fatty acid metabolisms were performed to unravel its molecular mechanisms. The deletion of seven genes in S. cerevisiae GFADENA caused a more significant effect on energy metabolism compared with amino acid and free fatty acid metabolisms based on the significantly different metabolites. Two metabolites α-ketoglutaric acid and fructose-1,6-bisphosphate were the most significantly different upregulation and downregulation metabolites, respectively (p < 0.05). Functions of metabolism, environmental information processing, and genetic information processing were related to sucrose tolerance enhancement and ethanol production increase in S. cerevisiae GFADENA by the regulation of significantly different metabolites. This study provided an effective pathway to increase ethanol yield and enhance sucrose tolerance in S. cerevisiae through bioengineering modification. KEY POINTS: • S. cerevisiae GFADENA with gene deletion was constructed by the CRISPR-Cas9 approach • S. cerevisiae GFADENA could produce ethanol using high-gravity fermentation condition • The ethanol yield of 145 g/L was produced using 400 g/L corn syrup by the SSF method.
酵母在高糖发酵条件下具有较强的糖耐受性和较高的生物乙醇产量，这在生物乙醇行业引起了广泛关注。在本研究中，利用成簇规律间隔短回文重复序列Cas9（CRISPR-Cas9）技术敲除酿酒酵母的GPD2、FPS1、ADH2、DLD3、ERG5、NTH1和AMS1基因，构建工程菌株酿酒酵母GFADENA。在400 g/L蔗糖的高糖发酵条件下，酿酒酵母GFADENA产生了135 g/L乙醇，与野生型菌株相比增加了17%。此外，酿酒酵母GFADENA利用400 g/L玉米糖浆通过同步糖化发酵（SSF）产生了145 g/L乙醇，糖-乙醇转化率为41.1%。进一步进行了涉及能量、氨基酸和游离脂肪酸代谢的靶向代谢组学研究，以揭示其分子机制。基于显著差异的代谢物，酿酒酵母GFADENA中七个基因的缺失对能量代谢的影响比对氨基酸和游离脂肪酸代谢的影响更为显著。两种代谢物α-酮戊二酸和果糖-1,6-二磷酸分别是上调和下调最显著的代谢物（p < 0.05）。通过对显著差异代谢物的调控，代谢、环境信息处理和遗传信息处理功能与酿酒酵母GFADENA中蔗糖耐受性增强和乙醇产量增加有关。本研究通过生物工程改造为提高酿酒酵母乙醇产量和增强蔗糖耐受性提供了一条有效途径。要点：• 通过CRISPR-Cas9方法构建了基因缺失的酿酒酵母GFADENA • 酿酒酵母GFADENA可在高糖发酵条件下生产乙醇 • 通过SSF方法利用400 g/L玉米糖浆生产的乙醇产量为145 g/L
