Yang Peizhou, Wu Wenjing, Chen Jianchao, Jiang Suwei, Zheng Zhi, Deng Yanhong, Lu Jiuling, Wang Hu, Zhou Yong, Geng Yuyou, Wang Kanglin
School 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.
Biotechnol Biofuels Bioprod. 2023 Apr 12;16(1):66. doi: 10.1186/s13068-023-02312-4.
The thermotolerant yeast is beneficial in terms of efficiency improvement of processes and reduction of costs, while Saccharomyces cerevisiae does not efficiently grow and ferment at high-temperature conditions. The sterol composition alteration from ergosterol to fecosterol in the cell membrane of S. cerevisiae affects the thermotolerant capability.
In this study, S. cerevisiae ERG5, ERG4, and ERG3 were knocked out using the CRISPR-Cas9 approach to impact the gene expression involved in ergosterol synthesis. The highest thermotolerant strain was S. cerevisiae ERG5ΔERG4ΔERG3Δ, which produced 22.1 g/L ethanol at 37 °C using the initial glucose concentration of 50 g/L with an increase by 9.4% compared with the wild type (20.2 g/L). The ethanol concentration of 9.4 g/L was produced at 42 ℃, which was 2.85-fold of the wild-type strain (3.3 g/L). The molecular mechanism of engineered S. cerevisiae at the RNA level was analyzed using the transcriptomics method. The simultaneous deletion of S. cerevisiae ERG5, ERG4, and ERG3 caused 278 up-regulated genes and 1892 down-regulated genes in comparison with the wild-type strain. KEGG pathway analysis indicated that the up-regulated genes relevant to ergosterol metabolism were ERG1, ERG11, and ERG5, while the down-regulated genes were ERG9 and ERG26. S. cerevisiae ERG5ΔERG4ΔERG3Δ produced 41.6 g/L of ethanol at 37 °C with 107.7 g/L of corn liquefied glucose as carbon source.
Simultaneous deletion of ERG5, ERG4, and ERG3 resulted in the thermotolerance improvement of S. cerevisiae ERG5ΔERG4ΔERG3Δ with cell viability improvement by 1.19-fold at 42 °C via modification of steroid metabolic pathway. S. cerevisiae ERG5ΔERG4ΔERG3Δ could effectively produce ethanol at 37 °C using corn liquefied glucose as carbon source. Therefore, S. cerevisiae ERG5ΔERG4ΔERG3Δ had potential in ethanol production at a large scale under supra-optimal temperature.
耐热酵母在提高工艺效率和降低成本方面具有优势，而酿酒酵母在高温条件下生长和发酵效率不高。酿酒酵母细胞膜中麦角固醇向粪甾醇的甾醇组成改变会影响其耐热能力。
在本研究中，采用CRISPR-Cas9方法敲除酿酒酵母的ERG5、ERG4和ERG3基因，以影响参与麦角固醇合成的基因表达。耐热性最强的菌株是酿酒酵母ERG5ΔERG4ΔERG3Δ，在初始葡萄糖浓度为50 g/L时，于37℃产生22.1 g/L乙醇，与野生型（20.2 g/L）相比增加了9.4%。在42℃时产生9.4 g/L乙醇，是野生型菌株（3.3 g/L）的2.85倍。采用转录组学方法分析了工程酿酒酵母在RNA水平的分子机制。与野生型菌株相比，酿酒酵母ERG5、ERG4和ERG3基因的同时缺失导致278个基因上调和1892个基因下调。KEGG通路分析表明，与麦角固醇代谢相关的上调基因是ERG1、ERG11和ERG5，而下调基因是ERG9和ERG26。酿酒酵母ERG5ΔERG4ΔERG3Δ以107.7 g/L玉米液化葡萄糖作为碳源，在37℃时产生41.6 g/L乙醇。
同时缺失ERG5、ERG4和ERG3基因导致酿酒酵母ERG5ΔERG4ΔERG3Δ耐热性提高，通过改变类固醇代谢途径，在42℃时细胞活力提高了1.19倍。酿酒酵母ERG5ΔERG4ΔERG3Δ以玉米液化葡萄糖作为碳源，在37℃时能有效产生乙醇。因此，酿酒酵母ERG5ΔERG4ΔERG3Δ在超最佳温度下大规模生产乙醇方面具有潜力。
