挖掘功能基因,提升菌株环境胁迫耐受性,对高效利用纤维素水解液产乙醇至关重要。产甘油假丝酵母(Candida glycerinogenes)是具有多重抗逆性的工业菌株,经基因组文库筛选,获得能够提高酵母乙酸耐受性的rRNA甲基转移酶基因CgBmt5。在酿酒酵母(Saccharomyces cerevisiae)中表达CgBmt5提高了乙酸耐受性,重组菌在胁迫下乙醇产量为60.5 g/L,提高17.7%。在C.glycerinogenes中过表达CgBmt5后,乙酸胁迫下乙醇产量提高17.6%,2种过表达的单位菌体产量、底物转化率、生产强度均有提高。进一步将C.glycerinogenes过表达菌应用于纤维素水解液发酵,乙醇产量提高71.7%,转化率提高65.0%,生产强度提高155.7%。乙酸胁迫下,过表达菌中脂质过氧化水平降低,且过氧化氢酶(catalase,CAT)和超氧化物歧化酶(superoxide dismutase,SOD)活性增加;转录分析发现,Pfk1和Arg3基因上调,Gpd1和Cox3下调,表明CgBmt5可能通过降低脂质过氧化水平、提高SOD和CAT活性及影响糖代谢和精氨酸合成促进菌株的乙酸耐受和胁迫下的发酵能力。该研究为酵母的胁迫耐受机制和纤维素乙醇技术发展提供了新的生物材料。
Improving stress tolerance by functional genes is very important for efficient utilization of cellulose hydrolysate to produce ethanol. Candida glycerinogenes is an industrial strain with multiple stress tolerance. The rRNA methyltransferase gene CgBmt5 was obtained by screening of the genomic library. Overexpression of CgBmt5 in Saccharomyces cerevisiae improved acetic acid tolerance, and the ethanol yield of the recombinant bacteria reached 60.5 g/L with an increase of 17.7% under acetic acid stress. In C. glycerinogenes, overexpression of CgBmt5 also enhanced ethanol yield by 17.6% under acetic acid stress. Besides, the ethanol yield per cell, glucose conversion, and production intensity of both recombinant strains were improved. The ethanol yield, glucose conversion rate, and production intensity were improved by 71.7%, 65.0%, and 155.7%, respectively, using cellulose hydrolysate as substrate. Under acetic acid stress, the lipid peroxidation level of overexpressed strain was decreased, and the activities of superoxide dismutase (SOD) and catalase (CAT) were increased. Transcriptional analysis showed that Pfk1 and Arg3 genes were up-regulated, and Gpd1 and Cox3 genes were down-regulated in the C. glycerinogenes CgBmt5, suggesting that CgBmt5 may promote acetic acid tolerance and fermentation performance by reducing lipid peroxidation levels, increasing SOD and CAT activities, and affecting glucose metabolism and arginine synthesis. This study provides new information for the stress tolerance of yeast and the development of cellulosic ethanol technology.
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