代谢工程与全基因组重组构建酿酒酵母抗逆高产乙醇菌株

将酿酒酵母海藻糖代谢工程与全基因组重组技术相结合,改良工业酿酒酵母菌株的抗逆性和乙醇发酵性能。对来源于二倍体出发菌株Zd4的两株优良单倍体Z1和Z2菌株进行杂交获得基因组重组菌株Z12,并对Z1和Z2先进行(1)过表达海藻糖-6-磷酸合成酶基因 (TPS1) ,(2)敲除海藻糖水解酶基因 (ATH1), (3)同时过表达 TPS1和敲除ATH1, 经此三种基因工程操作后再进行杂交获得代谢工程菌株的全基因组重组菌株Z12ptps1、Z12 Δath1和Z12pTΔA。与亲株Zd4相比,Z12及结合代谢工程获得的菌株在高糖、高乙醇浓度与高温条件下生长与乙醇发酵性能都有不同程度的改进。对比研究结果表明:在高糖发酵条件下,同时过表达 TPS1和敲除ATH1 的双基因操作工程菌株胞内海藻糖积累、乙醇主发酵速率和乙醇产量相对于亲株的提高幅度要大于只过表达 TPS1,或敲除ATH1 的工程菌。结合了全基因组重组后获得的二倍体工程菌株Z12pTΔA,与原始出发菌株Zd4及重组子Z12相比,主发酵速率分别提高11.4%和6.3%,乙醇产量提高7.0%和4.1%,与其胞内海藻糖含量高于其它菌株、在胁迫条件下具有更强耐逆境能力相一致。结果证明,海藻糖代谢工程与杂交介导的全基因组重组相结合,是提高酿酒酵母抗逆生长与乙醇发酵性能的有效策略与技术途径。

Construction of Saccharomyces cerevisiae Strains Improved Stress Tolerance and Ethanol Fermentation Performance through Metabolic Engineering and Genome Recombination

Some environmental stresses, i.e., high osmotic stress, fluctuating temperature and ethanol concentration, will greatly influence the viability and capability of the yeast S.cerevisiae strains during ethanol fermentation. In this study, we constructed a series of yeast strains with improved stress tolerances and ethanol fermentation performance through trehalose metabolism engineering combined with hybridization-based whole genome recombination. Firstly, two haploid strains , Z1 and Z2 isolated from the diploid strain Zd4, were engineered to enhance the intracellular trehalose by (1) overexpression of trehalose-6-phosphate synthase gene TPS1 (Z1ptps1 and Z2ptps1), (2) deletion of acidic trehalase gene ATH1 (Z1Δath1 and Z2Δath1), and (3) TPS1 overexpression combined with ATH1 deletion (Z1pTΔA and Z2pTΔA). We then obtained four recombination strains (Z12, Z12ptps1, Z12Δath1 and Z12 pTΔA) through the hybridization of Z1 and Z2, and their engineered strains. The results of high-gravity fermentation (270 g/L glucose) showed that TPS1 overexpression combined with ATH1 deletion had a distinct advantage in the improvement of stress tolerance over the single genetic manipulation. Compared to the original strain Zd4 and Z12, the strain Z12pTΔA(the hybrid of Z1pTΔA and Z2pTΔA) improved the fermentation rate by 11.4% and ethanol yield by 7.0%, while the strain Z12 without metabolic engineering only increased the main fermentation rate by 4.8% and ethanol yield by 2.8%. These improvements of fermentation performance consisted with their tolerances of the constructed strains under the conditions with osmotic pressure, high temperature and high concentration of ethanol. The combination of trehalose metabolic engineering and genome recombinant technology could effectively improve the stress tolerance and the ethanol fermentation performance of the industrial S.cerevisiae strains is demonstrated, and an innovative strategy for industrial yeast breeding is proposed.