利用基因工程提高酿酒酵母半乳糖代谢

Improvement of Galactose Metabolism of Saccharomyces cerevisiae via Gene Engineering

为了提高酿酒酵母半乳糖利用速率,减弱葡萄糖阻遏半乳糖代谢,研究了负代谢调控基因GAL80,GAL6以及MIG1基因对半乳糖代谢的影响。通过重复利用loxp-Kan MX-loxp抗性基因对GAL80,GAL6以及MIG1基因进行单敲除、双敲除及三敲除,共构建了7株工程菌株。结果表明,7株工程菌株的半乳糖利用速率、乙醇产率均加快。其中,GAL80和MIG1双敲除菌株FY-501α的半乳糖代谢速率为0.916 g·(L·h)^(-1),较亲本提高44.71%,乙醇产率为0.329 g·(L·h)^(-1),较亲本提高46.22%,而在FY-501α基础上继续敲除GAL6得到GY-501α,其半乳糖代谢速率以及乙醇产率均未见提高。7株菌的葡萄糖阻遏效应也得到减弱,其中FY-501α的葡萄糖阻遏效应较亲本减弱42.11%,其次为EY-502α,FY-502α和FY-503α,减弱31.57%。阻断半乳糖代谢途径的负调控基因可以加速半乳糖代谢、减弱葡萄糖阻遏效应。

The galactose metabolism productivity is much lower than that of glucose in Saccharomyces cerevisiae. An effective approach was undertaken to improve galactose utilization ability and ethanol productivity via deleting the negative regulated genes （ GALSO, GAL6 and MIGI ） that controlled the expression of the GAL genes. A resistance gene fragment Ioxp-KanMX-loxp was used repeatedly to knockout the negative regulated genes GAL80, GAL6 and MIG1, and seven engineered transformants were constructed. Compared with parent AY-5α, galactose metabolism rates increased in all seven engineered transformants. The galactose uptake rates and ethanol productivity of △gal80mig1 strain FY-501α reached as high as 0.916g·（L·h）^-1 and 0. 329 g·（L·h）^-1, respectively, which increased 44.71% and 46.22% with the wild-type strain AY-5α. The glucose repression was also relieved in seven engineered strains. The consumption time in galactose-glucose mixture media of FY-501α was 66 h, with 42.11% down-off. GALSO and MIG1 played more important roles in galactose fermentation of Saccharomyces cerevisiae than GAL6 did.