酿酒酵母YBR019C基因缺失突变的分析

【目的】研究目的基因YBR019C缺失对酿酒酵母(Saccharomyces cerevisiae)菌株糖代谢和乙醇发酵的影响。【方法】以酿酒酵母野生菌NF1002为出发菌株,选择2号染色体上的基因YBR019C为目的基因,以质粒pUG6和pUG66为模板进行PCR,构建带有Cre/loxP系统的酿酒酵母YBR019C基因敲除组件,并转化酿酒酵母NF1002,利用筛选标记Kan r和Ble r与YBR019C基因进行同源重组,筛选YBR019C双倍体缺陷型菌株。利用蔗糖和甘蔗糖蜜为碳源,对突变菌进行发酵特性的研究。【结果】成功获得YBR019C双倍体缺陷型菌株NFybr。碳源同化实验表明,突变株和野生菌均能利用葡萄糖和蔗糖,不能利用乳糖和木糖;但相比野生菌,突变株利用棉子糖和麦芽糖的能力有所下降,而且完全不能利用半乳糖。蔗糖发酵实验表明:突变株NF-ybr与野生菌株相比,在发酵终点乙醇浓度提高10.7%,发酵周期有所延长。按目前甘蔗糖蜜乙醇生产的发酵工艺,突变株在30℃发酵72h的醪液乙醇含量为12.52%,低于野生菌的13.89%。【结论】YBR019C基因的缺失影响了菌株对糖份的利用,导致乙醇发酵能力不及野生菌。本研究为菌株高效快捷的基因改造提供了参考。

Reseach on Saccharomyces cerevisiae Mutant Deficient in YBR019C

Objective]Our study focuses on the saccharometabolism and ethanol fermentation of Saccharomyces cerevisiae strain NF1002.Methods]YBR 019C on Chromosome Ⅱ was chosen to be modified by PCR with pUG6 and pUG66 plasmids as templates.After homologous recom-bination of Cre/loxP mediated marker and YBR 019C,YBR 019C deficient mutant of Saccharomyces cerevisiae strain NF1002 was constructed.Results]Glucose and sucrose can be utilized for metabolism at both mutant and wild strains except lactose and xylose.However,on-ly part of raffinose and maltose and few galactose can be utilized at the mutant strain.Results of sucrose fermentation at mutant strain NF-ybr displayed that ethanol content reached 13.68%（V/V）at the end of fermentation,which was 10. 7% higher than that for the wild strain.Further-more,according to the industrial processes for ethanol fermentation of sugarcan molasses,etha-nol content was 12.015%（V/V）at 30℃ for 72h, lower than that of the wild strain.Conclusion]Saccharometabolism ofSaccharomyces cerevisiae strain NF1002 was affected by deletion of gene＆amp;nbsp;YBR 019C ,showing less ethanol production.Our research provided advice on modification of Saccharomyces cerevisiae strains on an efficient way.