酿酒酵母YBR019C 基因缺失突变的分析

摘要

【目的】研究目的基因YBR 019C 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 YBR 019C 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YBR 019C 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YBR 019C 基因进行同源重组，筛选YBR 019C 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YBR 019C 双倍体缺陷型菌株 NF-ybr。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】YBR 019C 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。%[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 YBR 019C ,showing less ethanol production.Our research provided advice on modification of Saccharomyces cerevisiae strains on an efficient way.