酿酒酵母YBR019C基因缺失突变的分析

Reseach on Saccharomyces cerevisiae Mutant Deficient in YBR019C

关键词:  酿酒酵母  YBR019C  基因敲除  甘蔗糖蜜  乙醇

Key words:  Saccharomyces cerevisiae  YBR019C  gene deletion  sugarcan molasses  ethanol

摘要:

[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]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。

Abstract:

[Objective] Our study focuses on the saccharometabolism and ethanol fermentation of Saccharomyces cerevisiae strain NF1002.[Methods] YBR019C on Chromosome Ⅱ was chosen to be modified by PCR with pUG6 and pUG66 plasmids as templates.After homologous recombination of Cre/loxP mediated marker and YBR019C, YBR019C 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, only 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.Furthermore, according to the industrial processes for ethanol fermentation of sugarcan molasses, ethanol content was 12.015%(V/V)at 30℃ for 72h, lower than that of the wild strain.[Conclusion] Saccharometabolism of Saccharomyces cerevisiae strain NF1002 was affected by deletion of gene YBR019C, showing less ethanol production.Our research provided advice on modification of Saccharomyces cerevisiae strains on an efficient way.