酿酒酵母苏氨酸合成酶缺失对高级醇生成量的影响

Effects of THR4 Gene Deletion in S.cerevisiae on Higher Alcohols Yield

通过构建酿酒酵母苏氨酸合成酶基因(THR4)缺失的工程菌株,研究该基因对酿酒酵母高级醇生成量的影响。以质粒pUC-19为载体,KanMX抗性基因为筛选标记,构建了重组质粒pUC-TABK,经PCR扩增得到YAKanMX-YB重组盒,并以酿酒酵母AY15单倍体α5为出发菌株,通过醋酸锂转化和G418抗性筛选,获得THR4基因缺失的突变株α5-T7。将转化子和亲本菌株分别进行模拟酒精发酵及酒精浓醪发酵,发酵结束后进行发酵性能和高级醇生成量的测定。结果显示,与亲本菌株相比,突变株正丙醇生成量分别提高了1.61倍和2.6倍,异戊醇生成量分别提高了0.27倍和0.24倍,而异丁醇生成量没有明显差异,表明THR4基因缺失会使酿酒酵母高级醇特别是正丙醇生成量提高。

In this study, the engineering strain with the deletion of threonine synthase gene（THR4 gene） in industrial S.cerevisiae haploid α5 was constructed to investigate the effects of THR4 deletion strain on higher alcohols yield. Upper and down stream fragments of THR4 gene TA, TB and the resistance gene KanMX were orderly inserted into vector pUC-19 to construct recombinant plasmid pUC-TABK. The recombinant cassette YA-KanMX-TB was cloned from plasmid pUC-TABK by PCR and transformed into α5, then mutant THR4 gene-deletion α5-T7 was selected by YEPD agar plates containing 1000 μg/mL G418. The Fermenting performance and higher alcohols yield of parental haploid and THR4 mutant were determined at the end of simulation ethanol fermentation and high gravity ethanol fermentation. The results showed that, the production of the n-propanol in THR4 mutant was 1.61 and 2.6 fold higher than that of parental haploid, respectively, and the production of isoamyl alcohol was increased 0.27 and 0.24 fold, compared with parental haploid, and there was no significant difference in isobutanol yield between parental haploid and THR4 mutant. The results suggested that THR4 gene deletion could increase higher alcohols yield especially the yield of n-propanol in S. cerevisiae.