酿酒酵母类丙酮酸脱羧酶基因缺失对高级醇生成量的影响

Effect of YDL080C gene deletion on higher alcohols production in Saccharomyces cerevisiae haploids

摘要:

摘要：【目的】通过构建酿酒酵母类丙酮酸脱羧酶基因（YDL080C）缺失的工程菌株，研究该基因对酿酒酵母浓醪发酵产高级醇特别是异戊醇的影响。【方法】以酿酒酵母工业菌株AY-15的单倍体a-8或α-22的基因组DNA为模板，PCR分别扩增YDL080C上下游非编码区片段YA和YB；以pUG6质粒为模板，PCR扩增KanMX抗性基因片段。分别将YA、YB和KanMX片段连入pUC19载体，构建重组质粒pUC-YABK；并以其为模板，PCR扩增YA-KanMX-YB重组盒，分别电转化单倍体a-8和α-22。将转化子和亲本分别进行酒精浓醪发酵，发酵结束后测定其发酵性能和高级醇的生成量。【结果】筛选获得了YDL080C基因缺失突变株。酒精发酵后发酵性能和高级醇测定结果显示，转化子的异戊醇及总高级醇生成量与对应的单倍体亲本相比没有明显变化，但酒精度分别比亲本提高了0.6 (%, v/v) 和0.4 (%, v/v)。【结论】YDL080C基因缺失对降低酿酒酵母发酵产高级醇特别是异戊醇没有明显作用，但会使酒精度有所提高。

Abstract:

Abstract: [Objective] The deletion of pyruvate decarboxylase-like enzyme gene (YDL080C gene) in industrial Saccharomyces cerevisiae haploids a-8 and α-22 were respectively constructed, and the effect of YDL080C gene deletion on the production of higher alcohols, especially isoamyl alcohol, was investigated in high gravity ethanol fermentation. [Methods] Upper and down stream fragments YA (460 bp) and YB (630 bp) of YDL080C gene were amplified by PCR using the genomic DNA of S. cerevisiae haploids a-8 and α-22, respectively; KanMX marker for G418 resistance from plasmid pUG6 was cloned by PCR. Fragments YA, YB and KanMX were respectively inserted into the same plasmid pUC19 using EcoRI and KpnI, KpnI and BamHI, and KpnI sites in the order of YA-KanMX-YB to construct the recombined vector pUC-YABK. The recombinant cassette YA-KanMX-YB was cloned from plasmid pUC-YABK by PCR and respectively transformed into industrial yeast haploid a-8 and α-22. By the double homologous recombination, YDL080C gene deletion mutants were constructed and selected by the growth extent on YEPD agar plates containing 600 μg/mL G418. At the end of high gravity ethanol fermentation, fermentation performance and higher alcohols production of parental haploids and their mutants were determined. [Results] YDL080C deletion mutants were respectively selected from their corresponding parental haploid a-8 and α-22. The alcohol fermentation results showed that higher alcohols, especially isoamyl alcohol, were almost invariable among the mutants and their corresponding parental haploids. However, mutants yielded more amount of ethanol of 0.6 (%, v/v) and 0.4 (%, v/v) over its parental haploid, respectively. [Conclusion] Deletion of YDL080C gene in S. cerevisiae haploids has no noticeable effect on decreasing the production of higher alcohols, especially isoamyl alcohol, but it seems to somehow increase the production of ethanol.