发酵工程毕业论文完成版解说.doc

摘 要 高级醇是影响黄酒风味的重要物质之一，本文通过同源重组技术分别对支链氨基酸转氨酶的编码基因BAT1和BAT2，以及天冬氨酸β-半醛脱氢酶的编码基因HOM2进行敲除，研究基因对黄酒酵母高级醇生成量的影响。BAT1、BAT2双敲除突变株正丙醇、异丁醇和异戊醇含量BAT1突变株分别平均降低了10.12%、51.82%、25.28%，较BAT2突变株分别平均降低了10.76%、24.71%、15.72%。 （3）构建重组质粒pUC-HABK，PCR扩增重组质粒pUC-HABK上的重组盒HA-KanMX-HB并将其转化黄酒酵母二倍体，筛选获得HOM2基因一个等位基因的RY1-1。将突变株RY1-1与出发菌株RY1同时进行黄酒发酵实验，发酵结果显示：RY1-1产异戊醇的含量明显下降，比RY1降低了30.39%，并且其基本发酵性能无明显变化。RY1-1去除Kan抗性基因后，突变株相比基本保持不变。在突变株的基础上敲除基因基因个等位基因的RY1-3。将突变株RY1-3与突变株RY1-3的CO2总失重平均降低了5.0 g，酒精度平均下降了3.5度。同时，RY1-3产正丙醇、异丁醇和异戊醇含量关键词：酵母；支链氨基酸转氨酶编码基因；β-半醛脱氢酶编码基因；黄酒发酵ABSTRACT In this paper, higher alcohols as the main byproducts during yellow wine yeast fermentation were studied. BAT1 and BAT2 genes encoding branched-chain amino acid transaminase, HOM2 gene encoding aspartic β semi-aldehyde dehydrogenase were knocked out by the double homologous recombination, respectively. Then we studied the effect of gene deletion on the basic fermentation performances and higher alcohols production. (1) Recombinant plasmid pUC-BABK were constructed. The disruption cassette BA-KanMX-BB from the recombinant plasmid pUC-BABK was transformed into yellow wine yeast haploid and the BAT1 deletion strains were successfully obtained. The yellow rice wine fermentation test of BAT1 deletion strains and parental strains showed that the higher alcohols production and the basic fermentation performances were not affected by the deletion of BAT1 gene. (2) The disruption cassette BA-KanMX-BB was transformed into BAT2 deletion strains and the BAT1 and BAT2 double deletion strains were successfully obtained. The yellow rice wine fermentation test of BAT1 and BAT2 single and double deletions strains and parental strains showed that deletion of both BAT1 and BAT2 resulted in growth retardation and diminished higher alcohols production. The total loss of CO2 decreased by an average of 2.8 g, and ethanol production decreased by an average of 1.5 degrees. The content of propanol, isobutyl alcohol and isoamyl alcohol produced by BAT1 and B