Effect Of The Deletion Of BAT, HOM2 In Yellow Rice Wine Yeast On Production Of Higher Alcohols

Higher alcohols play an important part in the flavor of yellow rice wine. BAT1 and BAT2 genes encode branched-chain amino acid transaminases, and HOM2 gene encodes aspartic β semi-aldehyde dehydrogenase. In this paper, these genes were knocked out by the homologous recombination, respectively. Then we studied the effect of gene deletion on higher alcohols production. The main contents and results are as follows:(1) Recombinant plasmid pUC-BABK was constructed. The disruption cassette BA-KanMX-BB from the recombinant plasmid pUC-BABK was transformed into yellow rice wine yeast haploids 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 BAT2 double deletion strains decreased by an average of 10.46%,52.16% and 25.31% respectively compared with parental strains, and decreased by an average of 10.12%,51.82 % and 25.28% respectively compared with BAT1 deletion strains, and decreased by an average of 10.76%,24.71%,15.72% respectively compared with BAT2 deletion strains.(3) Recombinant plasmid pUC-HABK was constructed. The disruption cassette HA-KanMX-HB from the recombinant plasmid pUC-HABK was transformed into yellow rice wine yeast diploid and obtained one allele HOM2 gene deletion strain RY1-1. The yellow rice wine fermentation test of mutant strain RY1-1 and parental strain RY1 showed that the content of isoamyl alcohol produced by RY1-1 decreased 30.39% compared with RY1, and the basic fermentation performance of RY1-1 did not change. The KanMX of RY1-1 was deleted via Cre/Loxp system, then the mutant strain RY1-2 was obtained. The basic fermentation performances and the higher alcohols production of RY1-2 remained unchanged compared with RY1-1.(4) Recombinant plasmid pUC-HB1A1K was constructed. The disruption cassette HA1-KanMX-HBl from the recombinant plasmid pUC-HB1A1K was transformed into mutant strain RY1-2 and obtained two allele HOM2 gene deletion strain RY1-3. The yellow rice wine fermentation test of mutant strain RY1-3, RY1-2 and parental strain RY1 showed that the once again deletion of HOM2 resulted in severe growth retardation and diminished higher alcohols production greatly. The total loss of CO2 produced by RY1-3 decreased by an average of 5.0 g, and ethanol production decreased by an average of 3.5 degrees. The content of propanol, isobutyl alcohol and isoamyl alcohol produced by RY1-3 decreased 63.27%,46.80% and 66.98% respectively compared with RY1, and decreased 63.75%, 42.09% and 53.52% respectively compared with RY1-2.