Study On ALD4 Gene Disruption And GPD1 Silencing In Saccharomyces Cerevisiae Industrial Strain Y01

Acetic acid and glycerol are the major by-product in Saccharomyces cerevisiae ethanol fermentation.In this paper,S.cerevisiae Y01 aldehyde dehydrogenase ALD4 gene was disrupted.ALD4 gene deletion mutants DY01 was obtained.The production of S.cerevisiae ethanol was increased by GPDl silenced via antisense expression vector. The results were described as follows:1, PCR-based loxP-KanMX-loxP and Cre/loxP system apply to delete the industrial strain S.cerevisiae Y01 aldehyde dehydrogenase ALD4 gene.The production of ethanol was improved with 6.88% compared to the original strain Y01.2, Glycerol dehydrogenase GPDl gene 5'UTR Antisense expression vector pYES2.0-GPD1 was constructed for S.cerevisiae INVScl. When compared to the original strain, glycerol phosphate dehydrogenase (GPDH) activity 24.03% reduction and glycerol formation 25.43% reduction respectively.3, Antisense expression vector pYES2.0-GPDl/KanMX was constructed for glycerol dehydrogenase GPD1 gene 5'UTR of S.cerevisiae mutant strain DY01.When compared to the original strain DY01, S.cerevisiae DRY01glycerol dehydrogenase (GPDH) activity 20.04% reduction, glycerol formation 19.14% reduction respectively. At same time, the production of S.cerevisiae DRY01 ethanol fermentation increased by 9.74% compared with DY01.LoxP-KanMX-loxP gene disruption cassette and Cre/loxP system is an effective way to genetically modified via homologous integration,which breaks through difficulties of genetic engineers in industrial S.cerevisiae. We are confornted with lacking effective selectable marker and genetic diversity in industrial S.cerevisiae strain. ALD4 gene deleting is good for ethanol metabolism pathway in industrial S.cerevisiae.Antisence RNA complementary the 5'UTR of GPD1 mRNA triggers effective silencing in S.cerevisiae industrial strain DY01.