Effects Of Exogenous Addition And Gene Heterologous Expression On Aromatics Production By Yeast

Aromatic alcohol is a kind of aromatic substance with high market value including2-phenylethanol,tryptophol and tyrosol.2-phenylethanol is added to food,cosmetics,daily necessities and tobacco products as a flavoring substance due to its delicate rose aroma.Tryptophol is a multi-functional indole derivative that promotes sleep and inhibits the growth of pathogenic fungi.Both 2-phenylethanol and tryptophol can be applied to antisepsis by their bacteriostatic action,but the natural yield of both is very low.To improve the ability of Saccharomyces cerevisiae to produce aromatic alcohol,this study first analyzed the effect of adding different substances to the medium on the production of 2-phenylethanol and tryptophol by S.cerevisiae KMLY1-2.Then,based on literature and pre-transcriptional analysis,the plant-derived genes,bacteria-derived genes and KMLY1-2 differentially expressed genes were transferred to S.cerevisiae BY4741,for gene heterologous expression analysis.The main research contents and results are as follows:(1)It was found that gibberellic acid,tyrosine and tobacco stem could significantly improve the production capacity of 2-phenylethanol from KMLY1-2 by using HPLC,and the content of 2-phenylethanol was 122.059mg/L,22.685mg/Land 26.138mg/L,respectively,more than the control(3.558mg/L).(2)Taking PAAS rose source gene,periwinkle source IAId S gene and common sources of Proteus vulgaris ml-AAD amino acid decarboxylase genes as the research object,these three genes were chemically synthesized on the basis of codon optimization,and then connected to yeast expression vector p Y26TEF-GDP.After the transformation,screening and validation,the effects of these gene expressions on the ability of BY4741 to produce aromatic alcohols were verified.The results showed that the yield of2-phenylethanol of BY4741/ p Y26TEF-GPD-PAAS strain was 4.620 mg/L,10 times that of the control sample(BY4741),the yield of 2-phenylethanol of BY4741/p Y26TEF-GPD-ml-AAD strain was 2.002 mg/L,and the yield of tryptophol of BY4741/p Y26TEF-GPD-IAId S strain was 1.471 mg/L,which was 3 times higher than the control sample(0.474mg/L).(3)Determining the transcriptome of KMLY1-2 when producing different levels of2-phenylethanol and tryptophol by RNA-seq.It was found the expression levels of the key genes(aromatic amino acid aminotransferase gene ARO9,pyruvate decarboxylase gene ARO10,alcohol dehydrogenase gene ADH5,and ARO9 and ARO10 regulator genes ARO80,etc)were significantly increased under high-yield conditions.These genes can mediate the pathway from phenylalanine to 2-phenylethanol and tryptophol.(4)Six genes have significantly difference expressed including THI4,HAC1,TSA1,THI13,ULI1,and ZRT1 were selected for heterologous expression analysis in S.cerevisiae BY4741.At present,four expression vectors have been successfully constructed,which are p Y26TEF-GPD-HAC1,p Y26TEF-GPD-TSA1,p Y26TEF-GPD-THI4,p Y26TEF-GPD-ZRT1.