Screening Of The Yeast Strain For Resistance And Higher Production Of 2-phenylethanol With Uv Mutagenesis And GTME

2-phenylethanol(2-PE),an important flavour with a rose-like smell,is widely used in food,cosmetics and pharmaceutical industries.The production of natural 2-PE by yeast is the way to reach application value,but the inhibition of 2-phenylethanol to yeast is one of the most challenging problems in the production of 2-phenylethanol.Induced mutation breeding may improve tolerance and the production of 2-phenylethanol by yeast.In this study,UV mutation of S.cerevisiae CWY132 was carried out and two resistance mutants,UV-1 and UV-2,were screened out.They produced 7.74%and 11.3%more 2-phenylethanol than the control strain,respectively,and the molar conversion rate of L-phenylalanine to 2-phenylethanol is More than 97.9%.Global transcription machinery engineering(gTME)is a method for improving global transcription factors and cellular phenotype via error-prone PCR and DNA shuffling,which can modulate the transcriptome at a global level.In this study,effect of directed evolution of SPT15 on 2-phenylethanol tolerance of yeast was explored.We used error-prone PCR for the random mutagenesis of global transcription factor SPT15,which is coded for TATA-binding protein of yeast.Mutant library was constructed by ligating the error-prone PCR products with a pESC-TRP plasmid,and the expression plasmids were subsequently transformed to S.cerevisiae WHU2a.One resistance mutant strain PST 1 showing superior growth was screened out.The OD600 of PST1 growing in presence of 3.2 g/L 2-phenylethanol in liquid media for 40h was 1.6 fold of the control strain and it produced 32.28%more 2-phenylethanol than the control strain.Protein sequence analysis of the mutant SPT15 from mutant strain PST1 showed that two mutation sites,Ser176 Arg and Leu203 His,may be responsible for the improvement of 2-phenylethanol tolerance in PST1.This study revealed that SPT15 is an important global transcription factor for 2-phenylethanol tolerance of yeast.