Study On Screening The Saccharomyces Cerevisiae Mutant For 2-phenylethanol Resistance And Higher Production

2-phenylethanol is an aromatic alcohol,which is popular because of its sweet rose-like smell and widely used in food,cosmetics and pharmaceutical industries.The way to ferment2-phenylethanol of Saccharomyces cerevisiae is the main way of production of natural 2-phenylethanol,but the inhibition of2-phenylethanol to yeast is one of the most challenging problems in the production of 2-phenylethanol.Breeding of the2-phenylethanol tolerance strain of S.cerevisiae is an important way to overcome the inhibition and increase the 2-phenylethanol production.In this study,we carried out UV mutation of S.cerevisiae CWY132.We selected the conditions of 80%death rate to make10 batches of mutation breeding after analyzing lethal UV curve.A total of 150 flasks were carried out in this study and two tolerant mutants which were named YL-1 and YL-2 were screened out.We found that mutants YL-1 and YL-2 grew better than the control strain on 2.8 g/L concentration of 2-phenylethanol solid medium after subculture.The mutant YL-2showed about 10%increase of 2-phenylethanol than the control strain with High Performance Liquid Chromatography(HPLC).In this study,we constructed of the global transcription machinery engineering(gTME)method that gTME was applied to the breeding of 2-phenylethanol tolerance strain of S.cerevisiae.Firstly,we extracted genome of S.cerevisiae WHU2a and used error-prone PCR for amplifying spt15 gene fragments which encode one kind of TATA binding proteins.Secondly,the error-prone PCR producs were ligated to the vector PRS314 and which were transformed into E.coli DH5?to construct mutant library.In this study we obtained a total of 2.5×10~4 E.coli transformants.Lastly,the expression plasmids were transformed into the host S.cerevisiae WHU2a with lithium acetate method.The mutant named CY-1 was screened out in a medium containing a high concentration of 2-phenylethanol,which was showing superior tolerance.The mutant CY-1 grew better in liquid shake flasks with 3.2g/L concentration of2-phenylethanol and which was most obvious especially in the logarithmic growth phase.We found that there were three base mutations on gene mutation spt15 after TA cloning and sequencing.Following BLAST alignments of amino acid sequence which is SPT15protein of Saccharomyces cerevisiae,it can be seen that the amino acid sequence encoded by the mutant gene occurred two changes:D44E and Y53S.The mutant CY-1 showed about 13%increase of 2-phenylethanol than the control strain with High Performance Liquid Chromatography(HPLC).We speculated that mutanted amino acid sequence may be related with2-phenylethanol tolerance and mutated amino acid may be more involved in regulating the expression of 2-phenylethanol tolerance related genes.