Researches On The Strain Construction And Process Optimization For Cogenerating L-homophenylalanine And 2-phenyethanol

Purposes:L-homophenylalanine(L-HPA),an unnatural chiral a-amino acid,can be used as a precursor for production of angiotensin converting enzyme(ACE)inhibitor and 2-phenylethanol(2-PEA)is a widely used aroma compound with rose-like fragrance.They are two high-value-added productions.The purposes of this research are to breed a Saccharomyces cerevisiae to simultaneously synthesize L-HPA and 2-PEA and optimize the biotransformation processes to realize the goal of cogenerating L-HPA and 2-PEA.Methods:The aspartate aminotransferase(AAT)was encoded by aspC gene.ADH1 promoter and ADH1 terminator coming from pGBKT7 plasmid as regulatory elements,and aspC gene coming from E.coli BL21(DE3)were ligated and inserted into YEp352.Then the recombinant plasmid was constructed and transformed into Saccharomyces cerevisiae by the way of lithium acetate transformation.The recombinant yeast fermentation processes,including the reaction system,the induction conditions and reaction conditions,were optimized.And the productions of L-HPA and 2-PEA were detected by high performance liquid chromatography(HPLC).Results:1.An excellent yeast strain,Saccharomyces cerevisiae YS58,was screened from our laboratory yeast strain library which could synthesize both L-HPA and 2-PEA.2.The recombinant yeast named YS-4 was successfully constructed.AAT was expressed in YS-4.There was no significant difference in growth between YS-4 and YS58.However,the transaminase activity of YS-4 was about five times than that of YS58.3.The suitable concentration of ethanol inducing AAT to be expressed was 0.5%.The main components of optimal reaction system to cogenerate L-HPA and 2-PEA were composed of 4g/L peptone,lOg/L 2-oxo-4-phenylbutyric acid(OPBA),15g/L L-phenylalanine(L-Phe),60g/L initial glucose.The optimal reaction conditions were the reaction temperature of 30 ?,initial pH value of 9.4,liquid volume of 30%,reaction time of 48 hours.On the above conditions,the productions of L-HPA and 2-PEA in YS-4 cell were up to 43.7 mmol/L and 32.4 mmol/L,which were improved 78.9%and 6.52%comparing with those in the original strain YS58,respectively.Conclusions:S.cerevisiae YS58 was screened to simultaneously synthesize L-HPA and 2-PEA.As aspC gene being transferred into YS58,the recombinant S.cerevisiae YS-4 effectively improved the productions of L-HPA and 2-PEA.After optimizing the processes,the two pathways to synthesize L-HPA and 2-PEA were almost completely coupled.And we successfully realize the goal of cogenerating L-HPA and 2-PEA in Saccharomyces cerevisiae.Furthermore,Sac char omyces cerevisiae has fine coenzyme regeneration system and the reaction system required no additional expensive coenzyme.Therefore,our research possesses a potential value for industrilization.