Regulation of crucial enzymes and transcription factors on 2-phenylethanol biosynthesis via Ehrlich pathway in Saccharomyces cerevisiae.

Abstract

2-Phenylethanol (2-PE) is widely used in food, perfume and pharmaceutical industry, but lower production in microbes and less known regulatory mechanisms of 2-PE make further study necessary. In this study, crucial genes like ARO8 and ARO10 of Ehrlich pathway for 2-PE synthesis and key transcription factor ARO80 in Saccharomyces cerevisiae were re-regulated using constitutive promoter; in the meantime, the effect of nitrogen source in synthetic complete (SC) medium with L-phenylalanine (L-Phe) on Aro8/Aro9 and Aro10 was investigated. The results showed that aromatic aminotransferase activities of ARO8 over-expressing strains were seriously inhibited by ammonia sulfate in SC+Phe medium. Flask fermentation test demonstrated that over-expressing ARO8 or ARO10 led to about 42% increase in 2-PE production when compared with the control strain. Furthermore, influence of transcription factors Cat8 and Mig1 on 2-PE biosynthesis was explored. CAT8 over-expression or MIG1 deletion increased in the transcription of ARO9 and ARO10. 2-PE production of CAT8 over-expressing strain was 62% higher than that of control strain. Deletion of MIG1 also led to 2-PE biosynthesis enhancement. The strain of CAT8 over-expression and MIG1 deletion was most effective in regulating expression of ARO9 and ARO10. Analysis of mRNA levels and enzyme activities indicates that transaminase in Ehrlich pathway is the crucial target of Nitrogen Catabolize Repression (NCR). Among the engineering strains, the higher 3.73g/L 2-PE production in CAT8 over-expressing strain without in situ product recovery suggests that the robust strain has potentiality for commercial exploitation.