Reconstruction of metabolic module with improved promoter strength increases the productivity of 2-phenylethanol in Saccharomyces cerevisiae

Abstract

Background2-phenylethanol (2-PE) is an important aromatic compound with a lovely rose-like scent. Saccharomyces cerevisiae is a desirable microbe for 2-PE production but its natural yield is not high, and one or two crucial genes’ over-expression in S. cerevisiae did not improve 2-PE greatly.ResultsA new metabolic module was established here, in which, permease Gap1p for l-phenylalanine transportation, catalytic enzymes Aro8p, Aro10p and Adh2p in Ehrlich pathway respectively responsible for transamination, decarboxylation and reduction were assembled, besides, glutamate dehydrogenase Gdh2p was harbored for re-supplying another substrate 2-oxoglutarate, relieving product glutamate repression and regenerating cofactor NADH. Due to different promoter strengths, GAP1, ARO8, ARO9, ARO10, ADH2 and GDH2 in the new modularized YS58(G1-A8-A10-A2)-GDH strain enhanced 11.6-, 15.4-, 3.6-, 17.7-, 12.4- and 7.5-folds respectively, and crucial enzyme activities of aromatic aminotransferases and phenylpyruvate decarboxylase were 4.8- and 7-folds respectively higher than that of the control.ConclusionsUnder the optimum medium and cell density, YS58(G1-A8-A10-A2)-GDH presented efficient 2-PE synthesis ability with ~ 6.3 g L−1 of 2-PE titer in 5-L fermenter reaching 95% of conversation ratio. Under fed-batch fermentation, 2-PE productivity at 24 h increased 29% than that of single-batch fermentation. Metabolic modularization with promoter strategy provides a new prospective for efficient 2-PE production.

Biosynthesis of medicinal tropane alkaloids in yeast

“…We designed a third module (III) for production of the acyl donor 1-O-β-phenyllactoylglucose (PLA glucoside) from phenylalanine via aromatic aminotransferases Aro8 and Aro9, phenylpyruvate reductase (PPR) and PLA UDP-glucosyltransferase (UGT84A27) 16 . Yeast produce 3-phenylpyruvate from phenylalanine via Aro8 and Aro9 21 , and wild-type yeast and CSY1251 produce trace levels of PLA, potentially via nonspecific activity of a lactate dehydrogenase (LDH) acting on 3-phenylpyruvate 22 . We screened PPRs from E. coli 23 , Lactobacillus (UniProt A0A2U9AUW1), A. belladonna 24 and Wickerhamia fluorescens 25 and LDHs from Bacillus and Lactobacillus with reported activity on 3-phenylpyruvate 22,26,27 via expression from a plasmid in CSY1251.…”

Sensing, Uptake and Catabolism of L-Phenylalanine During 2-Phenylethanol Biosynthesis via the Ehrlich Pathway in Saccharomyces cerevisiae

“…In addition, deficiency of NADH and/or L-Phe may limit the efficiency of 2-PE synthesis in the Ehrlich pathway. It has been demonstrated that when GDH2, GAP1, ARO8, ARO10, and ADH2 were coexpressed in S. cerevisiae YS58, the intracellular level of L-Phe was increased and NADH was regenerated, leading to the concentration of 2-PE increasing to 6.3 g/L (Wang et al, 2018).…”

“…It has been demonstrated that overexpression of ARO80 can upregulate the transcription levels of ARO9 and ARO10, resulting in a significant increase in the 2-PE titre (Kim et al, 2014). Similarly, modification of the GATA factor can also effectively improve the efficiency of 2-PE synthesis; for example, overexpression of GLN3 and GAT1 in S. cerevisiae YS58 led to an increase in 2-PE production (Wang et al, 2018).…”

EngineeringYarrowia lipolyticaas a chassis forde novosynthesis of five aromatic-derived natural products and chemicals

“…Up to date, various metabolic engineering strategies have been implemented in E. coli and S. cerevisiae to improve aromatics production, including expression of the feedback-insensitive DAHP synthases 38 and chorismite synthase, enhancing the shikimate pathway 39 and modular microbial coculture 40–42 . A number of studies have been focused on optimizing the endogenous Ehrlich pathway to produce 2-phenylethanol up to 3-6 g/L 43–45 , however, the expensive precursor L-phenylalanine was fed into the bi-phasic bioreactors 46, 47 , restricting its economic potential for large-scale production. The highest titer of 2-phenylethanol production from the de novo shikimate pathway was reported recently as 1.58 g/L (13 mM) from S. cerevisiae 38 .…”