Transcriptomics and metabolomics analysis of L-phenylalanine overproduction in Escherichia coli

Abstract

Background: Highly efficient production of L-phenylalanine (L-Phe) in E. coli has been achieved by multiple rounds of random mutagenesis and modification of key genes of the shikimate (SHIK) and L-Phe branch pathways. In this study, we performed transcriptomic (16, 24 and 48 h) and metabolomic analyses (8, 16, 24, 32,40, and 48 h) based on time sequences in an engineered E. coli strain producing L-Phe, aiming to reveal the overall changes of metabolic activities during the fermentation process. Results: The largest biomass increase rate and the highest production rate were seen at 16 h and 24 h of fermentation, respectively reaching 5.9 h−1 and 2.76 g/L/h, while the maximal L-Phe titer of 60 g/L was accumulated after 48 h of fermentation. The DEGs and metabolites involved in the EMP, PP, TCA, SHIIK and L-Phe-branch pathways showed significant differences at different stages of fermentation. Specifically, the significant upregulation of genes encoding rate-limiting enzymes (aroD and yidB) and key genes (aroF, pheA and aspC) pushed more carbon flux toward the L-Phe synthesis. The RIA changes of a number of important metabolites (DAHP, DHS, DHQ, Glu and PPN) enabled the adequate supply of precursors for high-yield L-Phe production. In addition, other genes related to Glc transport and phosphate metabolism increased the absorption of Glc and contributed to rerouting the carbon flux into the L-Phe-branch. Conclusions: Transcriptomic and metabolomic analyses of an L-Phe overproducing strain of E. coli confirmed that precursor supply was not a major limiting factor in this strain, whereas the rational distribution of metabolic fluxes was achieved by redistributing the carbon flux (for example, the expression intensity of the genes tyrB, aspC, aroL and aroF/G/H or the activity of these enzymes is increased to some extent), which is the optimal strategy for enhancing L-Phe production.