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.

Keywords: Escherichia coli; L-phenylalanine production; Metabolic flux; Metabolomic analysis; Transcriptomic analysis.

Fig. 1

Biosynthetic pathway of L-Phe in E. coli. Single continuous arrows represent unique reactions catalyzed by one or more enzymes; two arrows represent two or more enzymatic reactions or incompletely characterized partial pathways. Details of the genes are listed in Additional file 1: Table S1

Fig. 2

Fermentation of the L-Phe-overproducing strain in a 7-L bioreactor. Triangles indicate OD₆₀₀, solid circles indicate the L-Phe titer, diamonds indicate the residual glucose concentration in fermentation broth. The results were comparable across the three replicates, and one was shown to illustrate the fermentation results

Fig. 3

a and b Volcano plot showing the differentially expressed genes in X16 vs. X24 and X24 vs. X48. Red dots indicate upregulated genes and blue dots indicate downregulated genes (padj < 0.05 and Fold change ≥ 2). padj: Corrected P value, generally less than 0.05 is considered to indicate significant difference. c and d Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed genes in X16 vs. X24 and X24 vs. X48

Fig. 4

Schematic representation of the transcriptional regulation of relevant genes involved in L-Phe biosynthesis. The two numbers indicate the fold-change in X16 vs. X24 and X24 vs. X48. Red numbers indicate significant upregulation, blue numbers indicate significant downregulation, and black numbers indicate no significant difference. Bars colored from red to blue indicates higher vs. lower gene expression, respectively

Fig. 5

Change trends of metabolites involved in central carbon metabolism and L-Phe biosynthesis at different time points. In the graph of metabolite abundance changes, the RIA value at the time point when the cell starts to accumulate the metabolite was defined as “1” to calculate the relative value of metabolites at each later time point