Analyzing the genetic characteristics of a tryptophan-overproducing Escherichia coli

Abstract

Background: L-tryptophan (L-trp) production in Escherichia coli has been developed by employing random mutagenesis and selection for a long time, but this approach produces an unclear genetic background. Results: We generated the L-trp overproducer TPD5 by combining an intracellular L-trp biosensor and uorescence-activated cell sorting (FACS) in E. coli, and succeeded in elucidating the genetic basis for Ltrp overproduction. The most signi cant identi ed positive mutations affected TnaA (deletion), AroG (S211F), TrpE (A63V), and RpoS (nonsense mutation Q33*). The underlying structure-function relationships of the feedback-resistant AroG (S211F) and TrpE (A63V) mutants were uncovered based on protein structure modeling and molecular dynamics simulations, respectively. According to transcriptomic analysis, the global regulator RpoS not only has a great in uence on cell growth and morphology, but also on carbon utilization and the direction of carbon ow. Finally, by balancing the concentrations of the L-trp precursors serine and glutamine based on the above analysis, we further increased the titer of L-trp to 3.18 g/L with a yield of 0.18 g/g. Conclusions: The analysis of the genetic characteristics of an L-trp overproducing E. coli provides valuable information on L-trp synthesis and elucidates the phenotype and complex cellular properties in a high-yielding strain, which opens the possibility to transfer bene cial mutations and reconstruct an overproducer with a clean genetic background.

Synergetic engineering of Escherichia coli for efficient production of l-tyrosine

“…These are the most used strategies for enhancing l -tyrosine production in E. coli . On this basis, the genes ppsA and tktA were expressed to increase the supply of phosphoenolpyruvate and erythrose 4-phosphate precursors of the shikimate pathway [ 30 ], the deletion of trpE blocked the branching metabolism from chorismic acid to tryptophan [ 31 ], and the deletion of pheA blocked the branching metabolism from prephenate to phenylalanine [ 32 ]. The final strain (HGA1) could produce 4.22 g/L of l -tyrosine in a shake flask after 48 h ( Fig.…”

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

“…Here, we used metabolomics and transcriptomics in an attempt to provide an answer. Time-series-based transcriptomic (16,24, and 48 h) and metabolomic (8,16,24,32,40, and 48 h) analysis were carried out in the strain. In the group comparisons X16 vs. X24 and X24 vs. X48, there were notable variations in EMP, PP, SHIK, L-Phe branch pathway, TCA cycle, and several other genes and metabolites, particularly in X16 vs. X24.…”

“…The specific ratios of mobile phases A and B in the injection process are 100% A (0-1.9 min), 43% A and 57% B (1.9-18.1 min), 100% B (18.1-22.3 min), and 100% A (22.3-26 min). The retention time of L-Phe was 13 min and the detection wavelength was 338 nm [16].…”