Abstract
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. Here, we generated the L-trp overproducer TPD5 by combining an intracellular L-trp biosensor and fluorescence-activated cell sorting (FACS) in E. coli, and succeeded in elucidating the genetic basis for L-trp overproduction. The most significant identified 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 influence on cell growth and morphology, but also on carbon utilization and the direction of carbon flow. 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 3.18g/L with a yield of 0.18g/g. 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 beneficial mutations and reconstruct an overproducer with a clean genetic background.
Highlights
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
To test the applicability of this biosensor, we introduced it into E. coli DH5α and induced GFPmut2 expression by adding different concentrations of the Ala-Trp dipeptide in vitro
The results showed that there was a good linear relationship between the Ala-Trp concentration and the fluorescence intensity when the dipeptide concentration was less than 2 mM (Fig. S2)
Summary
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. Due to the randomness of mutagenesis, strains developed using nontargeted strategies generally have an unclear genetic background, resulting in challenging and slow further improvement, especially in the later stages of strain improvement. Comparative genomic analysis of superior production strains with their parental ancestors can identify the underlying mutations, greatly increasing our understanding of the high-yield phenotype. This lays a foundation for avoiding unnecessary harmful mutations and identifying beneficial changes, making it possible to transfer these beneficial mutations to other strains and generate a more robust overproducer[10, 11]. Through complete genome and transcriptome sequencing, we were able to obtain comprehensive profiles of genomic changes, encompassing point mutations and indels, as well as transcriptomic changes, to guide the subsequent mutation analysis
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