Analyzing the genetic characteristics of a tryptophan-overproducing Escherichia coli

Autor: Jianping Lin, Zhu Yaru, Jinlong Li, Bai Danyang, Zhitao Mao, Dongqin Ding, Pi Liu, Hongwu Ma, Dawei Zhang
Rok vydání: 2021
Předmět:
Zdroj: Bioprocess and Biosystems Engineering. 44:1685-1697
ISSN: 1615-7605
1615-7591
DOI: 10.1007/s00449-021-02552-4
Popis: 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 3.18 g/L with a yield of 0.18 g/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.
Databáze: OpenAIRE
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