Metabolic engineering of Escherichia coli to enhance L-tryptophan biosynthesis

Abstract

L-tryptophan is an essential aromatic amino acid, which is also a precursor for the synthesis of multiple important bioactive compounds and is widely used in food additives, medicine and animal feed. There are many studies on the synthesis of L-tryptophan by microbial cell factories; however, further development has been limited by problems such as low conversion rates from glucose to L-tryptophan and dependence on antibiotics and inducers during the fermentation process. In this study, to enhance the L-tryptophan synthesis level for increasing demands, combinations of feedback-resistant enzymes AroG, TrpE and SerA were optimized, 13 synthesis-related genes (including ppsA, yddG and etc.) were overexpressed. And then the optimized aroG^S211F, trpE^{Q71K/S94N/C465Y}-trpABCD and serA^H344A/N364A expression cassette was integrated into the genome with the CRISPR-associated transposases system. The copy numbers of the expression cassette were optimized to balance the cell growth and L-tryptophan synthesis, yielding a producing strain without plasmids. To further optimize carbon flux and facilitate L-tryptophan biosynthesis, the yddG and prs^L135I was knocked in, and poxB was knocked out with CRISPR-Cas9 system. Finally, the accumulation of L-tryptophan reached 5.1 g/L in shake flask culture for 48 h, the total L-tryptophan production of the optimal strain reached 43.0 g/L (extracellular production was 30.9 g/L) under conditions of no antibiotics, inducers and other extra addition at 35 h in a 3 L bioreactor, and the total conversion rate reached 0.180 g L-tryptophan/g glucose.

Keywords

L-tryptophan / Escherichia coli / CRISPR-associated transposases system / Metabolic engineering

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Funding

National Natural Science Foundation of China(32272264)

Jiangsu Provincial Key Research and Development Program(BE2023686)

Fundamental Research Funds for the Central Universities(JUSRP122012)

Shenzhen Institute of Synthetic Biology Scientific Research Program(DWKF20210004)

RIGHTS & PERMISSIONS

Jiangnan University