Metabolic engineering of Escherichia coli for the efficient production of l-threonine

Abstract

l-Threonine is an important amino acid, which can be added in food, medicine or feed. In this paper, an l-threonine-producing strain was constructed using a modified CRISPR gene editing technology. Here, it was verified that the mutation of glycine at position 433 of aspartate kinase AKI to arginine (thrA ^G1297A) relieve effectively the feedback inhibition of AKI by l-threonine. The trc promoter replaced the native promoter of thrA in the Escherichia coli XQ-12 genome, and thus increasing its expression level. Moreover, by modifying the glycolytic pathway, disruption of phosphofructokinase encoded by the gene pfkA and pyruvate kinase encoded by the gene pykF increased l-threonine production. Then, the ppc gene encoding phosphoenolpyruvate carboxylase was overexpressed by replacing its native promoter with the core-trc promoter, which slightly improved the growth of the l-threonine-producing strain E. coli XQ-12 as well as the l-threonine production. In addition, it was found that further absence of the gene crr in the PTS system and the gene tdh encoding threonine dehydrogenase improved significantly l-threonine production. The final amounts of l-threonine produced by plasmid-free, antibiotic-free and inducer-free strains E. coli XQ-12.4 were 127.3 g/L and 3.536 g/L/h, respectively, in fed-batch fermentation.

Keywords

l-Threonine / Escherichia coli / Metabolic engineering / Aspartate kinase I

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Funding

the National Key Research and Development Program of China(2021YFC2100900); the National Natural Science Foundation of China(32271534); the Top-Notch Academic Programs Project of Jiangsu Higher Education Institutions(111-2-06)