[Metabolic engineering of Escherichia coli for high-level L-threonine production]

L-threonine, a member of the L-aspartic acid group, is one of the amino acids that cannot be synthesized by humans and livestock. It is widely used in feed, medicine, and food fields. The fermentation production of L-threonine faces the problems of a narrow substrate utilization spectrum, slow growth of strains, and low yields. In this study, the lab-stored Escherichia coli THRN1 was used as the chassis for metabolic engineering to construct an industrial strain capable of efficiently, stably, and continuously producing L-threonine. Firstly, comparative genomic analysis was performed on the mutated strains THRN1 and FMME1 to reveal the potential metabolic mechanism of excessive accumulation of L-threonine and identify the target of further metabolic modification. Subsequently, three unreported effective thrA mutants were obtained by the MutaT7 system of directed evolution in vivo, which increased the L-threonine synthesis flux, and strain THRN2 was obtained. Secondly, to improve the industrial applicability of the strain, we knocked out mlc and introduced the allogenic glvAC (Lentibacillus salicampi) to enhance the utilization of glucose and maltose from hydrolysis of industrial starch, and obtained strain THRN7. The strain was fermented in a 5 L bioreactor for 34 h, with the L-threonine titer of 121.26 g/L and the yield of 60.47%. Finally, through the optimization of fermentation process, strain THRN7 can produce 120.42 g/L L-threonine in a 50 L bioreactor within 32 h, with the yield and productivity reaching 60.88% and 3.76 g/(L·h), respectively. In this study, a high yield L-threonine strain with no resistance and no plasmid was constructed, which laid a solid foundation for the industrial production of L-threonine.