Abstract:[Background] Controllable cell proliferation is one of the important strategies for achieving automatic control of fermentation. It can be easily implemented by the controlled supply of key nutrients. [Objective] To analyze the characteristics of l-alanine biosynthesis in the l-lactic acid-producing strain Escherichia coli LG101 and explore the feasibility of taking l-alanine biosynthesis as a node to regulate cell proliferation. [Methods] We employed site-specific recombination to obtain mutants by deleting the corresponding genes (avtA, alaA, alaC) of the l-alanine biosynthesis pathway on the basis of comprehensively analyzing the l-alanine biosynthesis pathway. Shake-flask and bioreactor experiments were carried out to examine the physiological and metabolic differences between the original strain and the mutants. [Results] There were at least three l-alanine biosynthesis pathways in the genome of E.coli, two of which utilized pyruvate (also the precursor for the synthesis of lactic acid) as a precursor. The relevant coding genes were deleted, and mutants LG101A (LG101 DavtA), LG102 (LG101 DalaA DalaC) and LG103 (LG101 DalaA DalaC DavtA) were obtained. In shake-flask culture, the cell proliferation of the mutants LG101A and LG102 was not significantly different from that of the original strain. However, the mutant LG103 was l-alanine auxotrophic, and its proliferation presented a positive dose-dependent correlation with l-alanine supplementation. The physiological and metabolic properties of strain LG103 were further analyzed and evaluated in a 5 L fermenter. The strain LG103 restored cell proliferation after supplementation of l-alanine, and the accumulation of l-alanine (0.12 g/L) in the fermentation broth at the end of fermentation was significantly lower than that (0.92 g/L) of the original strain. [Conclusion] E.coli LG101 over-synthesizes and accumulates l-alanine through the reduction reaction with glutamate and valine as amino donors. The mutants with removal of the corresponding synthetic pathways became l-alanine auxotrophic and their proliferation showcased a dose-dependent relationship with l-alanine supplementation. The findings suggest that l-alanine synthesis can be utilized as a nutrient switch to control cell proliferation during the fermentation of E.coli for production of major compounds such as lactic acid.
