[Efficient production of l- lysine by Escherichia coli through systems metabolic engineering].

Abstract

As an essential amino acid, l- lysine is widely used in the feed, food, and pharmaceutical industries. However, the production of l- lysine viaEscherichia coli fermentation is still plagued by inadequate substrate utilization, severe carbon atom waste, and a low sugar-to-acid conversion rate. To address these issues, this study used E. coli M2019435 as the chassis strain to generate strain HM-01 by knocking out the global regulatory factor gene mlc and heterologously expressing malp and glvA derived from Bacillus subtilis as well as the sucrose metabolism gene cluster scrKYAB. On this basis, carbon-13 labeled metabolic flux analysis (¹³C-MFA) was employed to identify the bottlenecks of pyruvate accumulation and carbon atom loss. The pyruvate carboxylase gene pyc and carbonic anhydrase gene cynT were then introduced to construct a CO₂ refixation loop. Meanwhile, feedback inhibition was relieved and the l- lysine degradation gene cadA was knocked out, resulting in the generation of strain HM-05. Further optimization of the residual sugar concentration (0.50 g/L) and carbon-to-nitrogen ratio (1:1 to 1:2) during the fermentation of strain HM-05 was conducted in a 5-L fermenter to achieve the precise supply of carbon and nitrogen sources. After 30 h of fermentation, the l- lysine titer, sugar-to-acid conversion rate, and productivity reached 210.8 g/L, 0.768 g/g, and 7.02 g/(L · h), which were 44.8%, 27.5%, and 42.7% higher than those of the original strain, respectively. The strategy of enhanced substrate utilization-targeted carbon flux optimization-upgraded process optimization developed in this study provides important support for the industrial production of l- lysine.