Coordination of the metabolic pathway of Escherichia coli to efficiently synthesize L-alanine

• 1、Jiangnan University

Abstract： L-Alanine is the smallest chiral amino acid, which is widely used in industry, daily chemical, pharmaceutical, food and other fields. In the previous study, the authors constructed an Escherichia coli B0016-060BC strain that efficiently synthesized L-alanine through temperature control using glucose as the sole carbon source. In this study, the phosphotransacetylase-encoding gene (eutD) of this strain was further knocked out, and the synthesis of acetate as a by-product was reduced by 26.3%. The glycolysis regulatory system mediated by pyruvate sensing was used to dynamically coordinate the glucose uptake rate, and the L-alanine yield reached 182.6 g/L. The DAS+4 protein degradation tag was added to the LpdA subunit of pyruvate dehydrogenase of the recombinant strain to weaken the tricarboxylic acid cycle. The L-alanine yield of the resulting strain B0016-090BC reached 195.2 g/L, the total conversion rate was 88.6 g/100 g glucose, and the productivity was 3.07 g/L/h. The results effectively improved the synthesis of L-alanine and reduced the accumulation of by-products, which was helpful to reduce the fermentation cost of L-alanine and promote the large-scale fermentation of L-alanine.

Keywords： L-alanine, dynamic regulation, glycolysis, tricarboxylic acid cycle, scarless gene knockout