Flux redistribution of central carbon metabolism for efficient production of l-tryptophan in Escherichia coli

摘要

Microbial production of L-tryptophan (L-trp) has received considerable attention because of its diverse applications in food additives and pharmaceuticals. Overexpression of rate-limiting enzymes and blockage of competing pathways can effectively promote microbial production of L-trp. However, the biosynthetic process remains suboptimal due to imbalanced flux distribution between central carbon and tryptophan metabolism, presenting a major challenge to further improvement of L-trp yield. In this study, we redistributed central carbon metabolism to improve phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) pools in an L-trp producing strain of Escherichia coli for efficient L-trp synthesis. To do this, a phosphoketolase from Bifidobacterium adolescentis was introduced to strengthen E4P formation, and the L-trp titer and yield increased to 10.8 g/L and 0.148 g/g glucose, respectively. Next, the phosphotransferase system was substituted with PEP-independent glucose transport, meditated by a glucose facilitator from Zymomonas mobilis and native glucokinase. This modification improved L-trp yield to 0.164 g/g glucose, concomitant with 58% and 40% decreases of acetate and lactate accumulation, respectively. Then, to channel more central carbon flux to the tryptophan biosynthetic pathway, several metabolic engineering strategies were applied to rewire the PEP-pyruvate-oxaloacetate node. Finally, the constructed strain SX11 produced 41.7 g/L L-trp with an overall yield of 0.227 g/g glucose after 40 h fed-batch fermentation in 5-L bioreactor. This is the highest overall yield of L-trp ever reported from a rationally engineered strain. Our results suggest the flux redistribution of central carbon metabolism to maintain sufficient supply of PEP and E4P is a promising strategy for efficient L-trp biosynthesis, and this strategy would likely also increase the production of other aromatic amino acids and derivatives.