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

Abstract 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.