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.

Enhancing tryptophan production by balancing precursors in Escherichia coli

“…The production of 1 mol tryptophan requires the consumption of 1 mol indol‐3‐glycerol phosphate and 1 mol serine (Troendle et al, 2020; Xiong et al, 2021), suggesting that serine is another key precursor in tryptophan biosynthesis. To enhance the pool of intracellular serine, the genes encoding for 3‐phosphoglycerate dehydrogenase ( serA* ), phosphoserine aminotransferase ( serC ), and phosphoserine phosphatase ( serB ) were selected and overexpressed under medium‐strength promoters in strain TRP8.…”

“…Because of increasing concerns about the depletion of fossil resources, climate change, and the need for environmentally sustainable manufacturing, the fermentative conversion of renewable feedstocks into tryptophan by microbial cell factories has become increasingly attractive (Lee et al, 2019; Li et al, 2020; Noda & Kondo, 2017). Corynebacterium glutamicum and Escherichia coli ( E. coli ) have proven excellent biocatalysts for tryptophan production (Katsumata & Ikeda, 1993; Xiong et al, 2021). E. coli , in particular, benefits from rapid growth, easy cultivation, metabolic plasticity, a wealth of biochemical and physiological knowledge, and a wide array of tools for genetic and genomic engineering (Niu et al, 2019; Pontrelli et al, 2018; Yang et al, 2020).…”

“…Numerous strategies have been attempted to improve tryptophan production, such as eliminating negative regulatory factors (Y. Chen et al, 2018; Li et al, 2020; Niu et al, 2019; Troendle et al, 2018; Xiong et al, 2021), blocking competing pathways (Li et al, 2020), boosting tryptophan biosynthesis (Azuma et al, 1993), increasing the levels of intracellular precursors (Diao et al, 2020; Noda & Kondo, 2017), and modifying transporters (Liu et al, 2012). Although these approaches have significantly increased tryptophan yields, the carbon flux to the precursors phosphoenolpyruvate (PEP) and erythrose‐4‐phosphate (E4P) remains extremely imbalanced and limits overall productivity.…”

Adsorption Equilibria, Kinetics, and Column Dynamics of L-Tryptophan on Mixed-Mode Resin HD-1

“…Therefore, it has been the most commonly used industrial production method for l -Trp. 9 The complex component in the fermentation broth leads to the separation and purification of l -Trp being one of the keys to the industrial production of l -Trp. After a pretreatment for removing impurities such as the microbial cell, proteins, and pigments, the fermentation broth of l -Trp also contains soluble salts (mainly NaCl and NH 4 SO 4 ) and other amino acids (mainly l -glutamic acid ( l -Glu)).…”

Melatonin biosynthesis pathways in nature and its production in engineered microorganisms

“…Finally, they rewired the PEP-pyruvate-oxaloacetate node. Fed-batch fermentation in a 5-L bioreactor produced 41.7 g/L l -tryptophan, which is the highest yield reported to date [ 41 ].…”