Abstract
Optimizing the supply of biosynthetic precursors and cofactors is usually an effective metabolic strategy to improve the production of target compounds. Here, the combination of optimizing precursor synthesis and balancing cofactor metabolism was adopted to improve tryptophan production in Escherichia coli. First, glutamine synthesis was improved by expressing heterologous glutamine synthetase from Bacillus subtilis and Bacillus megaterium in the engineered Escherichia coli strain KW001, resulting in the best candidate strain TS-1. Then icd and gdhA were overexpressed in TS-1, which led to the accumulation of 1.060 g/L tryptophan. Subsequently, one more copy of prs was introduced on the chromosome to increase the flux of 5-phospho-α-d-ribose 1-diphosphate followed by the expression of mutated serA and thrA to increase the precursor supply of serine, resulting in the accumulation of 1.380 g/L tryptophan. Finally, to maintain cofactor balance, sthA and pntAB, encoding transhydrogenase, were overexpressed. With sufficient amounts of precursors and balanced cofactors, the engineered strain could produce 1.710 g/L tryptophan after 48 h of shake-flask fermentation, which was 2.76-times higher than the titer of the parent strain. Taken together, our results demonstrate that the combination of optimizing precursor supply and regulating cofactor metabolism is an effective approach for high-level production of tryptophan. Similar strategies could be applied to the production of other amino acids or related derivatives.
Highlights
Tryptophan, which is essential for mammals, is a critical aromatic amino acid that has been widely applied in food, pharmaceuticals, animal husbandry and other fields [1,2]
The glutamine biosynthesis pathway was optimized by overexpressing icd and gdhA, as well as a heterologous Bacillus subtilis glnA (BSglnA) in E. coli KW001
We developed a strategy based on the combined optimization of these three precursors biosynthesis and cofactor metabolism, which effectively improved tryptophan production in the engineered strains derived from KW001
Summary
Tryptophan, which is essential for mammals, is a critical aromatic amino acid that has been widely applied in food, pharmaceuticals, animal husbandry and other fields [1,2]. One strategy relies on the overexpression of ppsA [10,11], knockdown of pykAF [12] and knockout of ppc [13] This can increase the intracellular pool of PEP and strengthen the metabolic flux through the shikimate pathway, which is essential for aromatic amino acid synthesis. Liu et al introduced a heterologous phosphoketolase pathway into yeast to enhance E4P synthesis [17], a strategy that may be suited for E. coli as well It has been established in the literature that the optimization of PEP and E4P synthesis can improve tryptophan production [18], but the synthesis of another three precursors, glutamine, serine and 5-phospho-α-D-ribose 1-diphosphate (PRPP), was rarely optimized. The integrated strategy presented here can offer a reference for constructing efficient strains for the overproduction of tryptophan meeting the needs of industry
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