New strategy for removing acetic acid as a by-product during L-tryptophan production

Abstract

Acetic acid, an important by-product of L-tryptophan production by Escherichia coli, is detrimental to cell growth and tryptophan accumulation. To reduce acetic acid accumulation and further improve the production of L-tryptophan, an acetyl-coenzyme A (acetyl-CoA) synthase (ACS) with high synthetic activity is needed. In this study, E. coli ACS was found to have a superior catalytic capability compared to that of other bacterial strains. To reduce the accumulation of acetic acid by metabolic overflow, a higher expression level of ACS was obtained by promoter engineering and copy number optimisation. Furthermore, reconstruction of the tri-carboxylic acid cycle (TCA) not only enhanced the supply of phosphoenolpyruvate (PEP), which is one of the precursors required for tryptophan synthesis, but also improved bacterial growth. The final engineered strain T16 accumulated 54.6 g/L of L-tryptophan, 11.4% more than the original strain T03, with an accumulation of only 1.4 g/L acetic acid after fermentation, which reduced by 74.5% compared to T03.

Enhancing tryptophan production by balancing precursors in Escherichia coli

“…Enhancing the intracellular level of various precursors is one of the most common approaches for improving valuable chemical production. Numerous metabolic engineering strategies to boost the intracellular levels of PEP and E4P exist, such as overexpressing tktA and ppsA (Shen et al, 2012), inactivating the PTS system (Jian Wang et al, 2013) and pykA/F (encoding for pyruvate kinases) (Y. Chen et al, 2018), and deleting ppc (encoding for PEP carboxylase) (L. Du et al, 2019). In spite of higher tryptophan titers following these manipulations, the metabolic flux to PEP and E4P has remained extremely imbalanced, limiting tryptophan production.…”

“…Tryptophan fed-batch fermentation was performed as described previously (L. Du et al, 2019), with minor modifications. In brief, seed cultures for fermentation were grown transferring fresh colonies to a 100-ml flask containing 20 ml of seed medium.…”

Recent Advances in Metabolic Engineering for the Biosynthesis of Phosphoenol Pyruvate–Oxaloacetate–Pyruvate-Derived Amino Acids

“…An engineered E. coli expressed the PEP carboxylase gene pck , citric acid transport gene citT , aconitase gene acnBA , isocitrate dehydrogenase gene icd , and pyruvate carboxylase gene pyc via chromosomal integration or promoter replacement. L-Lrp production increased to 49 g/L at a yield of 0.186 g/g glucose in a 5 L bioreactor fed-batch fermentation [ 30 ]. In addition, knocking out the gene encoding the fructose repressor FruR ( fruR ), a regulatory factor of the glycolysis synthesis route of E. coli , in the engineered strain E. coli FB-04 ( Δtrp , ΔtnaA , ΔpheA , ΔtyrA [pACYC177- aroF fbr - trpE fbr D ]) increased L-Trp titer and yield by 62.5 and 52.4%, respectively, compared to those of the parental strain [ 89 ].…”