The improved l-tryptophan production in recombinant Escherichia coli by expressing the polyhydroxybutyrate synthesis pathway

Abstract

Polyhydroxybutyrate (PHB), the best known polyhydroxyalkanoates (PHA) has been believed to change intracellular metabolic flow and oxidation/reduction state, as well as enhance stress resistance of the host. In this study, a PHB biosynthesis pathway, which contains phaCAB operon genes from Ralstonia eutropha, was introduced into an L-tryptophan producing Escherichia coli strain GPT1002. The expression of the PHB biosynthesis genes resulted in PHB accumulation inside the cells and improved the L-tryptophan production. Quantitative real-time PCR analysis showed that the transcription of tryptophan operon genes in GPT2000 increased by 1.9 to 4.3 times compared with the control, indicating that PHB biosynthesis in engineered E. coli changed the physiological state of the host. Xylose was added into the medium as co-substrate to enhance the precursor supply for PHB biosynthesis. The addition of xylose improved both extracellular L-tryptophan production and intracellular PHB accumulation. Moreover, we obtained 14.4 g l(-1) L-tryptophan production and 9.7 % PHB (w/w) accumulation in GPT2000 via fed-batch cultivation.

Single-Cell Biorefinery

“…34 The positive effect of expression of PHB synthesis genes on the production of other metabolic products has also been reported in Streptococcus zooepidemicus 70 and C. glutamicum. Quantitative real-time PCR analysis showed that the transcription of tryptophan operon genes in engineered tryptophan-producing E. coli strain increased by 1.9-4.3 times when compared with the control.…”

Modulating the direction of carbon flow in Escherichia coli to improve l -tryptophan production by inactivating the global regulator FruR

“…In view of the length of the l-tryptophan biosynthetic pathway and its complicated regulation mechanism (Bongaerts et al, 2001;Flores et al, 1996;Gu et al, 2013), relying solely on the changes in metabolism caused by a fruR deletion may not achieve a sig- Fig. 4.…”

Application of fermentation process control to increase l‐tryptophan production in Escherichia coli

“…The pH of the culture medium also influences cellular homeostasis, and numerous intracellular enzymes are optimally active and stable only in a narrow pH range near neutrality . When the pH is low, the levels of TCA enzymes SucB and SucC are elevated, whereas those of acetate‐repressed proteins are decreased (e.g., Pta, TnaA, and AroK) . Given a lowered expression level of Pta and an increased capacity of the TCA cycle, lower concentrations of acetate were accumulated at low pH .…”

“…14,15 When the pH is low, the levels of TCA enzymes SucB and SucC are elevated, whereas those of acetate-repressed proteins are decreased (e.g., Pta, TnaA, and AroK). 16,17 Given a lowered expression level of Pta and an increased capacity of the TCA cycle, lower concentrations of acetate were accumulated at low pH. 14,18 Increased production of L-tryptophan was achieved using a three-stage pH strategy (pH 6.5, 0-12 hr; pH 6.8, 12-24 hr; and pH 7.2, 24-38 hr), in which a mixture of potassium hydroxide and ammonium hydroxide was used to adjust the pH level to prevent NH 4 + concentration above the inhibitory level.…”