Research On Deflection Of Carbon Flow In L-Tryptophan Biosynthesis By Central Metabolic Modification

L-tryptophan, an essential amino acid with great value in the food and pharmaceuticals industries, which could be transformed into a variety of physiological active substances. At present, more attentions has focused on improving L-tryptophan yield using microbe fermentation. In this study, according to the L-tryptophan metabolism pathway in Escherichia coli, molecular methods were performed to enhance the synthesis of phosphoenolpyruvate(PEP) and erythrose 4-phosphate(E4P), and reduce the carbon consumption, and finally redirect carbon flows into aromatic amino acid pathway.In order to redirect carbon flows to L-tryptophan biosynthesis, two genes(an untranslated small RNA, csr B, and phosphoenolpyruvate synthase, pps A) were selected to increase PEP levels, while, two genes(transaldolase, tal B, and transketolase, tkt A) to improve E4 P levels. The four recombinant plasmids were constructed and transformed into E. coli KT1306. The results showed that the strain overexpressed pps A gene yielded only 92.3% L-tryptophan compared with E. coli KT1306. However, overexpression of csr B, tkt A or tal B improved the production of L-tryptophan about 15%, 26.7% and 30.5% respectively. In conclusion, csr B, tkt A or tal B overexpression could improve central carbon flows into aromatic amino acid pathway.Recombinants co-express csr B-tkt A or csr B-tal B were constructed to improve L-tryptophan biosynthesis. E. coli KT1306/p RSF-tac-csr B-tac-tkt A produced more L-tryptophan and the yield represented a 38.7% enhancement over the control. Under the optimal conditons, 20 g·L-1 glucose, 10 g·L-1 ammonium sulfate and 20 m L/250 m L fluid volume, the biomass and L-tryptophan yield were 7.26 g·L-1 and 2.35 g·L-1 each, increased by 2.3 and 2.69 folds.The expression of csr B or tkt A genes independent or co-expression did not affect the biomass, but caused a reduce in the rate of gucose consumption, and made the L-tryptophan yields improved by 5.9%, 15.6% and 24.7% respectively. All these strains could reduce the yields of acetic acid, succinic acid and lactic acid, and the strain with csr B gene has the least organic acids which were 88%, 6.3% and 53% of the control. The results illustrated that csr B overpresstion reduced the excess carbon in glycolysis(EMP) via strengthened the sugar dysplasia. All these strains could improve L-tryptophan yield from glucose, the strain with csr B and tkt A genes produced 0.21 g·g-1 L-tryptophan at 0.1% glucose, which was closed to the maximum theoretical yield under a non-growing culture condition of E. coli.In 3 L fermentor incubation, E. coli KT1306/p RSF-tac-csr B-tac-tkt A produced 40.1 g·L-1 L-tyrptophan, overproduced 15.7% yield. And the conversion rate of glucose was 15.4% exceed literature reported. Acetic acid, lactic acid yields were in low levels about 12.5% and 46.5% of the control, L-phe and L-tyr were less than 2 g·L-1. This study provides a new ideal to improve the production of L-tryptophan by reforming central metabolic pathway and enhancing carbon flows into aromatic amino acid pathway.