Rational Modification Of Metabolic System For D-1,2,4-butanetriol Production By Recombinant Escherichia Coli

1,2,4-butanetriol(BT) is an important organic synthetic intermediate which is widely used in military industry, medicine, tobacco, cosmetics, paper making, agriculture, and polymer materials. Now commercial 1,2,4-butanetriol production mainly adopts reduction of malic acid ester catalyzed by NaBH4 or malic acid hydrogenation catalyzed by rubidium and carbon. But harsh reaction conditions, serious environmental pollution, low yield and difficulty of purification limit these methods’ application. Although biological catalysis can overcome the above disadvantages, its yield is relatively low. In our previous study, Escherichia coli was engineered for D-1,2,4-butanetriol synthesis. With the aim to further improve the efficiency of the recombinant strain, on the basis of the optimization of culture conditions, the metabolic system was optimized, the main conclusions are as follows:The effects of culture conditions on D-1,2,4-butanetriol were investigated. 10% inoculum size, 33oC, induce after cultured for 6 h after transferred, pH was controlled by adding CaCO3, 50 ml medium volume/250 ml, etc are appropriate shaking flask fermentation conditions.The relationship between energy supply, way of carbon source utilization and D-1,2,4-butanetriol synthesis were discussed. Right adding of carbon sources can promote D-1,2,4-butanetriol synthesis. Phosphoenolpyruvate: sugar phosphotransferase system(PTS system)was modified by knocking out ptsG. The reconstructed E. coli could convert D-xylose to D-1,2,4-butanetriol while growing utilizing glucose. By knocking out of gene mgsA which encodes methyl glyoxal synthase, engineered E. coli could uptake glucose and xylose simultaneously. Under the condition that the substrate concentration was 20 g/L, D-1,2,4-butanetriol titer could reach 9 g/L, the yield was about 64%. Xylose conversion is in harmony with glucose metabolism in mgsA mutant.The relationship between D-1,2,4-butenatriol synthesis pathway metabolic flux and D-1,2,4-butenatriol synthesis was investigated. On the basis of analyzing of fermentation process, the effect of xylonolactonase(xylC), xylonate dehydratase(xylD) from Caulobacter crescentus CB15 on 1,2,4-butanetriol production was investigated. The results show xylonolactone could be hydrolyzed efficiently by xylC. But the rapid accumulation of xylonate reduced the production capacity of engineering E. coli. When xylC, xylD was exploited at the same time the accumulation of xylonate significantly decreased, 1,2,4-butanetriol titer also increased to 7.9 g/L. When only xylD was exploited, 1,2,4-butanetriol titer was significantly increased and can reach 8.8 g/L, it was much higher than the control MJ133k-1’s 7.6 g/L. These results suggest that different rates of each step of reactions limit efficient synthesis of 1,2,4-butanetriol. The exploitation of xylC changed xylonolactone spontaneous hydrolysis into enzyme hydrolysis, the exploitation of xylD equipped the 1,2,4-butanetriol synthesis pathway with a specificed xylonate dehydratase, and perfected the 1,2,4-butanetriol synthesis pathway.The relationship between NADH and 1,2,4-butanetriol synthesis were investigated preliminarily. Lactic acid dehydrogenase gene ldh A was knocked out to reduce generation of competitive byproduct lactic acid which is NADH consumptive. Although 1,2,4-butanetriol production after 48 h were almost the same, the 1,2,4-butanetriol production of ldhA mutant is almost twice of MJ133k-1 when t=12h. These results indicate that the relationship between synthesis of 1,2,4-butanetriol and NADH are close.