Enhanced Production Of Xylitol From Hemicellulose Hydrolysate By Using Engineered Escherichia Coli

Xylitol is a five-carbon sugar alcohol, whose sweetness is similar to sucrose. Since its property of dental caries prevention and insulin-free metabolism, it has been widely used in food, light and pharmacy industry. In recent years, the need of xylitol was highly increased. The traditional method to produce xylitol is catalytic hydrogenation. However, this needs high temperature and high pressure, as well as using Raney nickel catalysts.The cost of this method is high and this method is not environmentally friendly. Currently, biological production of xylitol, especially metabolic engineering method, has received more attention. This paper selected an engineered E.coli strain as the host to produce xylitol. Hemicellulose hydrolysate was selected as the substrate and the conditions of fermentation were studied. Through these researches, we obtained a process which was suitable for industrial production.This paper selected an engineered E. coli W3110 Ptrc99a-rbs-xr6600,△ptsG, △xylA,△xylB,△F)as a target host and pure xylose as substrate. First, the best cultural conditions were determined in shake flask:seedage was 6h; ph was 7.0; Liquid volume was 50mL/250mL; Inoculation amount was 10%; rotation speed was 200r/min; Meanwhile, the induced conditions were determined:the induced time was 4h; IPTG concentration was 0.1mM; Induced temperature was 30℃. In the 5L fermenter, the conditions were determined as follows:dissolved oxygen was 30%; rotation speed was 500r/min and ventilation volume was 0.6vvm. The concentrations of substrates were determined as 100g/L xylose and 50g/L glucose. On this basis, we performed the fed-batch fermentation and determined the best fed-batch method. The concentration of xylitol reached 172g/L. The productivity reached 1.7g/L · h and the yield reached 0.95g/g.Using the pure xylose as substrate is not economical. Since the cost is too high, it is impossible to use this in large-scale production. Then, we used hemicellulose hydrolysate as substrate. We selected engineered E. coli W3110 (Ptrc99a-rbs-xr6600, △ptsG, △xylA,△xylB,△F) as the host to produce xylitol. First, we adjusted the component of medium, and successfully used corn syrup as nitrogen source, rather than yeast powder and peptone, which was decreased the cost a lot. Since the component of medium had changed, the induced conditions had also been adjusted: induced time was 6h; The IPTG concentration was 0.05mM; The induced temperature was 30℃. Because of the complex composition of hydrolysate, a lot of inhibitory factors exist in the hemicellulose hydrolysate. The concentrations of factors are high when the concentration of hemicellulose hydrolysate is high. Hence, the concentration of substrate was also adjusted. We determined 60g/L xylose and 30g/L glucose were the best concentrations of substrates. On this basis, we performed fed-batch fermentation and determined the best fed-batch method. The concentration of xylitol reached 166g/L. The productivity reached 1.7g/L·h and the yield reached 0.93g/g. This laid a foundation for the large-scale production of xylitol.