Efficient Production Of Xylitol From Hemicellulosic Hydrolysate Using Engineered Escherichia Coli

Xylitol is a five-carbon sugar alcohol and has sweetness similar to that of sucrose but with a much lower energy value. It has been reported to have many beneficial health properties including regulating insulin-independent metabolism in humans and preventing dental caries and a number of pediatric diseases. Industrially, xylitol is currently produced by chemical hydrogenation of D-xylose using Raney nickel catalysts. This chemical process requires expensive separation and purification steps as well as high pressure and temperature which lead to environmental pollution. Highly efficient biotechnological production of xylitol using microorganisms is gaining more attention and has been proposed as an alternative process. E. coli is an ideal host strain for efficient biotechnological production of various high-value chemical building blocks. It can be manipulated easily, grow rapidly in inexpensive media and gain high-level production of heterologous proteins and can utilize all the hemicellulosic sugars. Currently, xylitol production using E. coli from hemicellulosic hydrolyzate has been reported, but there are some problems to be solved as follows:1) soluble expression at higher temperatures; 2) the genetic stability of the host strain; 3) the by-product arabitol; 4) the cost problem. Faced with above problems, this study was carried out as follows:Firstly, to solve the problem of soluble expression at higher temperatures, a metabolically engineered Escherichia coli using plasmids has been constructed for the production of xylitol. An optimal plasmid was constructed to express xylose reductase from Neurospora crassa with almost no inclusion bodies at relatively high temperature; and the enzyme activity was increased by 5.68-fold. The phosphoenolpyruvate-dependent glucose phosphotransferase system (PTSGlc) was disrupted to eliminate catabolite repression and allow simultaneous uptake of glucose and xylose. The native pathway for D-xylose catabolism in E. coli W3110 was blocked by deleting the xylose isomerase (xylA) and xylulose kinase (xylB) genes. The putative pathway for xylitol phosphorylation was also blocked by disrupting the phosphoenolpyruvate-dependent fructose phosphotransferase system (PTSFru); and the xylitol productivity was increased by 8.71-fold. The xylitol producing recombinant E. coli allowed production of 172.4 g L-1 xylitol after 110 h of fed-batch cultivation with an average productivity of 1.57 g L-1 h-1. The molar yield of xylitol to glucose reached approximately 2.2 (mol xylitol mol-1 glucose). Furthermore, the recombinant strain also produced about 150 g L’ xylitol from hemicellulosic sugars in modified M9 minimal medium and the overall productivity was 1.40 g L-1 h-1.Secondly, a method designated as RecA-assisted chromosomal integration (RACI) for the production of valuable chemicals in Escherichia coli was developed. RACI relies on multiple-copy integration of the gene of interest into the chromosome, where their expression is controlled by the constitutive promoter P43. The IS5 sequence, which is present in multiple copies on E. coli W3110 chromosome, was chosen as the integration site to minimize unexpected effects of the knock-in on cell physiology and increase the likelihood of integration. In batch fermentation, the most successful integrated strain produced 110.1 g L-1 xylitol at 3.06 g L-1 h-1 in modified M9 minimal media from glucose-xylose mixture, which was slightly higher than that produced by strain expressing plasmids.To reduce the amount of L-arabinitol in the production of xylitol, we have made point mutation of XR to decrease L-arabinose reductase activity and a mutant with 27-fold lower activity toward L-arabinose was obtained. There were no L-arabinitol production in the shake-flask fermentation using this mutant and 47.34% of arabinose was reducted to arabinitol in the bioreator fermentation. Next, only 9.6% of arabinose was converted to L-arabinitol using the integrated strain with much lower XR activity, then 100% pure xylitol was produced using integrated strain with XR mutant from hemicellulosic hydrolysate. The combination of protein engineering and pathway engineering can lead to decrease undesired biocatalytic properties. The synergy manifested as increased selectivity such that L-arabinitol production was eliminated from an mixture of D-xylose, L-arabinose, and glucose in hemicellulosic hydrolysate.Finally, we have optimized the components of the culture media to get more actual conditions of xylitol production. We used industrial grade corn pulp powder as the nitrogen source, industrial grade glucose as the carbon source, hemicellulosic hydrolyzate as the substrate. The highest xylitol production was gained at 30℃ after evaluating the effect of temperature on the enzyme activity and xylitol productivity. Xylose and xylitol can affect the growth of the strain, so the concentration of the substrate and the opportunity for addition of substrate would directly affect xylitol productivity. After shake flask fermentation, the optimal substrate concentration was 20 g L-1 and the best time to adding the substrate was that the strain was cultured for four hours. The strain produced 143.8 g L-1 xylitol at 1.84 g L-1 h-1 in the fed-batch fermentation without byproduct arabitol.In a word, a recombinant E. coli that can produce high purity xylitol from hemicellulosic hydrolyzate using cheap medium has been achieved. This laid a good foundation for the industrial production of xylitol.