The Preliminary Study On The Mechanism Of Improving The Utilization Rate Of Xylose In Escherichia Coli

Lignocellulose is the largest source of renewable biomass resources in the world which can be used to produce biofuels and bulk chemicals. Lignocellulose can be hydrolyzed by acid or enzyme into pentose (xylose and arabinose) and hexose (glucose, galactose and mannose). The production of bio-energy and bio-bulk chemicals by fermentation of Lignocellulose degradation liquid can effectively reduce the cost of production and reduce the environmental pollution caused by the extraction of chemicals from petroleum. Therefore, it is the key to the development of cellulose industry to convert glucose and xylose by microbial fermentation. The current use of glucose by microorganisms has been greatly improved, but there are still many difficulties in the use of xylose. In this paper, the xylose metabolism pathway and xylose transport pathway were explored.The first part:The transformation of xylose metabolism. Objective:constructing the RBS library regulate the expression of xylose metabolism gene xylA and xylB; The construction glk genes RBS library construction. Analysis effect of glk on xylose metabolism;Determinating enzyme activity xylose metabolism gene xylA, xylB and tktA, talB. Methods:the RBS library was constructed by two-step homologous recombination; Enzyme activity was determined by enzyme coupling mechanism. Results:the xylA and xylB libraries were constructed, and it was confirmed that these two genes were not rate limiting steps in xylose metabolism. Proving the gene of glucose metabolism can also improve xylose utilization and glk enzyme live the lower active and better xylose metabolism and there is glucokinase isozyme in Escherichia coli. Xylose utilization rate and growth rate of SL002 are both increased to 77% through metabolic evolution.Then,the related protein of XylE were analyzed by directed evolution and site directed mutagenesis. Objective:The construction of a xylE mutation library; The construction of site directed mutagenesis in 171,175,383,388 of XylE; knocking out four potential xylose transporter GatC, araE, araG, man PTS. Methods:The xylE mutant library was constructed on the pSC102 plasmid using CPEC method, and the site directed mutagenesis was carried out using the Site-directed Mutagenesis PCR; The two-step homologous recombination was used to knockout gene. Results:the construction of pSC102-xylE mutation library through optimization of the concentration of Mn2,which was found optimal mutation rate of 0.3%in OmM Mn2 The point mutations from 171,175,383,388 to alanine analyze the influence on transporting glucose and xylose. It is found that these amino acid can be combined with glucose can not relieve the inhibition of glucose to xylose transport, but also does not affect the xylose transporters. These amino acids will not affect the spatial structure of XylE; knocking on the GatC, araE, araG, man PTS gene will not affect the xylose transport, four gene transfer capacity is limited.