Deregulation Of Gene Responsible For Xylose Metabolism In Bacillus Subtilis And Its Phenotypic Effects

As industrial fermentation materials, the degradation products of lignocellulose possess lots of advantages, such as optimization of the resource, environmental protection, avoiding food crisis and so on. It has become one of the most important trends in technological development. The most effective and cheapest method dealing with the lignocellulose leads to a mixture of pentose and hexose, which mainly include xylose and glucose. The practical application of lignocellulose has been limited, because most industrial strains can't utilize xylose or they can't use xylose and glucose at the same time. In this study, we manipulate genes responsible for xylose metabolism in Bacillus subtilis, in order to improve the xylose metabolism in Bacillus subtilis.We construct the plasmid T-AEE which is used for knocking out araE in B. subtilis 168 by homologous recombination. The plasmid T-AEE transform into the E. coli top10 to make it clone, and then transform into the 168 competent cells. Pick up the transformant through fastness. After a series of verification, the araE is inserted by ermC successfully to get 168 araE- strain. Besides we construct the plasmid T-XC which is used for knocking out xylR in 168C by homologous recombination. The plasmid T-XC transform into the 168C competent cells. Pick up the transformant, through PCR verification and digestion verification, the xylR is inserted by cat successfully to get 168C xylR-strain.Compare the growth condition of B. subtilis 168 with 168 araE-, 168C and 168C xylR-strains in different media. In solid minimal medium, there is no obviously difference among the four strains. After 24h's grow, the colony diameter is less than 1mm. In liquid minimal medium, the growth rate and the maximum biomass of the four strains have subtle differences. In xylose fermentation medium, the maximum biomass of 168 strain is lower than 168C and 168C xylR- strains, higher than 168 araE- obviously. Growth experiments show that it is feasible to improve xylose transportation by knocking out the araR in B. subtilis 168.