Metabolic Engineering Of Xylose Utilization In Bacillus Licheniformis

Currently, high-speed global economy and dwindling reserves of petroleum resources further exacerbated by energy shortages, human show greatly interested on the development and utilization of renewable cellulosic biomass resource. As xylose is the only monosaccharide less than glucose in lignocellulosic hydrolyzate, the efficiency biological conversion of xylose become one of the key factors affecting the industrial prospects of cellulosic biomass. It has become the academic consensus to use lignocellulose or its degradation products in the fermentation industry as raw materials for fermentation to reduce costs and resolve resource crisis, However, many microbes can not metabolize xylose efficiently, and when it ferments with mixture of the carbon source such as glucose and xylose fermentation, CCR will inhibit the effects of the uptake and utilization of xylose. In this study, Bacillus licheniformis was engineered to utilize xylose stability and efficiently with genetic engineering tools.we selected the laboratory stored Bacillus licheniformis WX-02as starting strain in this study, and fistyly, we constructed the ccpA knockout strain B. licheniformis WX-02△ccpA in order to relieve the CCR effect. For acetoin fermentation, compared to the wild strain, the growth of strain WX-02△ccpA was severely hampere. It showed us that it’s failed to relieve the CCR effect by deleting the ccpA gene. It has been reported that as many as5-10%of all bacterial genes are subject to CCR, and a similar phenomenon has been happened in other strains. This effect may be due to inhibition is extremely important genes which related to the growth and metabolism of strain.In order to prevent the production of CcpA binding protein HPr, then we constructed the ptsG and ptsH knockout strain B. licheniformis WX-02PTS-, leading to CcpA lose function indirectly. For acetoin fermentation as well as WX-02AccpA, also compared to the wild strain, the results show that deletion of this gene does not have an impact on the growth of strain, and it does not affect uptake and utilization of glucose either in glucose solo medium, and results also show no significant differences between acetoin and butanediol production; Futher we detected the above indicators in the medium with60 g/L glucose and30g/L xylose mixture as substrate for acetoin fermentation under the same condition. No difference was detected as above, so it’s failed to relief the CCR by delete the gene ptsGH.For these strategies of designing for relief the CCR could not work as expected, we tried to increase the specificity transport of xylose directly by overexpress heterologous protein AraE which is form Bacillus subtilis, then we constructed the AraE overexpression strains B. licheniformis WX-Q2pHYaraE with the commercial shuttle vector pHY300. The above indicators were detected in the medium with60g/L glucose and30g/L xylose mixture as substrate for acetoin fermentation under the same condition, however, the results showed that the xylose utilization of engineering strain did not increase either;Accordingly, we tried to enhance the key enzymes of xylose metabolism pathway to achieve the intended purpose, so we constructed the xylA and xylB overexpression strains B. licheniformis WX-02pT49xylA and B. licheniformis WX-02pT49xylB with the shuttle vector pT49separately. The fermentation conditions were the same as above, no difference was detected but the engineering strain grow weaker than the wild strain, this may be due to the containing of a plasmid vector.Moreover, this study also tried to modify regulation of xyl operon, we deleted the xylR gene which encods repressor protein XylR and replaced the inducible promoter of xylA by constitutive promoter P43in order to improve efficience of xylose utilization. But all the strategies failed, it seems that more about xylose utilization on B. licheniformis needs to be explored.