| Xylose is the most abundant monosaccharide except glucose in lignocellulosic hydrolyzate,and most microbes can not use or have low utilization rate for xylose,which limited the industrial use of lignocellulosic degradation products.Bacillus licheniformis was recognized as the safe strain,which can synthesize various products such as acetoin,bacitracin,Poly-γ-glutamic acid.Therefore,B.licheniformis WX-02 as the original strain,this aim is to construct engineering strains for high utilization of xylose efficiently around the regulatory factors,transporters,xylose center metabolic pathway and downstream pathway in this study.There were two regulatory factors,Ara R1 and Ara R2,presented in the B.licheniformis WX-02.In order to investigate the effects of xylose utilization,the mutant strains WX-02△ara R1 and WX-02△ara R2 were obtained.When the gene ara R2 was deleted,the mutant strain WX-02△ara R2 did not significantly affect the use of xylose,However,after the ara R1 gene was deleted,and the biomass and xylose utilization were destroyed by strain WX-02△ara R1,but the use of glucose was not affected.The ara R1replenishment strain WX-02△ara R1/p HY300-ara R1 recover use of xylose and the growth of the cells,it is speculated that regulatory factor Ara R1 may play a positive role in the regulation of xylose metabolism,so increase the copy number of gene ara R1 to obtain the expression strain WX-02/p HY300-ara R1,but the use of xylose did not was improved,so it could not achieve the effect of increasing the use of xylose when the regulation factor Ara R1 was expressioned directly.The H~+co-transporter and belongs to a MFS transporter,was encoded by gene ara T through the NCBI database in B.licheniformis ATCC 14580.In order to investigate the effects of xylose utilization,the expression strain WX-02/p HY300-ara T was obtained,xylose utilization was more 28.91%than the control strain WX-02/p HY300,more13.42%than wild strain WX-02,and acetoin and 2,3-butanediol were significantly improved by 91.76%,69.05%than control strain,and increased by 66.89%,57.78%than the WX-02 fermented with 40 g/L xylose as substrate.The integrated strain WX-02/ZT was that the gene ara T integrated into the B.licheniformis WX-02 chromosome at amylase amy L site,the xylose utilization was more 13.54%than the control strain WX-02,the acetoin and 2,3-butanediol were also significantly increased by 34.55%and 30.05%.In this study,xylose isomerase and xylulose kinase were two key enzymes in the xylose metabolism pathway,the over-expressed strain WX-02/p HY300-xylAB was constructed.The xylose utilization was more 11.83%than control WX-02/p HY300,but it was not significant compared with the wild strain WX-02,the content of fermentation products had also been improved by 4.34%,18.91%than control strain,and increased by12.16%and 13.80%than WX-02 after 40g/L xylose fermentation.The integrated strain WX-02/ZAB was that the gene xylAB was integrated into the phage capsid protein site xkd G,utilization of xylose was more 3.32%than control strain WX-02,acetoin did not effect much then WX-02,but 2,3-butanediol was significantly improved by 22.34%.In order to further enhance the use of xylose,the genes ara T and xylAB(gene ara T was integrated amy L,xylAB was integrated xkd G site)were integrated into the chromosome in different sites at the same time to obtain mutant strain WX-02/ZABT,xylose utilization was more 15.95%than control strain WX-02,product of acetoin and2,3-butanediol was also significantly increased by 37.40%and 30.85%。In addition,this study attempted to increase the use of xylose by enhancing the ABC transport system pathway and the pentose phosphate pathway.The substrate binding protein(Xyl F1 and Xyl F2)from ABC transport system was also overexpressed to get strains WX-02/p HY300-xyl F1 and WX-02/p HY300-xyl F2,the two enzymes transketolase(Txt A)and transaldolase(Ywj H)from pentose phosphate pathway were also over-expressed to get srains WX-02/p HY300-txt A and WX-02/p HY300-ywj H,but effect of the xylose utilization was not obvious. |
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