Excavation And Functional Analysis Of New Xylose Transporters

E.coli(Escherichia coli)is a good genetic engineering strain,it can use glucose,xylose,and other carbon sources,but at the same time with glucose and xylose as the substrate,because glucose and xylose utilization of sugar catabolite repression effect(CCR)effect,the substrate to consumption at the same time,the second growth phenomenon,limits the engineering bacterium e.coli with lignocellulose hydrolysate as raw material to produce commodity chemical applications.The key to solve this problem is:first,remove the inhibition of CCR effect on xylose metabolic pathway,and improve the metabolic rate of xylose;The second is to remove the inhibition of CCR effect on xylose transport and improve the transport rate of xylose.In the early stage,starting from Escherichia coli,our laboratory integrated the artificial promoter into the chromosome with the strategy of simultaneous editing of CRISPR/Cas9 genes,and optimized the expression of xylA,xylB,tktA and talB in xylitol metabolism pathway.The xylitol consumption rate of strain HQ304 was increased by three times.However,when strain HQ304 takes glucose and xylose as substrates,consumption of xylose is still limited.The main reason is that glucose can bind to the active site of xylose transporter XlyE,which blocks the transport of xylose into cells.Therefore,it is necessary to explore new xylose transporters to realize efficient co-utilization of glucose and xylose.In this paper,starting from HQ304,glucose metabolism pathway(pgi and ZWF were knocked out)was interrupted to make glucose become an inhibitor of xylose metabolism,and cell growth and xylose metabolism were coupled to obtain a strain HQ814 with xylose metabolism not inhibited by glucose.The resequencing results showed that the transporter XylC was mutated at N13S.Transcriptome analysis revealed a 76-fold increase in xylC*expression.Reverse metabolic engineering further demonstrated that the transporter xlyC*mutation relieved glucose inhibition and achieved xylose transport.After the recovery of pgi and ZWF genes in strain HQ814,strain HQ818 was obtained,which was able to use glucose and xylitol simultaneously.The glucose consumption rate was 0.71 g/gDWC/h,and the xylitol consumption was 0.63 g/gDWC/h.In the research process of this paper,the double-plasmid CRISPR/Cas9 gene editing system was optimized,and self-cutting module Ptrc-Cat-N20-gRNA was installed on the donor plasmid to obtain pV4 plasmid.After the current gene editing of the plasmid,the elimination of donor plasmids can be realized quickly,which is convenient for the transfer of donor plasmids and gene editing in the next round.Through the research of this thesis,the new xylose transporter XlyC*(N13S)and its function were systematically analyzed.In the course of the research,a new xylose transporter strategy was designed,which can be applied to the study of co-metabolism to relieve glucose metabolite repression.The engineering strain HQ818 obtained in this paper can simultaneously and efficiently utilize cellulose hydrolysate,expands the bio-fermentable raw materials,and provides an important platform for the production of fuel compounds and bulk chemicals.