Effect Of Overexpression Of PyrP Gene And NupC Gene On Cytidine Secretion By E.coli Fermentation

Cytosine nucleosides,also known as cytidines,are important raw materials for nucleoside health foods and pharmaceuticals,and are in great demand in the market.Cytidine is mainly obtained by microbial fermentation,but the cytidine fermentation level of the existing strain is not high and is affected by various factors.Optimizing the anabolic mode of cytidine in the cell and increasing the efficiency of cytidine secretion are the main ways to increase the fermentation unit.Cytidine may be transmembranely transported by a variety of transporters,uracil permease(pyrP,EC 3.1.3.104,encoded by the pyrP gene,molecular weight 48KDa)and nucleoside transporter(NupC,EC 2.3.1.256,by The NupC gene encoding,molecular weight 43KDa)may be involved in the cytidine secretion process.In this study,we constructed the uracil permease and nucleoside transporter NupC overexpression system to analyze the effects of overexpression of these two genes on cell growth and cytidine fermentation,and studied uracil permease and nucleoside transporter.The role of the cytidine secretion process,thereby continuously increasing the yield of bacterial cytidine fermentation.The specific research contents are as follows:1.Construction of heterologous pyrP gene overexpression vector and its effect on cytidine fermentation.The pyrP gene was amplified by PCR from Bacillus amyloliquefaciens BG-09 genomic DNA,and ligated with pSTV28 vector to construct plasmid vector pSTV28-pyrP.It was transferred to Escherichia coli BG-08 by chemical transformation to obtain recombinant strain E.coli BG-08P.After induction with 0.5 mmol/L IPTG for 6 h,the cells were collected by centrifugation,and the supernatant was sonicated and analyzed by SDS-PAGE.The results showed that the protein band was significantly enhanced at 48 kDa compared with the control group.The recombinant E.coli BG-08P was fermented at 36?,180 rpm for 40 hours,and analyzed for cell concentration,glucose consumption rate,pH,and cytidine production.The results showed that there was no significant difference in the growth of recombinant bacteria compared with the control group.Excessive expression of heterologous pyrP gene had no effect on the growth of the strain.The change trend of glucose consumption rate was consistent with the control strain.The concentration of cytidine in the fermentation broth was 0.91±0.03 g/L.,which is 1,30 times of the control strain,indicating that overexpression of the pyrP gene has a positive effect on the secretion of cytidine.2.Construction of heterologous NupC gene overexpression vector and its effect on cytidine fermentation.The NupC gene was PCR-amplified from Bacillus amyloliquefaciens BG-09 genomic DNA,ligated into the pSTV28 vector,and the plasmid vector pSTV28-NupC was constructed.The recombinant strain E.coli BG-08C was obtained by transferring it into E.coli BG-08 by chemical transformation.After induction with 0.5 mmol/L IPTG for 6 h,the cells were collected,and the supernatant was sonicated and analyzed by SDS-PAGE.The results showed that the protein band was significantly enhanced at 43 kDa compared with the control group.The recombinant E.coli BG-08C was fermented at 36?,180 rpm for 40 hours,and the cell concentration,glucose consumption rate,pH,and cytidine production during the fermentation were analyzed.The results showed that there was no significant difference in the growth of the recombinant bacteria compared with the control group.Excessive expression of the heterologous NupC gene did not affect the growth of the strain.The trend of glucose consumption rate was consistent with the control strain.The concentration of cytidine in the fermentation broth was 1.26±0.05 g/L g/L,which is 1.80 times of the control group,indicates that overexpression of the NupC gene has a positive effect on the increase of the cytidine fermentation unit.3.Construction of a tandem overexpression vector of heterologous pyrP and NupC genes and their effects on cytidine fermentation.The pyrP and NupC genes were ligated in tandem to the pSTV28 vector,and the plasmid vector pSTV28-pyrP-NupC was constructed and transformed into E.coli BG-08 to obtain recombinant E.coli BG-08PC.After induction with 0.5 mmol/L IPTG for 6 h,the cells were collected by centrifugation,and the supernatant was sonicated and analyzed by SDS-PAGE.Two distinct protein bands appeared at 43 kDa and 48 kDa.The recombinant strain E.coli BG-08PC was fermented at 36? and 180 rpm for 40 hours.The cell concentration,glucose consumption rate,pH and cytidine production in the fermentation process showed that there was no significant difference in the growth of the recombinant bacteria compared with the control group.It indicated that overexpression of heterologous tandem gene did not affect the growth of the strain.The trend of glucose consumption rate was consistent with that of the control strain.The concentration of cytidine in the fermentation broth was 1.59±0.05 g/L g/L,which was 2.27 times that of the control group.This indicates that the tandem expression of these two genes has a synergistic effect on the increase of cytidine concentration.4.The promotion of cytidine secretion by pyrP and NupC genes during fermentation.The analysis was carried out by comparing the constructed recombinant strains E.coli BG-08P,E.coli BG-08C,E.coli BG-08PC and control strains.The results showed that the concentration of bacteria,the rate of glucose consumption and the pH of the fennentation process were not significantly different from those of the control strains.The concentration of cytidine in the fermentation broth was increased to some extent,indicating that the pyrP gene was overexpressed and the uracil in the cells was increased.The formation of permease enhances the transport of pyrimidine and promotes the secretion of cytidine.Overexpression of NupC gene alone increases the synthetic dose of nucleoside transporter,triggering the alternate opening of cytidine binding sites on both sides of the cell membrane and enhancing the cell.The transport of glycosides;the expression of pyrP and NupC genes in tandem,the cytidine yield was significantly improved compared with the control strains,indicating that these two genes play a synergistic role in the cytosolic secretion process.