Identification And Functional Characterization Of Citrulline Transporters From Lactobacillus Brevis 2-34

Lactobacillus brevis 2-34 isolated from Huangjiu fermentation broth is capable of reabsorbing citrulline,which exerts potential applications to reduce the concentrations of citrulline and thereafter ethyl carbamate in Huangjiu.However,the mechanism of citrulline reabsorption yet to be illustrated hindered its proper utilization.Identification of genes encoding citrulline transporters in this strain will help get approach to this mechanism and shed light on its effective application in Huangjiu fermentation.The hypothetical citrulline transporter coding genes,dcuC and a/o antiporter,were knocked out using Cre-loxp system.It was found that the complete consumption of arginine was prolonged by the gene knockout strain,accompanied by certain amount of citrulline retained in the fermentation broth.In addition,the concentration of residual citrulline in the fermentation broth of the ?dcuC strain was higher than that of others,indicating that both of the DcuC and A/O antiporter are able to absorb the extracellular citrulline,and the former one with higher efficiency.The enzymes in arginine deiminase pathway of L.brevis 2-34 were expressed in Escherichia coli C43(DE3)using vectors pRSFDuet-1 and pETDuet-1.It was found that all recombinant E.coli strains harboring and expressing arginine deminase could degrade arginine and secrete citrulline.While,the fermentation broth of strains with plus DcuC or A/O antiporter contained less citrulline,which further indicated the roles of DcuC and A/O antiporter in transporting citrulline in L.brevis 2-34.During the citrulline reabsorbing process that happened in the mid-and late-logarithmic phase,the relative transcription levels of arc genes in L.brevis 2-34 was monitored using fluorescence quantitative PCR.It was found that the arcD gene encoding ArcD for arginine degradation was not expressed,by contrast,the dcuC and a/o antiporter genes were still highly expressed,which further proved that DcuC and A/O antiporter were positively correlated with citrulline reabsorption.The structure of DcuC and A/O antiporter were analyzed based on sequence homologous by bioinformatics methods.Both proteins were found to habour multiple transmembrance helics and a DcuC domain,which has been proved to be involved in citrulline absorbing.In addition,there were unique post-translational modification sites in each protein,which maybe correlated with the switch of secreting or absorbing of citrulline out or into the L.brevis 2-34 cell.