Cloning,expression And Functional Research Of Glucose Metabolism Transcription Factor Hex Of Pseudomonas Plecoglossicida

The transcriptional regulator HexR widely exists in Pseudomonas,and it mainly regulates the expression of genes related to glucose transport,glucose catabolism,and ED pathway.The glucose oxidation pathway in the periplasmic space of Pseudomonas produced the 2KGA,which was a crucial intermediate in the production of food antioxidant D-isoascorbic acid.Pseudomonas plecoglossicida JUIM01 is currently an industrial strain of 2KGA fermentation in China.Based on this background,this research was aimed to clone the gene of the transcription factor HexR of the strain using the PCR technology.Some relevant online analysis software and tools were used to analyze HexR protein in bioinformatics.The cloned gene was expressed by Escherichia coli expression system,and the expressed protein was further separated,purified,and identified.Then the hexR gene was knocked out by gene knockout technology and complemented.The effect of hexR gene knockout on the biosynthesis of 2KGA in P.plecoglossicida was analyzed by fermentation experiment.The main results are as follows:(1)The hexR gene fragment with a length of 873 bp was cloned from P.plecoglossicida JUIM01 using PCR technology,and the recombinant strain E.coli BL21(DE3)/p ET-28a(+)-hexR was successfully constructed.The recombinant plasmid was extracted and verified by the sequencing technology.The bioinformatics analysis results showed that the HexR protein contains 290 amino acid residues located in the cytoplasm.Besides,it was also a hydrophobic protein without transmembrane region and signal peptide.The HexR protein belongs to the Rpi R superfamily,and its theoretical molecular mass and isoelectric point were 31677.36 Da and 6.60,respectively.There were four different protein modification sites.The proportions of secondary structures such as random coil,beta-turn,extended strand,and alpha-helix were 25.17%,4.83%,11.72%,and 58.28%,respectively.(2)The constructed recombinant strain E.coli BL21(DE3)/p ET-28a(+)-hexR was induced to express,and the expression product was analyzed by SDS-PAGE and Western-blot.The results showed that the molecular weight of the protein was approximately 32.0 k Da,which was consistent with the theoretical molecular weight of the HexR protein predicted by bioinformatics methods.The HexR protein was separated and purified by affinity chromatography,dialysis,and ultrafiltration concentration,and a single-band recombinant HexR protein was obtained.The purified protein was identified by LC-MSMS and MALDI-TOF-MS.The results showed that the protein is HexR protein with a molecular weight of approximately31.914 k Da.(3)The mutant strain P.plecoglossicida JUIM01 ?hexR and the complementary strain P.plecoglossicida JUIM01 ?hexR/p BBR1MCS-2-hexR were successfully constructed,and their fermentation processes were compared at 32°C and 36°C.The results showed that when the fermentation temperature increased from 32°C to 36°C,the glucose metabolic flux shifted from the extracellular oxidation pathway to the intracellular phosphorylation pathway.The sugar-acid conversion rate of the three strains all decreased.The cell growth of the three strains was inhibited at 36°C,but the cell growth of the mutant strain was significantly better than that of the original strain and complementary strain,indicating that the knockout of the hexR gene is beneficial to cell growth.