Cloning And Expression Of 2-Ketogluconate Kinase And Its Roles In Pseudomonas Plecoglossicida JUIM01 Glucose Metabolism

2-ketogluconate(2KGA)is an important industrial organic acid,widely used in the food,pharmaceutical,cosmetic and environmental industries,and mainly used as the precursor for food antioxid erythorbic acid and/or sodium erythorbate production.In this study,the complete sequence of 2-ketogluconate kinase(KguK)was obtained by PCR from Pseudomonas plecoglossicida JUIM01,which is a major industrial strain of 2KGA production.The structures and functions of the KguK were predicted by on-line analysis tool and software.In view of that,the role of KguK protein in Pseudomonas plecoglossicida JUIM01 glucose metabolize was analysised by using E.coli expression system,Pichia pastoris expression system and Gene knockout technique respectively,which is helpful to provide the relevant theoretical basis for the improvement of 2KGA production strains.The obtained conclusions are listed as follows:(1)The gene of KguK was amplified from the strain Pseudomonas plecoglossicida JUIM01 by PCR.Then,the recombinant expression strains E.coli BL21(DE3)/pET-28a-kguK,E.coli BL21(DE3)/pET-42a-kguK,E.coli BL21(DE3)/pET-40b-kguK and E.coli Rosetta-gamiB(DE3)pLsS/pET-32a-kguK were constructed.The bioinformatics analysis showed that KguK in P.Plecoglossicida was located in the cytoplasm,containing 305 amino acid residues,having a low grand average of hydropathilic index and a conserved domain with similarity with the pfk B family.The results of recombinant strains induced by IPTG showed that KguK protein was expressed except in the E.coli BL21(DE3)/pET-40b-kguK with the form of inclusion bodies.The enzyme activity of KguK was 15.3 U/L after refolding by Chromatography which expressed in E.coli BL21(DE3)/pET-28a-kguK.(2)The codon-optimized gene fragment of KguK was obtained by the gene synthesis technique.Then,the recombinant strains P.pastoris X-33/pPICZ? B-kguK and P.pastoris X-33/pPICZ(??)B-kguK were constructed by the technology of digestion,seamless cloning,electroporation and homologous recombination.The KguK protein was expressed in the recombinant strains with methanol inducion was confirmed by SDS-PAGE and Western-Blot,but the extracellular secretion of KguK protein was extremely low in the strain P.pastoris X-33/pPICZ? B-kguK.The activity of KguK was 736.8 U/g after purified by nickel column,dialyzed and ultrafiltration.(3)According to the information of the kgu operon of Pseudomonas plecoglossicida JUIM01,the kguK in-frame deletion fragment was cloned by overlap-PCR.Then the recombinant knockout plasmid pK18mobsacB-?kguK and the kguK deletion mutant P.plecoglossicida JUIM01?kguK were constructed by molecular cloning technique and double exchange of homologous recombination,respectively.The differences of the characteristics of 2KGA fermentation between P.plecoglossicida JUIM01 and P.plecoglossicida JUIM01-?kguK showed that the phenomenon of 2KGA consumption appeared in both strains during the anaphase of the fermentation,but the consumption rates of 2KGA was lower in P.plecoglossicida JUIM01?kguK.The results showed that there may be another intracellular metabolic pathway of 2KGA in P.plecoglossicida JUIM01.