Preliminary Study Of 2-Ketogluconate Utilization Operon From Pseudomonas Plecoglossicida JUIM01

2-ketogluconate(2KGA) is one of bulk organic acids produced with fermentation, and used as the intermediate for erythorbic acid and/or sodium erythorbate production. The aim of present study is to clone 2KGA utilization operon(kgu operon) from an industrial 2KGA producer of Pseudomonas plecoglossicida JUIM01 and to find the gene compositions and biological information. On the basis of the above, the functions of the structural genes have been preliminarily verified by heterogeneous expression or gene disruption. The results of this study would provide an important theoretical foundation for further study about 2KGA metabolism and effectively increasing the 2KGA fermentation rate. The obtained conclusions are listed as follows:(1) The kgu operon with the entire nucleotide sequences of 6130 bp was firstly amplified from the strain Ps. plecoglossicida JUIM01 by LA-PCR. The sequences were predicted to be involved in the catabolism of 2KGA and revealed five open reading frames corresponding to the ptxS gene encoding 2KGA metabolism regulation protein, the kguE gene encoding 2KGA epimerase, the kguK gene encoding 2KGA kinase, the kguT gene encoding 2KGA transport and the kguD gene encoding 2KGA reductase. Analysis of the nucleotide sequences of the noncoding regions indicated that the corresponding fragment might be grouped into two transcriptional units, which were termed ptxS and kgu operon. Therefore, the kgu operon in Ps. plecoglossicida JUIM01 only consisted of 4 ORFs, which were kguE、kguK、kguT and kguD.(2) The complete sequences of kguE and kguD were cloned by TD-PCR based on the analysis results of the biological information of kgu operon, and the recombinant strains E. coli BL21(DE3)/pET-28a-kguE and E. coli BL21(DE3)/pET-28a-kguD were constructed. SDS-PAGE and Western-Blot analysis revealed that these genes were successfully expressed in the engineering bacterial strains. Two bands with molecular weights of about 29.0 ku and 36.0 ku could be detected and appeared to roughly coincide with the side of the predicted proteins encoded by kgu E and kguD respectively. Enzymatic assays revealed that KguD encoded by kguD may be required for 2KGA utilization.(3) According to the biological information of kgu operon, the kguT and kguT in-frame deletion fragment were cloned by TD-PCR and overlap-PCR, respectively. Then, the kguT deletion mutant Ps. plecoglossicida JUIM01△kguT and the kguT complementation Ps. plecoglossicida JUIM01△kguT-pBBR1MCS-2-kguT were obtained. In order to verify the functionality of the kgu T, growth ability of these strains were examined by using M9 medium containing 2KGA-Ca as the sole carbon source. The medium supported the growth of Ps. plecoglossicida JUIM01 and JUIM01△kguT- pBBR1MCS-2-kguT but not the JUIM01△kguT. The results suggested that kguT was essential for the catabolism of 2KGA and the kguT knock-out of Ps. plecoglossicida JUIM01 blocked branch catabolism of 2KGA. It will benefit for reducing the production costs with the relative advantages of 2KGA fermentation and applying for industrial production.