Study Of 2-keto-D-gluconic Acid Synthetic Pathway And Its Modification In Pseudomonas Plecoglossicida JUIM01

2-Keto-D-gluconic acid?2KGA?is a skeleton material for the synthesis of heterocyclic and chiral compounds,which is an important intermediate for the production of food-grade antioxidant D-isoascorbic acid approved by FDA.The industrial production of 2KGA by fermentation approach is the most economical,efficient and environmentally friendly method.In this study,an industrial 2KGA-producing strain Pseudomonas plecoglossicida JIUM01 was employed as a research object.The conversion rate of sugar and acid of the strain could reach to the value of 90%,but its productivity was relatively low,which could not meet the increasingly demanding requirements of energy saving and consumption reduction.By analyzing the synthetic pathway of 2KGA in P.plecoglossicida JUIM01,the main enzymes,coenzymes and electron transfer pathways involved in the synthetic pathway of 2KGA were identified.On this basis,P.plecoglossicida JUIM01 was metabolically engineered to improve the productivity of 2KGA.The main research results were as follows:?1?The synthetic pathway of 2KGA in P.plecoglossicida JUIM01 was analyzed.The whole genome of P.plecoglossicida JUIM01 was sequenced by high-throughput Illumina sequencing technique.The results showed that its chromosome size was 5,074,901 bp,the G+C content was 63.6%,the number of ORF was 4,592,and the ORF coding sequence accounted for 87.31%of the total sequence.The average length of ORF was 965 bp.87.9%of ORFs was annotated,there was no plasmid in the cells.P.plecoglossicida JUIM01 was a new species separated from Pseudomonas putida,which showed its unique characteristics in genome and was different from the model strains of Pseudomonas.Further comparative genomics analysis was carried out to identify the genes related to glucose metabolism pathway,2KGA synthetic pathway,coenzyme PQQ synthetic pathway and electron transport pathway,to map the basic structure of gene clusters related to these pathways,and to deduce the synthetic pathway of 2KGA.?2?The key enzymes of 2KGA synthetic pathway in P.plecoglossicida JUIM01 were identified.Membrane-bound glucose dehydrogenase from P.plecoglossicida JUIM01 was purified and characterized.The results showed that the wild PQQ-mGDH was a single subunit with a molecular weight of about 87 kDa and a specific enzyme activity of 16.85 U/mg.The substrate spectrum was relatively wide,and the optimum substrate was D-glucose,K_m and V_maxax values were 0.042 mM and 14.620?M/min,respectively.The optimum pH and temperature was 6.0 and 35°C,and the specific enzyme activity was 16.85 U/mg.Acidic tolerance and thermal stability of PQQ-mGDH were relatively poor?the enzyme activity was completely lost at 55°C for 2 h?.The metal ions Ca²⁺/Mg²⁺significantly promoted the enzymatic activity,while EDTA/EGTA inhibited the enzymatic activity.PQQ-mGDH?coding gene gcd?was knocked out and complemented.The results showed that the knockout of gcd blocked the synthesis of2KGA and reduced the growth rate by 19%.The role of mGDH in the synthesis of 2KGA was further verified by heterologous expression of mGDH in E.coli.Membrane-bound gluconate dehydrogenase was identified by the same method.The results showed that the specific activity of wild FAD-mGADH was 90.71 U/mg.It was composed of three subunits:?,flavoprotein and cytochrome c subunits with molecular weights of 27,65 and 47 kDa,respectively.Flavoprotein and cytochrome c subunits contained one assumed FAD binding subunit and three possible heme binding subunits?C-X-X-C-H?,respectively.The enzyme exhibited strict substrate specificity toward D-gluconic acid,with K_m and V_max values of 0.631 mM and 0.734 mM/min,respectively;the optimum pH and the optimum temperature of FAD-mGADH is 6.0 and 35°C,which possessed good acid resistance and thermal stability.Metal ions Ca²⁺and Mn²⁺can promote its enzyme activity.Furthermore,the role of FAD-mGADH in the synthesis of 2KGA was confirmed by its heterologous expression in E.coli.?3?The electron transfer pathway and coenzyme PQQ synthesis pathway of P.plecoglossicida JUIM01 were preliminarily identified.By method of biochemical inhibition for electron transfer chain,the types of terminal oxidases of electron transfer chain were identified as Cyo oxidase,CIO oxidase and three cytochrome c oxidases.It was preliminarily determined that the composition of the electron transfer chain system of P.plecoglossicida JUIM01 in the oxidation of glucose were PQQ-mGDH,ubiquinone and five terminal oxidases,in which CIO oxidase and Cyo oxidase played a major role.Further transcriptional level analysis and bioinformatics analysis were carried out to determine the gene composition of pqq operon including pqqF,pqqA,pqqB,pqqC,pqqD,pqqE,pqqM,pqqH,and pqqI which located on the reverse complementary chain.There were six promoter regions,seven terminator regions and six ribosome binding sites in the non-coding sequence.pqqF contained an independent promoter and terminator,pqqC,pqqD,pqqE and pqqM were confirmed to belong to the same transcription unit,pqqM and pqqH did not belong to the same transcription unit;the expression levels of pqqF,pqqA and pqqB were positively correlated with the expression level of gcd,of which pqqF was most correlated with the production of 2KGA,and can be used as a potential target for engineering to improve 2KGA productivity of P.glossicoplecida JUIM01.?4?Engineering of P.plecoglossicida JUIM01 for 2KGA production.The strain enhancing two key enzymes?PQQ-mGDH and FAD-mGADH?of 2KGA biosynthesis pathway was constructed.When 20 g/L of glucose was used as substrate,the expression of gcd in P.plecoglossicida JUIM01/pBBR1MCS-2-gcd-gad was increased by 3.21 times in 500 mL shaking flask,and enzyme activity was increased by 36%;the expression of gad gene and enzyme activity were increased by 5.66 times;the consumption rate of glucose and the 2KGA productivity both increased by 25%.When 140 g/L glucose was used as substrate,the consumption rate of glucose increased by 23.1%and the 2KGA productivity increased by 34%on the scale of 30 L fermentor.The inadequate supply of PQQ was solved by coenzyme modification,and the 2KGA productivity was further improved.The consumption rate of glucose and the 2KGA productivity in P.plecoglossicida JUIM01/pBBR1MCS-2-gcd-gad-pqqF which was cultivated in a 30 L fermentor with 140 g/L initial glucose concentration were21.28%and 21.41%higher than those of P.plecoglossicida JUIM01/pBBR1MCS-2-gcd-gad,respectively,and 61.23%and 62.61%higher than those of the original strain P.plecoglossicida JUIM01.