High-Efficient Production And The Metabolic Regulation Metabolism Of 2-Keto-D-gluconic Acid

2-Keto-D-gluconic acid(2KGA)is an important natural organic acid.Its derivant Disoascorbate(also called D-erythorbate)is a widely used food-safe antioxidant.Microbial fermentation is the commonly used 2KGA industrial production method.Pseudomonas is a group of widely used 2KGA producing bacteria.Technologies including batch,fed-batch,continuous and semi-continuous fermentation have been developed for 2KGA production.However,there are still some problems in 2KGA production processes such as much time spent on equipment sterilization to avoid contamination after reactor turnover,low productivity and unclear regulation mechanism of 2KGA matablolism in Pseudomonas species used as industrial2 KGA producers.In this study,the industrial 2KGA producing strain Pseudomonas plecoglossicida JUIM01 was chosen as the research strain from two candidates because of its high production and stability.Firstly,a novel two-stage semi-continuous fermentation process was developed on the basis of the existing 2KGA fermentation technologies.During the fermentation process,the changes in the expression of several 2KGA metabolism-related genes in P.plecoglossicida JUIM01 cells were investigated,and the key factor of 2KGA metabolism was explored.Then the regulation mechanism of 2KGA metabolism in P.plecoglossicida was clarified by gene knockout and complementation,protein heterologous expression and purification,electrophoretic mobility shift assay(EMSA),DNase I footprinting,isothermal titration calorimetry(ITC),molecular-docking simulation,etc.The main research results are summarized as follows:(1)Based on the control strategy of microbial physiological metabolic activity,a 2KGA two-stage semi-continuous fermentation technology was developed over the existing 2KGA fermentation technologies.By reducing the glucose concentration to an optimal level in the first-stage fermentation medium,the strain's bioactivity in the conversion of 2KGA was improved.A rational allocation of the first-stage fermentation broth was performed as seeds with high conversion activity for subsequent first-and second-stage fermentation.High concentration of glucose but little nitrogen sources were added in the second-stage fermentation medium to control the over-growth of the strain in the second-stage fermentation and reduce glucose consumption caused by strain growth.Three cycles of 2KGA two-stage semicontinuous fermentation were carried out under optimal conditions.The 2KGA yield and productivity of the first-stage fermentation reached 0.98 g/g and 9.12 g/(L·h),respectively,and those of the second-stage fermentation were 1.03 g/g and 8.11 g/(L·h).In the entire three-cycle two-stage semi-continuous fermentation process,the 2KGA titer reached over 215.2 g/L,the total yield was about 0.99 g/g,reaching over 91.6% of the theoretical yield,and the average productivity was 8.46 g/(L·h).(2)In order to clarify the metabolic regulation mechanism of 2KGA in P.plecoglossicida JUIM01,the expression-level changes of 9 key genes related to 2KGA metabolism were investigated.The transcriptional regulator PtxS encoded by ptxS was involved in 2KGA metabolism,and was speculated to play a key global regulatory role.The ptxS-knockout and complemented recombinants were constructed to study the effect of PtxS on their growth and2 KGA metabolism.The results showed the ptxS knockout had no significant effect on 2KGA titer,but could shorten the 2KGA fermentation period for about 4 h in the media containing20.0 g/L glucose.Additionally,the growth rate,glucose comsuption rate and 2KGA accumulation rate were negatively affected after ptxS complementation,indicating PtxS played an important role in 2KGA production.Comparison was made in the expression of 9 key genes related to 2KGA metabolism in P.plecoglossicida JUIM01 before and after ptxS knockout by real-time fluorescent quantitative PCR(RT-qPCR).The results showed that only gcd encoding glucose dehydrogenase remained similar expression level after ptxS was knocked out in P.plecoglossicida JUIM01,the expression levels of gndS,gndL,gndC,kguE,kguK,kguT and kguD were all significantly up-regulated by about 2.9?3.6 folds,and the expression of ?ptxS sequence was about 3-fold higher than that of ptxS gene before knockout.The negative regulatory control of PtxS on the transcription of the kgu operon,gad operon and ptxS gene in P.plecoglossicida was confirmed.(3)The transcriptional regulation mechanism of PtxS protein on the gluconate dehydrogenase operon(gad operon)in P.plecoglossicida was analyzed.The binding sites between PtxS and the gad operon promoter region were different from the reports on P.aeruginosa or P.putida,indicating that the P.plecoglossicida PtxS was capable of recognizing and binding to a wider range of sequences.The ptxS gene cloned from P.plecoglossicida JUIM01 was firstly expressed heterologously in Escherichia coli BL21(DE3).The consequent recombinant PtxS was purified,and its secondary structure was also analyzed.Subsequently,EMSA was used to verify the specific binding of the PtxS to the gad operon promoter region,and DNase ? footprinting analysis was used to clarify the binding sites between PtxS and the gad operon promoter region,which covered a ?37-bp sequence(5'-GAGCGGGCTTGCCCCG CGATGGAGGCTGGCGCGGATT-3'),which was located 156?192-bp upstream of the gnd S gene(the first ORF of the gad operon).Unlike the reported P.aeruginosa and P.putida,a typical14-bp palindrome sequence(5'-TGAAACCGGTTTCA-3')was not found in the binding region.Additionally,the transcriptional start site of the gad operon was determined by primer extension.The transcription of the gad operon started at a G/C base pair which was 214-bp upstream of the gnd S gene,and the subsequent transcriptional direction and partial sequence were shown as5'-GCTTGGGAGCGCAGGGCCAGT-3'.This site located 22-bp upstream of the binding region between the PtxS and the gad operon promoter region.In the area near the transcriptional start site,a sequence(5'-TGAATCCCATTGCT-3')highly similar with the typical 14-bp palindrome was found with 9-bp identity.Interestingly,the transcriptional start site(underlined)of the gad operon was located within this sequence.(4)The transcriptional regulation mechanism of PtxS on the ptxS gene in P.plecoglossicida was analyzed.Compared with gad operon,the transcriptional regulation of ptxS gene by PtxS was more similar to PtxS of P.aeruginosa and P.putida.The specific binding region between PtxS and the promoter region of ptxS gene covered a ?22-bp sequence(5'-TTATGAAAACGG TTTCAATAAA-3'),locating 37?58-bp upstream of the ptxS initiation codon(GTG).In addition,there was a highly similar sequence(5'-TGAAACCGTTTTCA-3')with only 1-bp difference(underlined)from the typical 14-bp palindrome sequence in this binding region.The transcription of ptxS gene started from a C/G base pair which was 46-bp upstream of the initiation codon,and the subsequent transcription direction and partial sequence were shown as5'-GTTTTCATAACCCTGCCCA-3'.The transcriptional start site located within the 22-bp binding region between the PtxS and the ptxS gene promoter region and even within the 14-bp palindrome-like sequence(5'-TGAAACCGTTTTCA-3',the underlined represented the transcription start site).Defining the transcription initiation site as “+1”,then the binding site of the PtxS protein and its coding gene promoter region ranged from “-12” to “+10”,the 14-bp palindrome-like sequence covered from “-7” to “+7”.(5)The product 2KGA was found to be the effector of P.plecoglossicida PtxS,and it could release the transcriptional inhibition of PtxS on the corresponding genes by binding to PtxS.ITC analysis showed 2KGA was the effector of PtxS,while compounds including glucose,gluconic acid,glucose-6-phosphate,pyruvate and 2-keto-L-gulonic acid were not.Then EMSA results confirmed that by binding to PtxS,the effector 2KGA could release the PtxS from the PtxS-DNA complexes,so as to release the transcriptional inhibition of PtxS on kgu operon and ptxS gene.The results of SEC-MALS analysis and molecular sieve filtration chromatography showed that P.plecoglossicida PtxS tended to be dimeric in solutions and per dimer bound to two 2KGA molecules.Based on these results,the tertiary structure of P.plecoglossicida PtxS and the molecular docking between the PtxS dimer and its effector 2KGA were simulated by SWISS-MODEL.The results showed that 2KGA mainly bound to the region close to the Cterminal of PtxS by interacting with the 296 th to the 301 st amino acids(Ala,Gln,Pro,Thr,Glu and Arg),and this region is the predicted protein kinase C phosphorylation site.In summary,a high-efficient production process for 2KGA fermentation was developed,and the mechanism of PtxS regulating 2KGA synthesis and metabolism in P.plecoglossicida JUIM01 was clarified in this study.The PtxS protein from P.plecoglossicida showed its specificities compared with the reported P.aeruginosa and P.putida PtxS proteins in terms of transcription regulation mechanism,especially in its atypical gad operon regulation mechanism,i.e.1)The binding region between PtxS and gad operon promoter region was atypical(neither the reported 14 bp typical palindrome sequence nor its similar sequences were found in this region);2)Unlike the transcriptional regulation of P.plecoglossicida PtxS on kgu operon and ptxS gene,the PtxS-gad promoter complex was insensitive to the effector 2KGA.The study also provided a theoretical basis for the synthetic-biology elementalization of the PtxS transcription regulation system.