Elucidation And Regulation Of Limiting Factors In The Biosynthesis Of 2-keto-L-gulonic Acid In Gluconobacter Oxydans ATCC9937

Vitamin C is one of the essential vitamins for the human body and is currently the best-selling vitamin in the market.It can eliminate free radicals produced by the human body and reduce the risk of various chronic metabolic diseases such as stroke,heart disease,and cancer.Currently,all commercial production of vitamin C is obtained through the biosynthesis of 2-keto-L-gulonic acid（2-KLG）and its esterification reaction.As an important precursor for the synthesis of vitamin C,2-KLG currently mainly relies on the classic three-step method using sorbitol as the substrate.With the increasing demand for vitamin C,it is increasingly urgent to develop more economical and efficient one-step fermentation processes with single bacteria.The direct one-step fermentation synthesis of2-KLG using glucose as the substrate is the most promising alternative approach,but there are currently no reports on mature process routes.Constructing an engineering strain that can directly utilize glucose for one-step fermentation to produce 2-KLG requires obtaining more suitable substrate strains,stronger catalytic activity rate-limiting enzymes,enhancing the intracellular transport capacity of substrate 2,5-diketo-D-gluconic acid（2,5-DKG）,and systematically optimizing the metabolic network of substrate strains.In this study,in order to address the relevant issues,we first re-screened Gluconobacter oxydans ATCC9937 as the substrate strain,analyzed the glucose metabolism process of G.oxydans ATCC9937,and obtained a 2,5-DKG reductase with stronger catalytic ability.In addition,to enhance the ability of engineering strains to synthesize 2-KLG,transport proteins with substrate 2,5-DKG function were screened.Subsequently,one-step synthesis of glucose to 2-KLG was achieved through fermentation optimization and adjustment of intracellular and extracellular glucose metabolism flux of engineering strains.The main research content is as follows:（1）Improved the ability of G.oxydans ATCC9937 to produce 2,5-DKG.In response to the difficulty in quantifying the intermediate product 2,5-DKG in the one-step fermentation pathway of glucose to 2-KLG,a 2,5-DKG quantification method based on enzyme conversion was developed.The ability of different strains to produce2,5-DKG was compared,and G.oxydans ATCC9937 was selected as the chassis strain.After analyzing the process products of producing 2,5-DKG by G.oxydans ATCC9937,it was found that the main reason for the low production of 2,5-DKG was the browning degradation caused by prolonged fermentation time.Finally,by optimizing the fermentation process,the inhibition of substrate on the strain was reduced,the fermentation time was shortened,and the ability of G.oxydans ATCC9937 to produce2,5-DKG was increased to 50.90 g·L^-1..（2）Identified the complete pathway and related enzymes for the synthesis of 2-KLG using glucose in G.oxydans ATCC9937.In response to the issue of unclear side effects of the glucose to 2-KLG pathway,a detailed analysis of G.oxydans ATCC9937glucose metabolism related genes were conducted through genome and transcriptome sequencing,and various enzyme coding genes of the glucose to 2-KLG pathway were identified.It was found that G.oxydans ATCC9937 has an endogenous 2,5-DKG reductase that can catalyze the production of 2-KLG from 2,5-DKG,and an aldo/keto reductase ps AKR with the function of degrading 2-KLG was also discovered.The detection method for 2-KLG was optimized,and the side reaction pathway was knocked out to obtain the engineering strain G.oxydans GKLG2 with a production capacity of2.61 g·L^-1.And a batch of promoters with different expression intensities were screened based on transcriptome data.（3）Obtained wild-type 2,5-DKG reductase with stronger catalytic ability.Eleven DKGRs and aldo/keto reductases from different sources were expressed by Escherichia coli BL21（DE3）.Eleven different sources of 2,5-DKG reductases and aldo/keto reductases were obtained through protein purification.By comparing the catalytic ability of 2,5-DKG reductase and aldo/keto reductase from different sources,it was found that G.oxydans ATCC9937 endogenous 2,5-DKG reductase has the strongest catalytic ability.Its optimal catalytic pH is 8.5,and the optimal catalytic temperature range is 20℃to 30℃.Na⁺,K⁺,and Mg²⁺have a promoting effect on the catalytic ability of enzymes,while Ca²⁺significantly inhibit their catalytic ability.Through evolutionary tree and amino acid sequence analysis,the key amino acid residues that affect enzyme activity were identified as Pro193,Leu194,Pro233,and Lys234.（4）Constructed a transport pathway for substrate 2,5-DKG of G.oxydans engineering strain.In response to the difficulty in transmembrane transport of substrate2,5-DKG,four strains of MFS family transporters that may have the ability to transport2,5-DKG were screened.Through sequence alignment,transmembrane domain prediction,homologous modeling,and molecular docking,it was confirmed that all four selected transporters were transmembrane proteins.The construction of a transformation pathway from 2,5-DKG to 2-KLG in E.coli BL（DE3）confirmed that Kgtp A has the strongest substrate transport ability.By comparing different expression methods,it was found that genomic integration expression can achieve the optimal expression effect of kgtp A,and an engineering strain G.oxydans GKLG7 with genomic integration expression of kptp A was obtained.Engineering strain G.oxydans GKLG9was obtained by replacing the NADPH consumption pathway ubi M,and the 2-KLG titer of G.oxydans GKLG9 was increased to 30.21 g·L^-1 through the fermentation process optimization award.（5）Redistributed intracellular and extracellular glucose metabolism flux of G.oxydans engineering strains.To address the issue of imbalanced glucose metabolism flux inside and outside G.oxydans cells,by inhibiting ineffective glucose metabolism pathways and strengthening the pentose phosphate pathway,the intracellular carbon metabolism flux is concentrated in the pentose phosphate pathway.The introduction of exogenous Galp/Glk glucose transport system increased intracellular glucose metabolism flux.Possible 2,5-DKG degradation gene 4kas was identified through metabolic analysis,and knocking out 4kas inhibited the browning degradation of 2,5-DKG,reducing extracellular carbon source loss.By expressing sorbitol dehydrogenase and sorbitone dehydrogenase,the engineered strain can simultaneously utilize sorbitol to synthesize 2-KLG,increasing the effective utilization of total carbon source and forming a cofactor cycle between 2,5-DKG reductase and sorbitone dehydrogenase.The 2-KLG titer of the engineering strain G.oxydans GKLG12 in the final 5 L fermentation tank reached 38.62 g·L^-1.