Structural And Functional Analysis And Engineering Of L-sorbosone/l-sorbose Dehydrogenases From Gluconobacter Oxydans

Vitamin C is an essential nutrient that humans must obtain from outside sources.Currently,a three-bacteria,two-step fermentation process is used to industrially produce 2-keto-L-gulonic acid（2-KLG）,a direct precursor of vitamin C.In this process,D-sorbitol is converted to 2-KLG by D-sorbitol dehydrogenase（SLDH）,L-sorbose dehydrogenase（SDH）and L-sorbosone dehydrogenase（SNDH）.In theory,by co-expressing the three enzymes in suitable strains,a one-strain,one-step fermentation method with lower production cost,more stable fermentation process,and more green environmental protection can be constructed.Currently,most strains producing 2-KLG by one-step fermentation are obtained through metabolic engineering,but the yield of 2-KLG does not meet the requirements of industrial production.With the vigorous development of enzyme engineering,it has become a feasible strategy to analyze the catalytic mechanism of enzymes and improve the catalytic activity of enzymes,which is beneficial to increasing the production of 2-KLG further.The conversion of L-sorbose to 2-KLG is considered the rate-limiting step in synthesizing2-KLG,which involves SNDH and SDH.This work examined SNDH and SDH in Gluconobacter oxydans WSH-004 in depth with regard to its enzymatic and physicochemical properties.Furthermore,the dynamic regulation mechanism and molecular catalysis mechanism of key enzymes were proposed based on crystal structure analysis,protein structure simulation and corresponding in vitro verification;Besides,the specific enzyme activity of key enzymes was effectively improved,and the yield of 2-KLG was preliminarily increased via semi-rational design.Finally,the recombinant strain that expressed mutant enzymes can reach a conversion rate of L-sorbose close to 50%.This work provides effective enzyme information and excellent enzyme elements for the final construction of 2-KLG strain by one-step fermentation,and has important reference significance for the innovation of industrial production methods of vitamin C.The specific research results are as follows:（1）Characterization of the enzymatic properties of SNDH.In view of the lack of enzymatic properties of SNDH in G.oxydans WSH-004,the enzymatic properties of SNDH were determined.The K_m for NADP⁺and NAD⁺were 0.14 m M and 0.42 m M,respectively,when using L-sorbosone as the substrate.The K_m for L-sorbosone is 0.21 m M and 0.53 m M,respectively,when using NADP⁺and NAD⁺as the cofactor.SNDH has strong substrate specificity,and the most suitable substrate is L-sorbosone.The optimum p H of SNDH is 10.5,the optimum reaction temperature is 50℃,and it has good thermal stability at 30℃,and Mg²⁺can promote the enzymatic activity of SNDH.（2）The crystal structure,dynamic regulation mechanism and catalytic mechanism analysis of SNDH.In view of the unclear catalytic mechanism of SNDH in G.oxydans WSH-004,the crystal structures of oxidized SNDH,reduced SNDH and mutant C296A were obtained by screening the crystallization conditions.The codes in the corresponding Protein Data Bank（PDB）are 7W5L,7W5N and 7W5K,respectively.Secondly,the dynamic regulation mechanism based on the redox of adjacent cysteine and catalytic mechanism of SNDH were proposed through structural analysis and multiple sequence alignment,and were verified in vitro with pure enzyme,respectively.（3）Semi-rational design enhances SNDH activity.In view of the current problem of insufficient production of 2-KLG in one step route,firstly,four strategies were used to improve the specific enzyme activity of SNDH:i)eliminating oxidized SNDH;ii)reducing steric hindrance at the entrance of the substrate pocket;iii)enhancing proton transport and iv)optimizing amino acids in the substrate pocket.Finally,mutants M167L,S453A,L460V,E471D and M167L/S453A/E471D were obtained,of which the mutant M167L had the highest specific enzyme activity,which was 2.7 times that of the wild type.Subsequently,the mechanism of the enhanced specific enzyme activity of the mutant was explained based on molecular dynamics simulations.（4）Semi-rational design enhances SDH activity.Aiming at the problem of the low conversion rate of L-sorbose in the one-step fermentation,a screening method based on yield-related SDH mutants was established.Subsequently,five SDH mutants were obtained that contributed to the increased production of 2-KLG through three strategies:i)Ala scanning,ii)iterative saturation mutation,and iii)surface charge optimization.The recombinant strain with the mutant V336I/V368A could produce 3.39 g/L of 2-KLG in a 24-well plate,which was 1.9times that of the wild type.（5）Improve 2-KLG production by fermentation optimization,and identification of genes related to L-sorbose consumption.Firstly,the fermentation optimization of RS-M167L-V336I/V368A was carried out.The recombinant strain was fermented in a shake flask for 16 h,and the 2-KLG reached the highest:5.03 g/L,and the conversion rate reached 50%.Second,4candidate genes were found in E.coli BL21（DE3）which were proposed to be related to L-sorbose metabolism.And E.coli BL21（DE3）was genetically modified by i)blocking L-sorbose phosphorylation and ii)knocking out the L-sorbose phosphate transport system,and the recombinant strain was constructed and verified by fermentation.Finally,the genes aga V(EIIB^sor),aga W(EIIC^sor),aga E(EIID^sor)and aga F(EIIA^sor)were determined to be related to the transport of L-sorbose.The recombinant strains not only did not increase the production of2-KLG,but also could still consume L-sorbose.It indicated that there are other L-sorbose utilization systems to be explored in E.coli BL21（DE3）.