Systemaric Regulation Of Gluconobacter Oxydans Dehydrogenases For The Synthesis Of 2-keto-L-gulonic Acid

2-Keto-L-gulonic acid（2-KLG）is a precursor of vitamin C,which is directly converted into vitamin C by a lactonization reaction.At present,industrial production of 2-KLG still utilizes the traditional“two-step fermentation process”.There are many problems in this fermentation process,such as complex processes,many influencing factors,and the challenges of precise control.At the same time,because this process involves three bacteria,lots of energy is wasted in the secondary sterilization process,and many raw materials are consumed in the growth of the strains,which reduces the substrate conversion.In order to achieve the goals of“carbon peak”and“carbon neutrality”,it is urgent to improve the traditional fermentation method and upgrade the fermentation industry for vitamin C.In our laboratory’s previous study,a 2-KLG producing strain G.oxydans WSH-004 was obtained,whereas the conversion rate was low from D-sorbitol to 2-KLG.Thus the metabolic pathway of 2-KLG in G.oxydans was studied.Related dehydrogenases forming side products in the pathway were characterized and gene knockout was performed.Finally,identification and overexpression of genes for rate limiting steps of the 2-KLG synthesis pathway were performance to increase the production of2-KLG.The main results are as follows:1.A novel xylitol dehydrogenase was screened from G.oxydans.The unknown dehydrogenases of G.oxydans were expressed in E.coli,and DCIP+PMS were used to screen the activity of these dehydrogenases utilizing D-sorbitol as the substrate.Finally,a xylitol dehydrogenase converting D-sorbitol to D-fructose was obtained.Subsequently,xylitol dehydrogenase was purified,and enzymatic properties were identified.It was found that the cofactor of xylitol dehydrogenase was NAD⁺,the optimum temperature was 57°C,and the optimum p H was 12.And the xylitol dehydrogenase had no dependence on metal ions.Metal ions of Zn²⁺,Mn²⁺and Co²⁺could improve the catalytic activity of xylitol dehydrogenase,but the addition of Cu²⁺had a significant inhibition.The substrate spectrum of the xylitol dehydrogenase was identified and it was found to be catalytically active toward D-sorbitol and xylitol and inactive toward other common sugar alcohol substrates..2.Membrane-bound D-sorbitol dehydrogenase and side-product formation PQQ-dependent D-glucose dehydrogenase were identified in G.oxydans WSH-003.Three D-sorbitol dehydrogenase genes（sld BA1,sld BA2,and sld SLC）were obtained according to the analysis results of G.oxydans WSH-003 genome.These D-sorbitol dehydrogenases were knocked out individually and combined.The results found that D-sorbitol dehydrogenases Sld BA1 and Sld SLC had catalytic activity,whereas Sld BA2 did not.Subsequently,38 unknown dehydrogenases were successfully knocked out in G.oxydans WSH-003,and 23 of the knockout strains had higher yields of sorbose than the wild strain.Meanwhile,the side-products were significantly reduced when the PQQ-dependent D-glucose dehydrogenase was knocked out.And then 27 dehydrogenases were continuously knocked out in G.oxydans WSH-003 by using upp gene as reverse screening marker.The multi-knockout strain G.oxydans MD-16produced 149.5 g/L of L-sorbose in shake flask with the conversion rate 99.6%,and 298.6 g/L in 5-L fermentor with a conversion rate of 99.5%.3.Construction of high-efficiency shuttle plasmids for G.oxydans.The replicons of endogenous plasmids were predicted by analyzing the sequences,and 15 shuttle vectors were constructed utilizing the replicons.All 15 shuttle vectors could express m Cherry in G.oxydans WSH-003,and the highest fluorescence intensity(p15-K-P₀₂₉₅-m Cherry)was 10 times higher than the lowest fluorescence intensity(p5-k-P₀₂₉₅-mcherry).Then the relative copy numbers of these plasmids were detected by q PCR,and the relative copy number of p15-K-P₀₂₉₅-m Cherry reached 19.Then,the resistance screening tag of the shuttle plasmid was extended,and five shuttle plasmids with different resistances were successfully transformed into G.oxydans WSH-003.Finally,these constructed shuttle plasmids were verified in other strains of Gluconobacter.The results shown that these shuttle vectors were practical in genetic operations for Gluconobacter strains to produce valuable compounds.4.The substrate and high-temperature tolerance of G.oxydans WSH-004 were significantly improved through adaptive evolution.To improve the tolerance of G.oxydans WSH-004 to a higher concentration of D-sorbitol,G.oxydans was adaptively evolved by the continuous microdroplet culture.Finally,adaptive evolution strains G.oxydans MMC10 and G.oxydans 2-KLG5 were obtained.Between them,G.oxydans MMC10 can tolerate a higher D-sorbitol concentration（300 g/L）and a higher temperature（40°C）.Validation by fermentor fermentation further demonstrated that high concentrations of D-sorbitol had no obvious inhibitory effect on both its growth and the conversion of D-sorbitol to L-sorbose by fermentor level verification of these strains.The 2-KLG yield of G.oxydans 2-KLG5 improved to 5.6 g/L,1.6 times higher than the wild strain.Genome sequencing analysis of G.oxydans 2-KLG5revealed that the mutant genes of the evolutionary strain were mainly concentrated on the ribosomal protein genes involved in the protein translation system,suggesting that the tolerance improvement of the strain was related to the changes of translation level.5.The yield of 2-KLG was increased by enhancing the expression of sndh-sdh.The genome analysis revealed that G.oxydans WSH-004 had a key gene cluster sndh-sdh for 2-KLG synthesis.Subsequently,the gene cluster of sndh-sdh was knockout,and the results proved that it was essential for 2-KLG synthesis.The SDH from G.oxydans WSH-004 and SNDH from G.cerinus 1.110 were screened for a higher yield of 2-KLG.Then these dehydrogenases were overexpressed in G.oxydans 2-KLG5,and cytochrome oxidase expression was improved to enhance the electron transport.The results showed that the 2-KLG yield of engineered strains reached 18.3 g/L in shaking flask,and the conversion rate of 2-KLG from D-sorbitol was 22.9%.Finally,the yield of 2-KLG reached 45.1 g/L in a 5-L fermentor with the conversion rate of 30.1%.