The Study Of Characterization Of Dehydrogenases And Their Catalysis Capability For 2-KLG Synthesis

2-keto-L-gulonic acid(2-KLG)is the direct precursor of vitamin C,which can be converted into vitamin C through esterification.2-KLG is mainly produced by the classical two-step fermentation route in industry.The classical two-step fermentation route includes the two-step fermentation process performed by three bacteria.Compared with single-step and pure fermentation,the classical two-step fermentation route has many disadvantages,such as high energy consumption,long fermentation time and difficult regulation.With development of modern fermentation industry,the improvement of the classical two-step fermentation route has become particularly urgent.At present,no microbes with great capabilities of synthesis of vitamin C or 2-KLG were found in nature.Construction of one-step fermentational strain is the most potentional strategy for 2-KLG production.However,construction of 2-KLG synthetic pathway based on indentification and analysis of 2-KLG synthetic enzymes is the most important primary work.Based on the enzymatic catalytic process involved in the classical twostep fermentation method,this study characterized the dehydrogenase related to 2-KLG synthesis in the classical two-step fermentation route and demonstrated key dehydrogenases in 2-KLG synthesis pathway.Finally,the one-step mix-cultural fermentation for 2-KLG production from D-sorbitol in Escherichia coli was achieved.The main research results are as follows:1.The synthesis of PQQ was achieved by recombinational E.coli BL21(DE3)that expressed PQQ synthetic gene cluster.PQQ is the third kind coenzyme except for cofactors of flavine and nucleotides.It's also a coenzyme of dehydrogenase related to 2-KLG synthesis pathway in the classical two-step route,especially sorbose/sorbosone dehydrogenases(SSDHs)and sorbosone dehydrogenases(SNDHs)in Ketogulonigenium vulgare.In this study,the four PQQ synthetic gene clusters were obtained from four PQQ synthesis strains including Gluconobacter oxydans WSH-003,Methylobacterium extorquens AM1,Methylovorus sp.MP688 and Klebsiella pneumoniae strain HS11286 by genome analysis.The four gene clusters were expressed in E.coli BL21(DE3)respectively to produce PQQ.E.coli BL21(DE3)cannot synthesize PQQ.The recombinational E.coli BL21(DE3)expressed PQQ synthetic gene cluster from K.pneumonia strain HS11286 produced 357.5 ?g/L PQQ through IPTG induction in 250 mL flask.2.The production of L-sorbose from D-sorbitol was realized by recombinational E.coli BL21(DE3)that overexpressed sorbitol dehydrogenases and PQQ synthesis gene cluster.In this study,three sorbitol dehydrogenases,SldBA1,SldBA2 and SldSLC,were identified by overexpression and activity test.SldBA1 and SldBA2 can catalyze D-sorbitol to L-sorbose with PQQ as cofactors.SldSLC maybe catalyze D-sorbitol to D-fructose.The recombinational strains with capacity of L-sorbose synthesis were constructed by overexpressed SldBA1 or SldBA2 respectively in the E.coli BL21(DE3).The SldBA1 could possibly be main dehydrogenase which catalyze D-sorbitol to L-sorbose in G.oxydans WSH-003 according to catalytic ability of the recombinational E.coli BL21(DE3)strains.The recombiantional E.coli BL21(DE3)with SldBA1 can transform D-sorbitol to L-sorbose with adding PQQ.Moreover,the production of L-sorbose from D-sorbitol was achieved by recombinant E.coli BL21(DE3)through overexpression of SldBA1 and PQQ synthetic gene cluster from K.pneumonia.The highest titer of L-sorbose is 3.8 g/L.3.The dehydrogenases related to 2-KLG synthesis in K.vulgare were overexpressed,purified and characterized respectively.In the study,5 sorbose/sorbosone dehydrogenases genes,ssda1,ssda1-p,ssda2,ssda3,ssdb and two sorbosone dehydrogenases genes,gsndh and sndh,were analyzed and overexpressed in E.coli BL21(DE3).At first,these 7 enzymes were purified by ammonium sulfate precipitation and Ni-affinity column chromatography,and then their enzymatic properties were demonstrated in detail.We found that all 7 dehydrogenase are PQQ dependent.The 5 SSDHs also have high activities for 1-propanol and glyoxal,and they also can catalyze some common sugars and alcohols.However,only SSDA1 and SSDA3 can catalyze L-sorbose to 2-KLG directly.The two SNDHs have similar substrates spectrum and have remarkable activities for D-glucose,D-galactose,D-mannose,D-xylose,L-rhamnose and D-lactose besides glyoxal.The optimal reaction temperature of 7 dehydrogenases are about 30°C,the optimal pH are alkaline.Calcium or magnesium ions have significant improvement for activities of these dehydrogenases and different electron acceptors have great influences on enzyme activities.4.Synthesis of 2-KLG from L-sorbose was achieved by whole-cell catalysis and the condition of catalysis was optimized.In this study,2-KLG can be produced by whole-cell catalysis using recombinational E.coli BL21(DE3)overexpressed SSDA1 or SSDA3 with adding PQQ and electron acceptor(PMS or DCIP).It was found that low pH value and 2-KLG have strong inhibitory effect on the catalytic reaction.However,the 2-KLG production strain was constructed by introducing the PQQ synthetic gene cluster into the recombinant bacteria expressed SSDA1 or SSDA3.The 2-KLG concentrations were 11.2 g/L and 12.4 g/L with the conversion rate of 56.8% and 61.7% respectively by whole-cell catalysis with adding PMS and DCIP in the flask.5.Production of 2-KLG from D-sorbitol was achieved by mix-cultural fermentation of G.oxydans and recombinational E.coli BL21(DE3)in 5 L fermentor.In this study,2-KLG was synthesized by controlling pH and adding electron acceptors in 5 L fermentor,and the highest titer of 2-KLG reached 72.4 g/L and the conversion rate reached 71.2%.G.oxydans and recombinational E.coli BL21(DE3)/ssda3-pET-pqq was co-cultured and pH was controlled during the mix-cultural fermentation.The concentration of 2-KLG was 16.8 g/L,and the conversion rate was 33.6% when the inoculation ratio of G.oxydans and recombinational E.coli BL21(DE3)was 2:1 and the pH was controlled as pH7.5 after adding electron acceptors(PMS and DCIP).