Enhance 2-keto-L-gulonic Acid Biosynthesis In Gluconobacter Oxydans WSH-003 By Protein Compartment Strategy

Gluconobacter oxydans(G.oxydans)is the first bacteria in the process of 2-keto-L-gulonic acid(2-KLG)production by two step fermentation,which has the capability to convert D-sorbitol to L-sorbose quickly and efficiently.In this study,applying an industrial G.oxydans WSH-003 as the experimental strain,the key enzymes(L-sorbose dehydrogenase and L-sorbosone dehydrogenase)involved in the second step of 2-KLG production by two step fermentation,were heterogenous expressed in G.oxydans WSH-003.The conversion efficiency from L-sorbose to 2-KLG was strengthened by strategy of protein compartment.In addition,the enzymatic properties of L-sorbose dehydrogenase(SDH)and L-sorbosone dehydrogenase(SNDH)were studied.And a step-wise pH controlling method was employed in the fermentation process to futher enhance the 2-KLG synthesis ability of G.oxydans.The main results are listed as following.(1)L-sorbose dehydrogenase and L-sorbosone dehydrogenase were expressed and purified and their enzymatic properties were analyzed.SDH,SNDH,SNDH with SH3 domian in the N terminal and SDH with SH3 ligands in the C terminal were expressed in Escherichia coli BL21(DE3)respectively,and the enzyme activity was determined.The purified enzymes were obtained by affinity chromatography and gel filtration chromatography.Results showed that SH3 domain could enhance the specific activity of SNDH,while SH3 ligands could weaken the specific activity of SDH.The optimal temperature and pH for SDH were 30°C and 8.0;while the optimal temperature and pH for SNDH were 35°C and 8.0.(2)The feasibility of protein compartment strategy was tested and 2-KLG biosynthesis in G.oxydans WSH-003 was strengthened by protein compartment strategy.Firstly,the interaction between SH3 domain and SH3 ligand in G.oxydans WSH-003 was verified by the bimolecular fluorescent complementation.SH3 domain was fused to the N terminal of SNDH,whilst SH3 ligand was fused to the C terminal of SDH.The interaction of SH3 domain and SH3 ligand could make SDH and SNDH close in the spatial position.With the changing of amount of SH3 ligand and the adjusting the ratio of SDH and SNDH in the synthetic protein scaffolds,the metabolic flux of SNDH could be increased,and therefore more 2-KLG was abtained by converting from sorbosone.The results showed that the 2-KLG production was 25.91 g·L-1 when two SH3 ligands were fused to the C terminal of SDH.Compared with the case without SH3 ligand,2-KLG production was increased by 72.39%.(3)The nitrogen source and metal ion addition were optimized in the level of shaking flask.Constant pH fermentation was carried out in a 3 L fermentor and a step-wise pH control strategy was proposed.The optimal medium was composed of(g·L-1): D-sorbitol 150,yeast extract 10,MgSO4 0.5,CaCO3 0.5,KH2PO4 1.0,K2HPO4 0.5.Based on the optimized fermentation medium,batch fermentation was carried out in a 3-L fermentor.The results showed that,lower value of pH,which was cased by gradually accumulation of 2-KLG,is not suitable for the activities of SDH and SNDH.After investigation of the effect of pH controlling in the fermentation process,it was found that when the pH was controlled at 4.0 and 5.0,D-sorbtol was converted to L-sorbose efficiently,and when the pH was controlled at 7.0,the conversion form L-sorbose to 2-KLG was more efficiently.The titer of 2-KLG was increased to 33.15 g·L-1 by using a step-wise p H controlling strategy.Compared with the batch fermentation procedure without pH controlling,2-KLG production was increased by 31.75%.