The Engineered Ketogulonigenium Vulgare Enhanced Interaction With Companion Bacillus Strain

Increasing the interaction between Ketogulonigenium vulgare and companion Bacillus strain is an important way to optimize the industrial production of Vitamin C.In this study,the interaction between the engineered K.vulgare and companion Bacillus strain was enhanced,and the fermentation level of the microbial consortium was improved.Firstly,we reconstructed the synthetic pathways of threonine,proline and histine via expressing absent genes encoding homoserine kinase,pyroline-5-carboxylate reductase and histidinol-phosphate phosphatase,respectively.When mono-cultured in shake flasks,the strain SyBE_Kv02080002 expressing hsk from Gluconobacter oxydans in threonine biosynthetic pathway achieved the highest biomass and the titer increased by 25.13%.When co-cultured with B.endophyticus,the fermentation cycle decreased by 16.67% in shake flasks and 28.57% in 5-L fermenter.Furthermore,we investigated the mechanism of engineered SyBE_Kv02080002 with high 2-KGA yield.First,we found the cell morphology of SyBE_Kv02080002 changed and exhibited 2 folds longer than original K.vulgare through scanning electron microscope observation.Moreover,reconstruction of threonine biosynthetic pathway resulted in up-regulation of genes encoding sorbosone/idonate dehydrogenase via transcriptional level analysis.In this study,the transcriptional levels of sndh and idh were 3.78 times and 1.54 times higher than original K.vulgare,respectively.Then,we co-expressed the genes encoding lipoyl synthase(LipA)and lipoyl transferase(LipB)from G.oxydans in K.vulgare,named SyBE_Kv02080002.We found 2-KGA productivity of engineered strain SyBE_Kv02080002 was higher than that of exogenous lipoic acid addition,and increased by 5.25% than original K.vulgare in shake flasks.When mono-cultured in 5-L fermenter,the fermentation cycle decreased by 5 h.When co-cultured with B.endophyticus and B.thuringiensis,the 2-KGA production increased by 7.4% and 3.0%,respectively.Moreover,the transcriptional level of genes encoding keto acid dehydrogenase complex(E2)subunit and the glycine cleavage system(H)(lipoic acids are coenzymes of these proteins)were found to be 2.54 times and 2.51 times higher than that of the original strain by transcriptional level analysis.Finally,we compared the companion ability of two Bacillus strains via analyzing the extracellular metabolites.We found the levels of most substances secreted by B.endophyticus and B.thuringiensis were different,which led to the different companion abilities for helping K.vulgare to produce 2-KGA.