Construction Of A Shuttle Vector For Use In E.coli And Gluconobacter Oxydans

Gluconobacter oxydans is characterized by its ability to oxidize a lot of carbohydrates and alcohol. The products acetaldehyde, ketones and organic acids accumulate in the culture medium. As most reactions are catalyzed by the membrane-bound dehydrogenases whose reaction centers locate in the peripheral cytoplasm space, transfer of the substrates into the cell is unnecessary. Because of the above characteristics, Gluconobacter oxydans has been widely used in industrial production, such as in dihydroxyacetone and vitamin C production. With the growing interest in the study of the important strains of Gluconobacter oxydans, genetic engineering has been increasingly used for strain improvement. Transformation of Gluconobacter oxydans with multiple copy plasmids carrying dehydrogenase genes can significantly increase the industrial productivity of 5-keto-D-gluconic acid and esters.The present work aimed to construct a shuttle vector pZL1, which can replicate both in E.coli and Goxydans. After 144h subculture, the shuttle vector still maintained a high stability. The relative copy number of the shuttle vector pZL1 in Gluconobacter oxydans was determined by real-time quantitative fluorescent PCR. In order to test the efficiency to express heterologous genes with pZL1, the expression vector pZL4 which contained a reporter gene and an upstream strong promoter tufB was constructed. The expression of the reporter gene was detected by Microplate Reader and Fluorescence Microscope. In addition, the fluorescence signal of Goxydans DSM2003 harboring pZL4 was comparable with that of the reference Goxydans DSM2003 harboring pBBR1MCS5-tufB-wasabi.