Construction And Application Of Gene Expression Vectors For Gluconobacter Oxydans

Gluconobacter oxydans has various membrane-bound dehydrogenases, which can oxidize various alcohols and sugar alcohols incompletely to the corresponding aldehydes, ketones and acids. The unique property of incomplete oxidation makes it as a biocatalyst widely used in industrial biotechnology. Overexpressing dehydrogenases genes in G. oxydans by gene engineering can improve the enzyme activity and then enhance the product yield in a great extent. However, the vectors used in G. oxydans often have low copy numbers, which limits the application potential of G. oxydans in industrial production. In this study, we aimed to increase the copy number of the broad-host-range plasmid pBBR1MCS-5 by site-directed mutagenesis. And the obtained mutant plasmids with increased copy number were used to efficiently express homologous and heterologous genes in G. oxydans. Among the engineered constructs, the G. oxydans/pBBR-3510-ga2dh strain with a very high productivity of 2KGA was obtained.Firstly, six plasmids named pBBR-10、pBBR-35、pBBR-3510、pBBR-RBS、pBBR-R35、 pBBR-R31 were constructed by mutating the upstream of rep gene, which is the replication control region of pBBR1MCS-5.RT-PCR analysis showed that the copy numbers of all the mutant plasmids were all higher than that of the parental plasmid pBBR1MCS-5.To identify the application ability of these mutant vectors, GFP gene was used as a reporter. Under fluorescence microscope, all the recombinant strains showed fluorescence signal clearly, while the wild strain G. oxydans DSM 2003 showed no fluorescence signal, which meant these vectors could be used to express genes in G. oxydans.Secondly, in order to construct a 2KGA high-yielding strain, ga2dh gene encoding gluconate-2-dehydrogenase which can oxidize GA to 2KGA was overexpressed in G. oxydans DSM 2003 using these mutant plasmids. According to the relative transcriptional level of the ga2dh gene in the recombinant G. oxydans strains and the production of 2KGA in flasks, the G. oxydans/pBBR-3510-ga2dh strain exhibited the greatest increase in 2KGA production.In a batch biotransformation process in a 7-1 fermenter, approximately 320 g/12KGA was accumulated from 320 g/1 GA by 30 g/1 G. oxydans/pBBR-3510-ga2dh cells in 17 h, generating a space-time yield of 18.86 g/1/h, which enhanced by about 60% and 184%, respectively, compared to that of ga2dh-overexpressing G. oxydans harboring the control plasmid pBBR1MCS-5 or the parental strain containing the empty vector. When the initial GA concentration was increased to 480 g/1,30 g/1 cells of G. oxydans/pBBR-3510-ga2dh still completely exhausted the supplied GA in 45 h and produced 486 g/12KGA when the continuous oxygen was supplied, corresponding to a space-time yield of 10.80 g/l/h, which was higher than the highest record.