Simultaneous transformation of five vectors in Gluconobacter oxydans

Gluconobacter oxydans is an obligate Gram-negative bacterium that belongs to the family Acetobacteraceae. It is one of the most frequently used microorganisms in industrial biotechnology to produce chemicals related to incomplete oxidation. However, the fine-tuning of G. oxydans is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. Thus, a series of shuttle vectors for G. oxydans inspired by a series of wild-type plasmids in different G. oxydans strains were constructed. Fifteen shuttle vectors were employed to express mCherry in G. oxydans WSH-003 using the replication origin of these wild-type plasmids. Among them, the intensity of fluorescent proteins expressed by p15-K-mCherry was about 10 times that of fluorescent proteins expressed by p5-K-mCherry. Quantitative real-time polymerase chain reaction showed that the relative copy number of p15-K-mCherry reached 19 and had high stability. In contrast, some of the plasmids had a relative copy number of less than 10. The co-expression of multiple shuttle vectors revealed five shuttle vectors that could be transformed into G. oxydans WSH-003 and could express five different fluorescent proteins. The shuttle vectors will facilitate genetic operations for Gluconobacter strains to produce useful compounds more efficiently.