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.

Gluconobacter oxydans是一种专性革兰氏阴性细菌，属于醋杆菌科。它是工业生物技术中最常用的微生物之一，用于生产与不完全氧化相关的化学品。然而，由于缺乏有效的遗传工具来实现复杂的代谢操作，阻碍了G. oxydans的微调。因此，受不同G. oxydans菌株中的一系列野生型质粒的启发，构建了一系列用于G. oxydans的穿梭载体。使用 15 个穿梭载体在G. oxydans中表达 mCherryWSH-003 使用这些野生型质粒的复制起点。其中，p15-K-mCherry表达的荧光蛋白强度约为p5-K-mCherry表达荧光蛋白的10倍。实时定量聚合酶链反应表明p15-K-mCherry的相对拷贝数达到19，具有较高的稳定性。相比之下，一些质粒的相对拷贝数小于 10。多个穿梭载体的共表达揭示了五种穿梭载体，它们可以转化为G. oxydans WSH-003，并可以表达五种不同的荧光蛋白。穿梭载体将促进葡萄糖杆菌菌株的遗传操作，以更有效地生产有用的化合物。