Characterization of two cryptic plasmids from Kocuria palustris IPUFS-1 and construction of novel Escherichia coli-Kocuria shuttle vector for biocatalysis.

Two cryptic plasmids, designated pKPAL1 and pKPAL2, were identified from the gram-positive bacterium Kocuria palustris IPUFS-1, which was isolated from a fish source. The 2251-bp and 2488-bp circular genomes of pKPAL1 and pKPAL2, respectively, were sequenced. Subsequent open reading frame (ORF) and homology search analyses suggested that pKPAL1 and pKPAL2 possess two and three ORFs, respectively, and encode the putative replication proteins, RepA and RepB, like the genomes of several plasmids in gram-positive bacteria. Thus, pKPAL1 and pKPAL2 were inferred to belong to the ColE2 plasmid family. We constructed novel Escherichia coli-Kocuria shuttle vectors pKITE101-103 based on pKPAL1. The constructed shuttle vector was stably maintained in Kocuria transformant cells, and vector copy number was estimated to be about 60 per cell. Leifsonia sp. S749 alcohol dehydrogenase (LSADH) was efficiently expressed in Kocuria rhizophila DC2201 using the pKITE103P vector under the control of the promoter of glyceraldehyde 3-phosphate dehydrogenase (gapdh). Herein, we demonstrate that the novel shuttle vector is a useful tool for developing biocatalysts based on organic solvent-tolerant Kocuria cells.