Abstract
The novel cryptic pKPAL3 plasmid was isolated from the Gram-positive microorganism Kocuria palustris IPUFS-1 and characterized in detail. pKPAL3 is a circular plasmid that is 4,443 bp in length. Open reading frame (ORF) and homology search analyses indicated that pKPAL3 possesses four ORFs; however, there were no replication protein coding genes predicted in the plasmid. Instead, there were two nucleotide sequence regions that showed significant identities with untranslated regions of K. rhizophila DC2201 (NBRC 103217) genomic sequences, and these sequences were essential for autonomous replication of pKPAL3 in Kocuria cells. Based on these findings, we constructed the novel Escherichia coli–Kocuria shuttle vectors pKITE301 (kanamycin resistant) and pKITE303 (thiostrepton resistant) from pKPAL3. The copy numbers of the constructed shuttle vectors were estimated to be 20 per cell, and they exhibited low segregation stability in Kocuria transformant cells in the absence of antibiotics. Moreover, constructed vectors showed compatibility with the other K. rhizophila shuttle vector pKITE103. We successfully expressed multiple heterologous genes, including the styrene monooxygenase gene from Rhodococcus sp. ST-10 (rhsmo) and alcohol dehydrogenase gene from Leifsonia sp. S749 (lsadh), in K. rhizophila DC2201 using the pKITE301P and pKITE103P vectors under the control of the glyceraldehyde 3-phosphate dehydrogenase (gapdh) promotor. The RhSMO–LSADH co-expressing K. rhizophila was used as a biocatalyst in an organic solvent–water biphasic reaction system to efficiently convert styrene into (S)-styrene oxide with 99% ee in the presence of 2-propanol as a hydrogen donor. The product concentration of the reaction in the organic solvent reached 235 mM after 30 h under optimum conditions. Thus, we demonstrated that this novel shuttle vector is useful for developing biocatalysts based on organic solvent-tolerant Kocuria cells.
Highlights
Enantiopure epoxides are useful building blocks for synthesizing various chiral materials, including pharmaceuticals, agrochemicals, and fine chemicals (Farina et al, 2006; Patel, 2008)
We reported the isolation and characterization of the two cryptic plasmids pKPAL1 and pKPAL2 from K. palustris IPUFS-1 and the construction of the E. coli–Kocuria shuttle vector pKITE101 series based on pKPAL1 (Toda et al, 2017)
K. rhizophila DC2201 (NBRC 103217), K. kristinae NBRC 15354, K. varians NBRC15358, K. palustris NBRC 16318, K. rhizophila NBRC 16319, K. polaris NBRC 103063, K. flava HO-9041 (NBRC 107626), and K. turfanensis HO-9042 (NBRC 107627) were used as transformation hosts for the constructed shuttle vectors, while E. coli JM109 and EC100D pir-116 were used for cloning. pHSG298 and pUC118 were used to construct the E. coli–Kocuria shuttle vectors. pGEM-T Easy Vector (Promega Corp., Fitchburg, WI, United States) was used to construct control plasmids for quantitative polymerase chain reaction
Summary
Enantiopure epoxides are useful building blocks for synthesizing various chiral materials, including pharmaceuticals, agrochemicals, and fine chemicals (Farina et al, 2006; Patel, 2008). Direct enantioselective epoxidation of prochiral alkenes is a straightforward strategy for producing chiral epoxides, and many chemical approaches have been developed to achieve this objective (Shi, 2004). Many chemical catalysts that contain transition metals, such as metal–salen complexes and chiral metalloporphyrins, have been developed for the enantioselective epoxidation of alkenes (Katsuki and Sharpless, 1980; Hanson and Sharpless, 1986; Irie et al, 1991; Chang et al, 1994; Tu et al, 1996). Biological synthesis of epoxides has several advantages over chemical synthesis, including superior chemoselectivity, regioselectivity, and enantioselectivity, as well as improved environmental sustainability
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