A novel regio-specific cyclosporin hydroxylase gene revealed through the genome mining of Pseudonocardia autotrophica. (1st May 2014)

• Record Type: Journal Article
• Authors: Ban, Jun-Gyu; Woo, Min-Woo; Lee, Bo-Ram; Lee, Mi-Jin; Choi, Si-Sun; Kim, Eung-Soo
• Is Part Of: Journal of industrial microbiology & biotechnology. Volume 41:Number 5(2014)
• Page Start: 879
• Page End: 886
• Publication Date: 2014-05-01
• Subjects: Cytochrome P450 hydroxylase -- Cyclosporine A -- Regio-selectivity -- Pseudonocardia autotrophica; Industrial microbiology -- Periodicals; 660.62
• DOI: 10.1007/s10295-014-1432-5
• Languages: English
• ISSNs: 1367-5435

Abstract

Abstract: The regio-specific hydroxylation at the 4th N -methyl leucine of the immunosuppressive agent cyclosporin A (CsA) was previously proposed to be mediated by a unique cytochrome P450 hydroxylase (CYP), CYP-sb21 from the rare actinomycetes Sebekia benihana. Interestingly, a different rare actinomycetes species, Pseudonocardia autotrophica, was found to possess a different regio-selectivity, the preferential hydroxylation at the 9th N -methyl leucine of CsA. Through an in silico analysis of the whole genome of P. autotrophica, we describe here the classification of 31 total CYPs in P. autotrophica . Three putative CsA CYP genes, showing the highest sequence homologies with CYP-sb21, were successfully inactivated using PCR-targeted gene disruption. Only one knock-out mutant, ΔCYP-pa1, failed to convert CsA to its hydroxylated forms. The hydroxylation activity of CsA by CYP-pa1 was confirmed by CYP-pa1 gene complementation as well as heterologous expression in the CsA non-hydroxylating Streptomyces coelicolor . Moreover, the cyclosporine regio-selectivity of CYP-pa1 expressed in the ∆CYP-sb21 S. benihana mutant strain was also confirmed unchanged through cross complementation. These results show that preferential regio-specific hydroxylation at the 9th N -methyl leucine of CsA is carried out by a specific P450 hydroxylase gene in P. autotrophica, CYP-pa1, setting the stage for the biotechnological application of CsA regio-selective hydroxylation.