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In vitro reconstitution of the cyclosporine specific P450 hydroxylases using heterologous redox partner proteins

Journal of Industrial Microbiology & Biotechnology

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The cytochrome P450 enzymes (CYPs) CYP-sb21 from Sebekia benihana and CYP-pa1 from Pseudonocardia autotrophica are able to hydroxylate the immunosuppressant cyclosporin A (CsA) in a regioselective manner, giving rise to the production of two hair-stimulating agents (with dramatically attenuated immunosuppressant activity), γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH) and γ-hydroxy-N-methyl-l-Leu9-CsA (CsA-9-OH). Recently, the in vitro activity of CYP-sb21 was identified using several surrogate redox partner proteins. Herein, we reconstituted the in vitro activity of CYP-pa1 for the first time via a similar strategy. Moreover, the supporting activities of a set of ferredoxin (Fdx)/ferredoxin reductase (FdR) pairs from the cyanobacterium Synechococcus elongatus PCC 7942 were comparatively analyzed to identify the optimal redox systems for these two CsA hydroxylases. The results suggest the great value of cyanobacterial redox partner proteins for both academic research and industrial application of P450 biocatalysts.

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This work was supported by National Natural Science Foundation of China under Grant Nos. NSFC 31422002 (S. Li) and 31300663 (D. Han), the Natural Science Foundation of Shandong Province, China (No. ZR2016CQ05) (L. Ma), and the National Research Foundation (NRF) of Korea (No. NRF-2014R1A2A1A11052236) (E.-S. Kim). We are grateful to the financial support from the Applied Basic Research Programs of Science and Technology of Qingdao (14-2-4-10-jch). We thank Prof. Xuefeng Lu at Qingdao Institute of Bioenergy and Biotechnology, Chinese Academy of Sciences, for providing us with the S. elongatus PCC7942 genome DNA.

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Correspondence to Eung-Soo Kim or Shengying Li.

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Y. Sun, L. Ma and D. Han contributed equally to this work.

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Sun, Y., Ma, L., Han, D. et al. In vitro reconstitution of the cyclosporine specific P450 hydroxylases using heterologous redox partner proteins. J Ind Microbiol Biotechnol 44, 161–166 (2017).

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