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Production of ω-hydroxy palmitic acid using CYP153A35 and comparison of cytochrome P450 electron transfer system in vivo

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Abstract

Bacterial cytochrome P450 enzymes in cytochrome P450 (CYP)153 family were recently reported as fatty acid ω-hydroxylase. Among them, CYP153As from Marinobacter aquaeolei VT8 (CYP153A33), Alcanivorax borkumensis SK2 (CYP153A13), and Gordonia alkanivorans (CYP153A35) were selected, and their specific activities and product yields of ω-hydroxy palmitic acid based on whole cell reactions toward palmitic acid were compared. Using CamAB as redox partner, CYP153A35 and CYP153A13 showed the highest product yields of ω-hydroxy palmitic acid in whole cell and in vitro reactions, respectively. Artificial self-sufficient CYP153A35-BMR was constructed by fusing it to the reductase domain of CYP102A1 (i.e., BM3) from Bacillus megaterium, and its catalytic activity was compared with CYP153A35 and CamAB systems. Unexpectedly, the system with CamAB resulted in a 1.5-fold higher yield of ω-hydroxy palmitic acid than that using A35-BMR in whole cell reactions, whereas the electron coupling efficiency of CYP153A35-BM3 reductase was 4-fold higher than that of CYP153A35 and CamAB system. Furthermore, various CamAB expression systems according to gene arrangements of the three proteins and promoter strength in their gene expression were compared in terms of product yields and productivities. Tricistronic expression of the three proteins in the order of putidaredoxin (CamB), CYP153A35, and putidaredoxin reductase (CamA), i.e., A35-AB2, showed the highest product yield from 5 mM palmitic acid for 9 h in batch reaction owing to the concentration of CamB, which is the rate-limiting factor for the activity of CYP153A35. However, in fed-batch reaction, A35-AB1, which expressed the three proteins individually using three T7 promoters, resulted with the highest product yield of 17.0 mM (4.6 g/L) ω-hydroxy palmitic acid from 20 mM (5.1 g/L) palmitic acid for 30 h.

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Acknowledgments

This paper is dedicated to Romas Kazlauskas on the occasion of his 60th birthday. This study was funded by a grant of the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea. (HN12C0055) and Seoul R&BD Program (PA140024).

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Authors and Affiliations

  1. School of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea

    Eunok Jung, Beom Gi Park, Joonwon Kim & Byung-Gee Kim

  2. Department of Bioscience and Biotechnology, Konkuk University, Seoul, South Korea

    Md. Murshidul Ahsan & Hyungdon Yun

  3. Department of Environmental Engineering, Ajou University, Gyeonggi-do, Suwon, South Korea

    Kwon-Young Choi

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  1. Eunok Jung
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Correspondence to Byung-Gee Kim.

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Jung, E., Park, B.G., Ahsan, M.M. et al. Production of ω-hydroxy palmitic acid using CYP153A35 and comparison of cytochrome P450 electron transfer system in vivo. Appl Microbiol Biotechnol 100, 10375–10384 (2016). https://doi.org/10.1007/s00253-016-7675-5

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