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Front. Plant Sci. | doi: 10.3389/fpls.2019.01520

Molecular basis of C-30 product regioselectivity of legume oxidases involved in high-value triterpenoid biosynthesis

 Much Z. Fanani1,  Ery O. Fukushima2, 3,  Satoru Sawai1, 4, 5, 6, Jianwei Tang4, Masato Ishimori7, Hiroshi Sudo6, 8, Kiyoshi Ohyama4, 9, 10,  Hikaru Seki1, 4,  Kazuki Saito4, 5 and  Toshiya Muranaka2, 4*
  • 1Department of Biotechnology, Graduate School of Engineering Science, Osaka University, Japan
  • 2Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan
  • 3Regional College Amazon Ikiam, Ecuador
  • 4RIKEN Center for Sustainable Resource Science (CSRS), Japan
  • 5Graduate School of Pharmaceutical Sciences, Chiba University, Japan
  • 6Tokiwa Phytochemical Co., Ltd., Japan
  • 7Chiba University , Graduate School of Pharmaceutical Sciences, Japan
  • 8School of Pharmacy and Pharmaceutical Sciences, Hoshi University, Japan
  • 9Department of Chemistry and Materials Science, Tokyo Institute of Technology, Japan
  • 10Leaf Tobacco Research Center, Japan Tobacco (Japan), Japan

The triterpenes are structurally diverse group of specialized metabolites with important roles in plant defense and human health. Glycyrrhizin, with a carboxyl group at C-30 of its aglycone moiety, is a valuable triterpene glycoside, the production of which is restricted to legume medicinal plants belonging to the Glycyrrhiza species. Cytochrome P450 monooxygenases (P450s) are important for generating triterpene chemodiversity by catalyzing site-specific oxidation of the triterpene scaffold. CYP72A154 was previously identified from the glycyrrhizin-producing plant Glycyrrhiza uralensis as a C-30 oxidase in glycyrrhizin biosynthesis, but its regioselectivity is rather low. In contrast, CYP72A63 from Medicago truncatula showed superior regioselectivity in C-30 oxidation, improving the production of glycyrrhizin aglycone in engineered yeast. The underlying molecular basis of C-30 product regioselectivity is not well understood. Here, we identified two amino acid residues that control C-30 product regioselectivity and contribute to the chemodiversity of triterpenes accumulated in legumes. Amino acid sequence comparison combined with structural analysis of the protein model identified Leu149 and Leu398 as important amino acid residues for C-30 product regioselectivity. These results were further confirmed by mutagenesis of CYP72A154 homologs from glycyrrhizin-producing species, functional phylogenomics analyses, and comparison of corresponding residues of C-30 oxidase homologs in other legumes. These findings could be combined with metabolic engineering to further enhance the production of high-value triterpene compounds.

Keywords: chemodiversity, Cytochrome P450 monooxygenase, legume, product regioselectivity, triterpene

Received: 15 Jun 2019; Accepted: 31 Oct 2019.

Copyright: © 2019 Fanani, Fukushima, Sawai, Tang, Ishimori, Sudo, Ohyama, Seki, Saito and Muranaka. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Toshiya Muranaka, Osaka University, Department of Biotechnology, Graduate School of Engineering, Suita, 565-0871, Ōsaka, Japan, muranaka@bio.eng.osaka-u.ac.jp