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Applied Microbiology and Biotechnology

, Volume 99, Issue 18, pp 7549–7558 | Cite as

Optimization of recombinant expression enables discovery of novel cytochrome P450 activity in rice diterpenoid biosynthesis

  • Naoki Kitaoka
  • Yisheng Wu
  • Meimei Xu
  • Reuben J. PetersEmail author
Biotechnologically relevant enzymes and proteins

Abstract

The oxygenation reactions catalyzed by cytochromes P450 (CYPs) play critical roles in plant natural products biosynthesis. At the same time, CYPs are one of most challenging enzymes to functionally characterize due to the difficulty of recombinantly expressing these membrane-associated monooxygenases. In the course of investigating rice diterpenoid biosynthesis, we have developed a synthetic biology approach for functional expression of relevant CYPs in Escherichia coli. In certain cases, activity was observed for only one of two closely related paralogs although it seems clear that related reactions are required for production of the known diterpenoids. Here, we report that optimization of the recombinant expression system enabled characterization of not only these previously recalcitrant CYPs, but also discovery of additional activity relevant to rice diterpenoid biosynthesis. Of particular interest, CYP701A8 was found to catalyze 3β-hydroxylation of syn-pimaradiene, which is presumably relevant to momilactone biosynthesis, while CYP71Z6 & 7 were found to catalyze multiple reactions, with CYP71Z6 catalyzing the production of 2α,3α-dihydroxy-ent-isokaurene via 2α-hydroxy-ent-isokaurene, and CYP71Z7 catalyzing the production of 3α-hydroxy-ent-cassadien-2-one via 2α-hydroxy-ent-cassadiene and ent-cassadien-2-one, which may be relevant to oryzadione and phytocassane biosynthesis, respectively.

Keywords

Metabolic engineering Natural products Labdane-related diterpenoids Phytoalexin Antibiotic Evolution 

Notes

Funding

This work was supported by grants from the USDA (AFRI-NIFA 2014-67013-21720) and NIH (GM076324), and funds from Iowa State University, to R.J.P.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2015_6496_MOESM1_ESM.pdf (377 kb)
ESM 1 (PDF 377 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Naoki Kitaoka
    • 1
    • 2
  • Yisheng Wu
    • 1
    • 3
  • Meimei Xu
    • 1
  • Reuben J. Peters
    • 1
    Email author
  1. 1.Department of Biochemistry, Biophysics, and Molecular BiologyIowa State UniversityAmesUSA
  2. 2.Tohoku UniversitySendaiJapan
  3. 3.Conagen Inc.BedfordUSA

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