Skip to main content
SpringerLink
Log in

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

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Atawong A, Hasegawa M, Kodama O (2002) Biosynthesis of rice phytoalexin: enzymatic conversion of 3ß-hydroxy-9ß-pimara-7,15-dien-19,6ß-olide to momilactone A. Biosci Biotechnol Biochem 66(3):566–570

    Article  CAS  PubMed  Google Scholar 

  • Cyr A, Wilderman PR, Determan M, Peters RJ (2007) A modular approach for facile biosynthesis of labdane-related diterpenes. J Am Chem Soc 129:6684–6685

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Hamberger B, Bak S (2013) Plant P450s as versatile drivers for evolution of species-specific chemical diversity. Philos Trans R Soc Lond Ser B Biol Sci 368(1612):20120426 doi:10.1098/rstb.2012.0426

  • Itoh H, Tatsumi T, Sakamoto T, Otomo K, Toyomasu T, Kitano H, Ashikari M, Ichihara S, Matsuoka M (2004) A rice semi-dwarf gene, Tan-Ginbozu (D35), encodes the gibberellin biosynthesis enzyme, ent-kaurene oxidase. Plant Mol Biol 54:533–547

    Article  CAS  PubMed  Google Scholar 

  • Kitaoka N, Lu X, Yang B, Peters RJ (2015) The application of synthetic biology to elucidation of plant mono-, sesqui-, and diterpenoid metabolism. Mol Plant 8(1):6–16

  • Kono Y, Kojima A, Nagai R, Watanabe M, Kawashima T, Onizawa T, Teraoka T, Watanab M, Koshino H, Uzawa J, Suzuki Y, Sakurai A (2004) Antibacterial diterpenes and their fatty acid conjugates from rice leaves. Phytochemistry 65(9):1291–1298. doi:10.1016/j.phytochem.2004.03.016

    Article  CAS  PubMed  Google Scholar 

  • Mizutani M, Ohta D (1998) Two isoforms of NADPH: cytochrome P450 reductase in Arabidopsis thaliana. Gene structure, heterologous expression in insect cells, and differential regulation. Plant Physiol 116:357–367

  • Mizutani M, Sato F (2011) Unusual P450 reactions in plant secondary metabolism. Arch Biochem Biophys 507(1):194–203. doi:10.1016/j.abb.2010.09.026

    Article  CAS  PubMed  Google Scholar 

  • Morrone D, Chen X, Coates RM, Peters RJ (2010a) Characterization of the kaurene oxidase CYP701A3, a multifunctional cytochrome P450 from gibberellin biosynthesis. Biochem J 431(3):337–344. doi:10.1042/BJ20100597

    Article  CAS  PubMed  Google Scholar 

  • Morrone D, Lowry L, Determan MK, Hershey DM, Xu M, Peters RJ (2010b) Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering. Appl Microbiol Biotechnol 85:1893–1906

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Okada K (2011) The biosynthesis of isoprenoids and the mechanisms regulating it in plants. Biosci Biotechnol Biochem 75(7):1219–1225

    Article  CAS  PubMed  Google Scholar 

  • Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver microsome II. Solubilization, purification, and properties. J Biol Chem 239:2379–2385

    CAS  PubMed  Google Scholar 

  • Peters RJ (2010) Two rings in them all: the labdane-related diterpenoids. Nat Prod Rep 27(11):1521–1530. doi:10.1039/C0NP00019A

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Podust LM, Sherman DH (2012) Diversity of P450 enzymes in the biosynthesis of natural products. Nat Prod Rep 29(10):1251–1266. doi:10.1039/c2np20020a

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Prisic S, Xu M, Wilderman PR, Peters RJ (2004) Rice contains two disparate ent-copalyl diphosphate synthases with distinct metabolic functions. Plant Physiol 136(4):4228–4236

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Schmelz EA, Huffaker A, Sims JW, Christensen SA, Lu X, Okada K, Peters RJ (2014) Biosynthesis, elicitation and roles of monocot terpenoid phytoalexins. Plant J 79(4):659–678. doi:10.1111/tpj.12436

    Article  CAS  PubMed  Google Scholar 

  • Shimura K, Okada A, Okada K, Jikumaru Y, Ko K-W, Toyomasu T, Sassa T, Hasegawa M, Kodama O, Shibuya N, Koga J, Nojiri H, Yamane H (2007) Identification of a biosynthetic gene cluster in rice for momilactones. J Biol Chem 282:34013–34018

    Article  CAS  PubMed  Google Scholar 

  • Sono M, Roach MP, Coulter ED, Dawson JH (1996) Heme-containing oxygenases. Chem Rev 96(7):2841–2888

    Article  CAS  PubMed  Google Scholar 

  • Swaminathan S, Morrone D, Wang Q, Fulton DB, Peters RJ (2009) CYP76M7 is an ent-cassadiene C11α-hydroxylase defining a second multifunctional diterpenoid biosynthetic gene cluster in rice. Plant Cell 21:3315–3325

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Urban P, Mignotte C, Kazmaier M, Delorme F, Pompon D (1997) Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5. J Biol Chem 272(31):19176–19186

    Article  CAS  PubMed  Google Scholar 

  • Wang Q, Hillwig ML, Peters RJ (2011) CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice. Plant J 65(1):87–95

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wang Q, Hillwig ML, Okada K, Yamazaki K, Wu Y, Swaminathan S, Yamane H, Peters RJ (2012a) Characterization of CYP76M5-8 indicates metabolic plasticity within a plant biosynthetic gene cluster. J Biol Chem 287(9):6159–6168

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wang Q, Hillwig ML, Wu Y, Peters RJ (2012b) CYP701A8: a rice ent-kaurene oxidase paralog diverted to more specialized diterpenoid metabolism. Plant Physiol 158(3):1418–1425

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Watanabe M, Kono Y, Uzawa J, Teraoka T, Hosokawa D, Suzuki Y, Sakurai A, Teraguchi M (1992) Structures of oryzalic acid B and three related compounds, a group of novel antibacterial diterpenes, isolated from leaves of a bacterial leaf blight-resistant cultivar of rice. Biosci Biotechnol Biochem 56:113–117

    Article  CAS  Google Scholar 

  • Wilderman PR, Xu M, Jin Y, Coates RM, Peters RJ (2004) Identification of syn-pimara-7,15-diene synthase reveals functional clustering of terpene synthases involved in rice phytoalexin/allelochemical biosynthesis. Plant Physiol 135(4):2098–2105

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wu Y, Hillwig ML, Wang Q, Peters RJ (2011) Parsing a multifunctional biosynthetic gene cluster from rice: biochemical characterization of CYP71Z6 & 7. FEBS Lett 585:3446–3451

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wu Y, Wang Q, Hillwig ML, Peters RJ (2013) Picking sides: distinct roles for CYP76M6 and -8 in rice oryzalexin biosynthesis. Biochem J 454(2):209–216

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Zi J, Peters RJ (2013) Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the Lamiaceae. Org Biomol Chem 11(44):7650–7652. doi:10.1039/c3ob41885e

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Zi J, Mafu S, Peters RJ (2014a) To gibberellins and beyond! Surveying the evolution of (Di)terpenoid metabolism. Annu Rev Plant Biol 65:259–286. doi:10.1146/annurev-arplant-050213-035705

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Zi J, Matsuba Y, Hong Y, Jackson A, Pichersky E, Tantillo DJ, Peters RJ (2014b) Biosynthesis of lycosantalonol, a cis-prenyl derived diterpenoid. J Am Chem Soc 136:16951–16953

    Article  CAS  PubMed  Google Scholar 

Download references

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.

Author information

Author notes

  1. Naoki Kitaoka

    Present address: Tohoku University, Sendai, 980-8577, Japan

  2. Yisheng Wu

    Present address: Conagen Inc., Bedford, MA, 01730, USA

Authors and Affiliations

  1. Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, 50011, USA

    Naoki Kitaoka, Yisheng Wu, Meimei Xu & Reuben J. Peters

Authors
  1. Naoki Kitaoka
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yisheng Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Meimei Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Reuben J. Peters
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Reuben J. Peters.

Additional information

Naoki Kitaoka and Yisheng Wu contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 377 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kitaoka, N., Wu, Y., Xu, M. et al. Optimization of recombinant expression enables discovery of novel cytochrome P450 activity in rice diterpenoid biosynthesis. Appl Microbiol Biotechnol 99, 7549–7558 (2015). https://doi.org/10.1007/s00253-015-6496-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-015-6496-2

Keywords

Search

Navigation