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. 2011 Mar;68(6):1033-40.
doi: 10.1007/s00018-010-0506-4. Epub 2010 Aug 22.

Zingiber zerumbet CYP71BA1 catalyzes the conversion of α-humulene to 8-hydroxy-α-humulene in zerumbone biosynthesis

Fengnian Yu  1 , Sho Okamoto, Hisashi Harada, Kazuhisa Yamasaki, Norihiko Misawa, Ryutaro Utsumi
Affiliations

Zingiber zerumbet CYP71BA1 catalyzes the conversion of α-humulene to 8-hydroxy-α-humulene in zerumbone biosynthesis

Fengnian Yu et al. Cell Mol Life Sci. 2011 Mar.

Abstract

Plant cytochrome P450s are involved in the biosynthesis of various classes of secondary metabolites. To elucidate the biosynthesis of zerumbone, a sesquiterpenoid with multiple potential anticancer properties, a family of P450 genes expressed in rhizomes of Zingiber zerumbet Smith, were cloned using a PCR-based cloning strategy. After functional expression in yeast, one of these P450s was found to convert α-humulene into 8-hydroxy-α-humulene, a proposed intermediate of zerumbone biosynthesis. This P450 has been designated CYP71BA1, a new member of the CYP71 family. CYP71BA1 transcripts were detected almost exclusively in rhizomes and showed a similar expression pattern to ZSS1 transcripts during rhizome development. Coexpression of a gene cluster encoding four enzymes of the mevalonate pathway with CYP71BA1 and ZSS1 in Escherichia coli leads to the production of 8-hydroxy-α-humulene in the presence of mevalonate, suggesting the possibility of microbial production of this zerumbone intermediate from a relatively simple carbon source by metabolic engineering.

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Figures

Fig. 1
Fig. 1
Proposed pathway for zerumbone biosynthesis in Z. Zerumbet
Fig. 2
Fig. 2
Phylogenetic tree of Z. zerumbet CYP71BA1 and related functionally characterized terpene-modifying P450s. CYP706B1, G. arboreum (+)-δ-cadinene-8-hydroxylase (AF332974); CYP76B6, C. roseus geraniol 10-hydroxylase (AJ251269); CYP71A-like, Mentha x piperita (+)-menthofuran synthase (AF346833); CYP71AV1, A. annua amorpha-4,11-diene monooxygenase (DQ315671); HPO, H. muticus premnaspirodiene oxygenase (EF569601); CYP71D20, N. tabacum 5-epiaristolochene-1,3-dihydroxylase (AF368376); CYP71D13, Mentha x piperita (−)-limonene-3-hydroxylase (AY281027); CYP71D18, Mentha spicata (−)-limonene-6-hydroxylase (AF124815); GAO, B. spinosa germacrene A oxidase (GU256647); CYP720B1, Pinus taeda abietadienol/abietadienal oxidase (AY779537); CYP701A3, A. thaliana ent-kaurene oxidase (AF047719)
Fig. 3
Fig. 3
GC-MS analysis of products formed in in vitro enzyme assays. a Chromatogram of products obtained from assay with yeast microsomes harboring empty expression vector (pYeDP60). b Chromatogram of products generated by incubation of α-humulene with yeast microsomes harboring CYP71BA1 and a chromatogram of authentic 8-hydroxy-α-humulene. c Mass spectrum of product formed by incubation of α-humulene with yeast microsomes expressing CYP71BA1. d Mass spectrum of authentic 8-hydroxy-α-humulene
Fig. 4
Fig. 4
Transcript expression analysis of CYP71BA1 and ZSS1. a RT-PCR analysis of ZSS1 and CYP71BA1 transcripts in leaves, stems and rhizomes. b Temporal expression of ZSS1 and CYP71BA1 during rhizome development (2007)
Fig. 5
Fig. 5
Schematic structure of plasmid pAC-MvATR2Hum. MK mevalonate kinase, PMK phosphomevalonate kinase, MPPD mevalonate diphosphate decarboxylase, IPPI isopentenyl diphosphate isomerase, ATR2 Arabidopsis P450 reductase 2 gene, SD Shine-Dalgarno sequence
Fig. 6
Fig. 6
Identification of in vivo 8-hydroxy-α-humulene production in metabolically engineered E. coli. a Chromatogram of the products obtained from the control E. coli containing pAC-MvATR2Hum and the empty vector pET101. b Chromatogram of the products formed from engineered E. coli harboring pAC-MvATR2Hum and pET-CYP71BA
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