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

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.

Fig. 1

Proposed pathway for zerumbone biosynthesis in Z. Zerumbet

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

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

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

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

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