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Molecular cloning and functional characterization of α-humulene synthase, a possible key enzyme of zerumbone biosynthesis in shampoo ginger (Zingiber zerumbet Smith)

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Abstract

Shampoo ginger (Zingiber zerumbet Smith) has a high content and large variety of terpenoids in the essential oil of its rhizome. Here, we report on the isolation of a cDNA clone (ZSS1) encoding α-humulene synthase, a possible key enzyme of zerumbone biosynthesis. This clone contains an open reading frame of 1,644 bp and is predicted to encode a protein of 548 amino acids with a calculated molecular mass of 64.5 kDa. The deduced amino acid sequence shows 34–63% identity with known sesquiterpene synthases of other angiosperm species. Based on exon-intron organization, ZSS1 is classified as the terpene synthase-III (TPS-III) subfamily. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of a major product, α-humulene (95%) and a minor by-product, β-caryophyllene (5%). Introduction of ZSS1 and the foreign mevalonate pathway involved in FPP synthesis into E. coli results in in vivo production of α-humulene. Transcript of ZSS1 was detected almost exclusively in rhizomes and was up-regulated in both leaves and rhizomes after treatment with methyl jasmonate (MeJA), suggesting its ecological function in shampoo ginger.

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Abbreviations

DMAPP:

Dimethylallyl diphosphate

DXP:

Deoxyxylulose 5-phosphate

FPP:

Farnesyl diphosphate

GC-MS:

Gas chromatography-mass spectrometry

HMGS:

Hydroxymethylglutaryl CoA synthase

HMGR:

Hydroxymethylglutaryl CoA reductase

IPP:

Isopentenyl diphosphate

IPPI:

Isopentenyl diphosphate isomerase

MK:

Mevalonate kinase

MeJA:

Methyl jasmonate

MPD:

Mevalonate diphosphate decarboxylase

ORF:

Open reading frame

PMK:

Phosphomevalonate kinase

RACE:

Rapid Amplification of cDNA Ends

RT-PCR:

Reverse transcription PCR

TPS:

Terpene synthase

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Acknowledgments

We thank Mr. T. Ishida, Ms. R. Sawa and Mr. H. Miyawaki for providing the ginger plants. We are also grateful to Professor R.Croteau for his helpful advice. This work was supported in part by the Research and Development Program for New Bio-industry Initiatives (2006–2010) from the Bio-oriented Technology Research Advancement Institution (BRAIN), by the “Academic Frontier” Project for Private Universities: matching fund subsidy from Ministry of Education, Culture, Sports, Science and Technology (2004–2008) and by the Sasagawa Scientific Research Grant from the Japan Science Society.

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Authors and Affiliations

  1. Department of Bioscience, Graduate School of Agriculture, Kinki University, 3327-204 Nakamachi, Nara, 631-8505, Japan

    Fengnian Yu, Sho Okamto & Ryutaro Utsumi

  2. Department of Biotechnology and Chemistry, Graduate School of Engineering, Kinki University, Higashihiroshima, Hiroshima, 739-2116, Japan

    Kaoru Nakasone

  3. Marine Biotechnology Institute, Heita, Kamaishi, Iwate, 026-0001, Japan

    Kyoko Adachi, Satoru Matsuda, Hisashi Harada & Norihiko Misawa

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  1. Fengnian Yu
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  2. Sho Okamto
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  5. Satoru Matsuda
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  6. Hisashi Harada
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  8. Ryutaro Utsumi
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Corresponding author

Correspondence to Ryutaro Utsumi.

Additional information

The nucleotide sequences reported in this article has been deposited in DDBJ/GenBank/EMBL under accession number(s) AB 247331 and AB263736.

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Yu, F., Okamto, S., Nakasone, K. et al. Molecular cloning and functional characterization of α-humulene synthase, a possible key enzyme of zerumbone biosynthesis in shampoo ginger (Zingiber zerumbet Smith). Planta 227, 1291–1299 (2008). https://doi.org/10.1007/s00425-008-0700-x

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  • DOI: https://doi.org/10.1007/s00425-008-0700-x

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