Applied Microbiology and Biotechnology

, Volume 87, Issue 3, pp 1077–1088 | Cite as

Cloning and functional analysis of the second geranylgeranyl diphosphate synthase gene influencing helvolic acid biosynthesis in Metarhizium anisopliae

  • Suthitar Singkaravanit
  • Hiroshi Kinoshita
  • Fumio Ihara
  • Takuya Nihira
Applied Genetics and Molecular Biotechnology
First Online:
Received:
Revised:
Accepted:

Abstract

A gene (ggs2) having high similarity to the geranylgeranyl diphosphate synthase (GGPP synthase) gene was cloned from Metarhizium anisopliae NAFF635007. The ggs2 gene (1,239-bp open reading frame with no intron) encoded a protein of 412 amino acids, and the transcription occurred only after late log-phase during the growth. Gene disruption of ggs2, performed to clarify the function in M. anisopliae, resulted in decreased GGPP synthase activity together with a slight delay of sporulation. An high performance liquid chromatography (HPLC) comparison of compound profiles between the wild-type strain and the disruptant revealed that a compound was abolished by the ggs2 disruption. Purification and structural elucidation by 1H-NMR and mass spectrometry analyses revealed that the lost compound is helvolic acid. Furthermore, the pathogenicity assay against two species of insect larvae revealed that the ggs2-disruptant possessed much weaker toxicity than the wild-type strain. Based on these results, it was concluded that ggs2 encodes the GGPP synthase influencing the biosynthesis of secondary metabolites in various species, including helvolic acid in M. anisopliae. To the best of our knowledge, this is the first report to identify a GGPP synthase gene related to secondary metabolism in entomopathogenic fungi.

Keywords

Geranylgeranyl diphosphate synthase Metarhizium anisopliae Helvolic acid Secondary metabolism 
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Notes

Acknowledgements

This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT; to S. S.), by a grant for a “Research Project in the Field of Biotechnology” from the Japan Society for the Promotion of Science (JSPS), by grants from the National Research Council of Thailand and the National Science and Technology Development Agency of Thailand (to T. N.) and by a Grant-in-Aid for Scientific Research from JSPS (to H. K. and T. N.). This paper is part of a Ph.D. dissertation by S. Singkaravanit.

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Suthitar Singkaravanit
    • 3
  • Hiroshi Kinoshita
    • 3
  • Fumio Ihara
    • 2
  • Takuya Nihira
    • 3
    • 1
  1. 1.MU-OU Collaborative Research Center for Bioscience and Biotechnology, Faculty of ScienceMahidol UniversityBangkokThailand
  2. 2.Apple Pest Control Research SubteamNational Institute of Fruit Tree ScienceMoriokaJapan
  3. 3.International Center for BiotechnologyOsaka UniversityOsakaJapan

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