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

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.

Genetic basis of destruxin production in the entomopathogen Metarhizium robertsii

“…In contrast, mutants of B. bassiana with targeted disruptions of the peptide synthetase genes responsible for the biosynthesis of beauvericin and bassianolide were morphologically indistinguishable from WT, but the KO strains showed decreased virulence when tested against S. frugiperda, Helicoverpa zeae, and G. mellonella larvae (Xu et al 2008(Xu et al , 2009. Similarly, targeted gene knockout of a geranylgeranyl diphosphate synthase abolished the production of helvolic acid and reduced virulence of M. anisopliae NAFF635007 against two genera of insect larvae (Singkaravanit et al 2010).…”

Metabolic Conservation and Diversification of Metarhizium Species Correlate with Fungal Host-Specificity

“…The biosynthetic mechanism of helvolic acid has not been fully understood. A loss-of-function study revealed that a geranylgeranyl diphosphate synthase (BAJ05823) is involved in the production of helvolic acid in M. anisopliae (Singkaravanit et al, 2010). Our genome survey indicated that the homolog of this protein is present not only in the non-specialists (e.g., MAA_03020 of M. robertsii , 82% identity) but also in M. acridum (MAC_02619, 80%) and M. album (MAM_05601, 75%).…”

Secondary metabolite arsenal of an opportunistic pathogenic fungus

“…Following on trypacidin, helvolic acid also exhibits antiprotozoal activity [67] and is produced by several fungal spp. in addition to A. fumigatus, including several plant and insect pathogens and many endophytes [28,68,69]. Along with gliotoxin and fumagillin, helvolic acid exhibits cilioinhibitory activity [70] and is a potent antimicrobial [71].…”