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.
Abstracte seed oil of jatropha (Jatropha curcas L.) is a source of biodiesel fuel. Although jatropha can grow in semiarid lands unsuitable for the food production, its oil productivity in such conditions is unsatisfactory at present. erefore, it is desirable to improve the oil productivity of jatropha even in semi-arid lands by enhancing its drought tolerance. Genetic engineering is promising to dramatically improve plant traits. Although we previously reported a transformation method, which involves wounding of tissue explants in order to increase the chance of Agrobacterium infection, for jatropha, it remains a challenge to enhance the shoot regeneration and root induction processes. Here, we report the generation of three kinds of transgenic jatropha plants in an attempt to improve their drought tolerance. e rst one overexpresses the PPAT gene, which encodes an enzyme that catalyzes the CoA biosynthetic pathway; the second overexpresses the NF-YB gene, which encodes a subunit of the NF-Y transcription factor; and the last overexpresses the GSMT and DMT genes, which encode enzymes that catalyze production of glycine betaine. We also report a modi ed protocol that improves the e ciency of shoot regeneration and root induction in transgenic jatropha plantlets.
Based on comparative amino-acid sequence alignment of geranylgeranyl diphosphate (GGPP) synthase from filamentous fungi, degenerated oligonucleotide primers were designed for searching GGPP synthase gene(s) in entomopathogenic fungi. Polymerase chain reaction with the designed primers amplified GGPP synthase homologues from five representative entomopathogenic fungi: Metarhizium anisopliae, Beauveria bassiana, Verticillium lecanii, Paecilomyces farinosus, and Nomuraea rileyi. Sequence comparison of the amplified of GGPP synthase homologue fragments revealed that M. anisopliae and B. bassiana have at least two different types of the GGPP synthase gene homologues. The first type (designated as ggs1), which is highly conserved among the five strains, has a unique Ser-rich region, SSXSSVSGSSS (X refers to L, A, V, or S), and is constitutively expressed throughout growth. In contrast, the second type of GGPP synthase gene homologue (ggs2) was discovered only in some strains, and genes of this type possessed high similarity to each other but showed relatively weak similarity to the ggs1 genes, with no detectable transcription under the cultivation conditions applied in this experiment. The ggs1 cloned from M. anisopliae, which encoded a putative protein of 359 amino acid residues, was heterologously expressed in E. coli. The recombinant protein showed activity to synthesize GGPP from farnesyl diphosphate and isopentenyl diphosphate. These results strongly suggested that the ggs1 gene encodes a GGPP synthase involved in primary metabolism.
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