, 1992). A 1370-bp EcoRV–BamHI fragment internal to orf4 was cloned into the same sites of pKC1139 to pSK1-dKS, an orf4-disruption plasmid.

Fermentation of S. galbus and extraction of galbonolides were conducted by following the previously documented procedure, with minor modifications (Fauth et al., 1986). Briefly, mycelium was collected from the culture by centrifugation and submerged in a minimal volume of methanol for extraction. The CB-839 manufacturer culture supernatant was extracted with an equal volume of ethylacetate. The assay organism, C. neoformans IFO 40092, was obtained from the Culture Collection of the Research Centre for Pathogenic Fungi & Microbial Toxicoses, Chiba University, Chiba, Japan. Cryptococcus neoformans was maintained in

GYM agar and cultured in Bennett medium at 28 °C in a rotary shaker. The Bennett medium culture (OD600 nm, 2.0) was added to GYM soft agar (0.4% w/v agar) at a 0.01% dilution and overlaid on GYM agar to prepare the assay plate. For TLC analysis, a silica gel 60 F254 TLC-plate (Merck, Darmstadt, Germany) was developed with a solvent system composed of ethylacetate and benzene (1 : 3) (Abe et al., 1985) and placed on the assay plate upside down. The assay plates were incubated at 28 °C until the assay organism grew to selleck products form a confluent lawn of cells. An Agilent 1100 series LC system (Santa Clara, CA) was used for HPLC-MS analysis. A Bruker HCT 3000 ion trap mass spectrometer (Billerica, those MA) was coupled with the HPLC column, and the mass scan range was m/z 100–500. The dry temperature was 350 °C, the nebulizer gas was 40 p.s.i., and the dry gas was 9 L min−1. The separation was performed on a Gemini C-18 column (150 × 3.0 mm, 5.0 μm; Phenomenex, Torrance, CA) by isocratic elution. The column temperature was maintained at 25 °C. The flow rate was maintained at 0.5 mL min−1. The mobile phase was composed of methanol and 25 mM ammonium acetate in water (3 : 1). A

Varian HPLC ProStar system (Lake Forest, CA) was used for HPLC analysis with UV detection. The separation was performed on a Varian Pursuit XRs C-18 column (250 × 4.6 mm, 5.0 μm) and monitored at 230 nm. The flow rate was maintained at 0.75 mL min−1. A mobile phase consisting of 25 mM ammonium acetate in water, pH 5.5 (A), and methanol (B) was run with gradient elution: 100% A for 30 min; from 100% A to 5% A for 10 min; and then maintained at 5% A for 10 min. Cloning of an fkbI homologue led to the isolation of the cosmid clone pHJK1011, which contains a methoxymalonyl-ACP biosynthesis locus, from S. galbus. Complete nucleotide sequence determination of pHJK1011 confirmed the presence of a complete set of methoxymalonyl-ACP biosynthetic genes, galGHJIK (Fig. 2). The nucleotide sequence of the cosmid clone (41 591 bp insert) was deposited in the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank) under the accession number of CP000868.