Biosynthetic gene-based secondary metabolite screening: a new diterpene, methyl phomopsenonate, from the fungus Phomopsis amygdali.

The presence of the geranylgeranyl diphosphate synthase (GGS) gene is a common feature of gene clusters for diterpene biosynthesis. We demonstrated identification of a diterpene gene cluster using homology-based PCR of GGS genes and the subsequent genome walking in the fungus Phomopsis amygdali N2. Structure determination of a novel diterpene hydrocarbon phomopsene provided by enzymatic synthesis with the recombinant terpene synthase PaPS and screening of fungal broth extracts with reference to characteristic NMR signals of phomopsene allowed us to isolate a new diterpene, methyl phomopsenonate. The versatility of the gene-based screening of unidentified diterpenes is discussed in regard to fungal genomic data.

Spectrophotometric determination of quinolone antibiotics by an association complex formation with aluminum(III) and erythrosin.

A simple and sensitive spectrophotometric method for the determination of quinolone antibiotics was established based on an association complex formation with aluminum(III) and erythrosin. In the determination of ofloxacin as a quinolone antibiotic, Beer's law is obeyed in the range of 0.1 - 3.2 microg ml(-1), with an effective molar absorptivity at 555 nm and the relative standard deviation being 1.2 x 10(5) L mol(-1) cm(-1) and 0.9% (n = 6). This method was successfully applied to the assay of quinolone antibiotics in pharmaceutical preparations.

Functional analysis of eubacterial ent-copalyl diphosphate synthase and pimara-9(11),15-diene synthase with unique primary sequences.

We have previously cloned a DNA fragment that contained four ORFs and was confirmed to participate in viguiepinol {3-hydroxypimara-9(11),15-diene} biosynthesis by a heterologous expression experiment, from Streptomyces sp. strain KO-3988. Of the four ORFs, ORF2 and ORF4 were confirmed to encode an ent-CDP synthase and a GGDP synthase, respectively, by experiments using recombinant enzymes. In this study, ORF3, that did not show similarities with any other known proteins was expressed in Escherichia coli and used for functional analysis. The purified ORF3 product clearly converted ent-CDP into PMD. Since ORF2 and ORF3 are the first examples of enzymes with these biosynthetic functions from prokaryotes, enzymatic properties of both enzymes were investigated. ORF2 is likely to be a dimer and requires a divalent cation such as Mg2+ and Zn2+ for its activity. The optimum pH and temperature were 5.5 and 35 degrees C. The Km value was calculated to be 13.7 +/- 1.0 microM for GGDP and the kcat value was 3.3 x 10(-2)/sec. ORF3 is likely to be a monomer and also requires a divalent cation. The optimum pH and temperature were 7.0 and 30 degrees C. The Km value for ent-CDP was estimated to be 2.6 +/- 0.2 microM and the kcat value was 1.4 x 10(-3)/sec.