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KS-505a (longestin), produced by Streptomyces argenteolus, has a unique structure that consists of a tetraterpene (C40) skeleton, to which a 2-O-methylglucuronic acid and an o-succinyl benzoate moiety are attached. It is a novel inhibitor of calmodulin-dependent cyclic-nucleotide phosphodiesterase, which is representative of a potent anti-amnesia drug. As a first step to understanding the biosynthetic machinery of this unique and pharmaceutically useful compound, we cloned a KS505a biosynthetic gene cluster. First we searched for a gene encoding octaprenyl diphosphates, which yielded a C40 precursor by PCR, and four candidate genes were obtained. Among these, one was confirmed to have the expected enzyme activity by recombinant enzyme assay. On the basis of an analysis of the flanking regions of the gene, a putative KS-505a biosynthetic gene cluster consisting of 24 ORFs was judged perhaps to be present on a 28-kb DNA fragment. A gene disruption experiment was also employed to confirm that the cluster indeed participated in KS-505a biosynthesis. This is believed to be the first report detailing the gene cluster of a cyclized tetraterpenoid.

Furaquinocin (FQ) A, produced by Streptomyces sp. strain KO-3988, is a natural polyketide-isoprenoid hybrid compound that exhibits a potent antitumor activity. As a first step toward understanding the biosynthetic machinery of this unique and pharmaceutically useful compound, we have cloned an FQ A biosynthetic gene cluster by taking advantage of the fact that an isoprenoid biosynthetic gene cluster generally exists in flanking regions of the mevalonate (MV) pathway gene cluster in actinomycetes. Interestingly, Streptomyces sp. strain KO-3988 was the first example of a microorganism equipped with two distinct mevalonate pathway gene clusters. We were able to localize a 25-kb DNA region that harbored FQ A biosynthetic genes (fur genes) in both the upstream and downstream regions of one of the MV pathway gene clusters (MV2) by using heterologous expression in Streptomyces lividans TK23. This was the first example of a gene cluster responsible for the biosynthesis of a polyketide-isoprenoid hybrid compound. We have also confirmed that four genes responsible for viguiepinol [3-hydroxypimara-9(11),15-diene] biosynthesis exist in the upstream region of the other MV pathway gene cluster (MV1), which had previously been cloned from strain KO-3988. This was the first example of prokaryotic enzymes with these biosynthetic functions. By phylogenetic analysis, these two MV pathway clusters were identified as probably being independently distributed in strain KO-3988 (orthologs), rather than one cluster being generated by the duplication of the other cluster (paralogs).

We have previously shown that gene clusters for biosyntheses of terpentecin and BE-40644, a diterpene antibiotic and a sesquiterpene antibiotic, respectively, were located in the adjacent mevalonate pathway gene clusters. In this study, a mevalonate pathway gene cluster was cloned from Streptomyces sp. strain KO-3988, which was known to produce furaquinocin A, employing a hybridization experiment using a 3-hydroxy-3-methyl glutaryl CoA (HMG-CoA) reductase gene, which had been previously cloned from the strain KO-3988, as a probe. By sequencing flanking regions, we found four open reading frames that could encode a putative cytochrome P450 (ORF1), an isoprenoid cyclase (ORF2), an unknown protein (ORF3), and a polyprenyl diphosphate synthase gene (ORF4) in the upstream region of the mevalonate pathway gene cluster, though we did not find any genes related to furaquinocin A biosynthesis. The two ORFs (ORF2 and 4) were expressed as recombinant enzymes in E. coli and used for studies to investigate functions of these products. The ORF4 product was confirmed to be a geranylgeranyl diphosphate (GGDP, C20) synthase. The ORF2 product proved to catalyze a conversion of GGDP into copalyl diphosphate, the first example of an enzyme with this function of prokaryotic origin. These results again showed that actinomycetes possessing the mevalonate pathway usually produce an isoprenoid and that its biosynthetic gene cluster exists in adjacent the mevalonate pathway gene cluster.

Streptomyces griseolosporeus MF730-N6, a terpenoid antibiotic-terpentecin (Tp) producer, has both the nonmevalonate and mevalonate pathways for the formation of IPP. The Tp biosynthetic gene (ter) and the mevalonate pathway gene cluster (mev) including an HMG-CoA reductase gene (hmgr) had previously been cloned from strain MF730-N6. In this study, two distinct dxs genes (dxs 1 and dxs 2) and a dxr gene, which encode DXP synthases and DXP reductoisomerase, and participate in the initial and the second step of the nonmevalonate pathway, respectively, were cloned. These gene products were expressed in E. coli and confirmed to have the expected activities. The dxs 1, dxs 2, dxr, mev, and ter genes were used for Northern blot and primer extension analyses to examine temporal expression of these genes together with a gap gene coding for GAP dehydrogenase, which was also cloned in this study and used as an internal control. Transcripts of the dxs 1, dxs 2, dxr, and gap genes were detected throughout the cultivation. On the other hand, messages of the mev and ter genes were not detected at early growth phase but appeared when Tp production started. These results suggested that the nonmevalonate pathway and the mevalonate pathway were mainly used for the primary metabolism and the secondary metabolism, respectively, and that both of the two dxs genes were actually transcribed in this strain.

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°C. The Km value was calculated to be 13.7 ± 1.0 μM for GGDP and the kcat value was 3.3 × 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°C. The Km value for ent-CDP was estimated to be 2.6 ± 0.2 μM and the kcat value was 1.4 × 10−3/sec. [ABSTRACT FROM PUBLISHER]

Prenyltransferases catalyze the sequential condensation of isopentenyl diphosphate into prenyl diphosphates with specific chain lengths. Pioneering studies demonstrated that the product specificities of type I prenyltransferases were mainly determined by the amino acid residues at the 4th and 5th positions before the first aspartate-rich motif (FARM) of the prenyltransferases. We previously cloned a type I geranylgeranyl diphosphate synthase (GGDPSase) gene from Streptomyces griseolosporeus MF730-N6 [Hamano, Y., Dairi, T., Yamamoto, M., Kawasaki, T., Kaneda, K., Kuzuyama, T., Itoh, N., and Seto, H. (2001) Biosci. Biotechnol. Biochem. 65, 1627–1635]. In this study, a prenyltransferase gene was cloned from Streptomyces argenteolus A-2 and was confirmed to encode a type I farnesyl diphosphate synthase (FDPSase). Interestingly, the amino acid residues at the 4th and 5th positions before the FARM were the same in these two enzymes. To identify the amino acid that determines the product chain length, mutated enzymes, GGDPSase (L-50S), FDPSase (S-50L), GGDPSase (V-8A), FDPSase (A-8V), GGDPSase (A+57L), and FDPSase (L+58A), in which the amino acid residue at the –50th, –8th, and +57th (58th) position before or after the FARM was substituted with the corresponding amino acid of the other enzyme, were constructed. The GGDPSase (A+57L) and FDPSase (L+58A) produced farnesyl diphosphate and geranylgeranyl diphosphate, respectively. On the other hand, the other mutated enzymes produced prenyl diphosphates with the same chain lengths as the wild type enzymes did. These results showed that the amino acid residue at the 57th (58th) position after the FARM also played an important role in determination of the product specificity. [ABSTRACT FROM AUTHOR]

Examines the role of eubacterial diterpine cyclase genes in the production of isoprenoid antibiotic terpentecin. Details on the sequence analysis in the upstream region of gene cluster; Expression of cyclase genes in Streptomyces lividans; Discussion on the biosynthesis of isoprenoid antibiotics produced by actinomycetes.

The article presents a study which reveals a new biosynthetic pathway for ubiquinone and menaquinone (MK). Researchers have conducted 13C-labeling experiments using Streptomyces CL190 to reveal the origin of isopentenyl diphosphate (IPP) and have established an equivocal assignment of the naphthoquinone moiety of MK with a highly symmetric structure. Results of the analysis show that there is a new pathway for the biosynthesis of MK in Streptomyces and in several pathogenic bacteria.