Presence of Copalyl Diphosphate Synthase Gene in an Actinomycete Possessing the Mevalonate Pathway

Abstract

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 (HMGCoA) 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 argenteolus that produce the isoprenoid compounds, pentalenolactone4), brasilicardin A5) and KS505a3), respectively. On the other hand, we have previously showed that both the mevalonate and MEP pathways were operating in some Streptomyces strains such as Kitasatospora griseola (terpentecin producer)6,7), Actinoplanes sp. strain A40644 (BE-40644 producer)7-9), Streptomyces sp. strain CLI90 (naphterpin producer)7,10,11),

Studies on Biosynthetic Genes and Enzymes of Isoprenoids Produced by Actinomycetes

“…10, although the absolute stereochemistry of the product remains unclear. However, this is the first example of an enzyme of prokaryotic origin with this function [79].…”

“…First, we analyzed the flanking regions of one of the MV clusters (MV1) and found 4 ORFs that could encode a putative cytochrome P450 (ORF1), a diterpene cyclase (ORF2), an unknown protein (ORF3), and a GGDP (C 20 ) synthase gene (ORF4) in the upstream region of the MV1 cluster [79] (Fig. 5C), although we did not find genes related to the biosynthesis of furaquinocin A.…”

Gibberellin biosynthesis in bacteria: Separate ent‐copalyl diphosphate and ent‐kaurene synthases in Bradyrhizobium japonicum

“…In particular, the ent-CPP synthase from Streptomyces sp. strain KO-3988 (SkoCPS) [30], the terpentediene diphosphate synthase from Streptomyces griseolosporeus (SgTPS) [31], and the halimadienyl diphosphate synthase from Mycobacterium tuberculosis (MtHPS) [32], which are 499-511 aa in length. Further, in the initial characterization of the associated operon it was suggested that the corresponding gene encoded a 516 aa length protein [27], and this was the construct analyzed here.…”

Cloning of the Gene Cluster Responsible for the Biosynthesis of Brasilicardin A, a Unique Diterpenoid

“…We found recently that biosynthetic genes of terpentecin [12] and 3-hydroxypimeradiene (viguiepinol) [14,15], both of which are diterpene compounds produced by actinomycetes, were located in flanking regions of the genes encoding geranylgeranyl diphosphate synthase (GGPPS), which supplies a direct precursor of diterpene compounds. Hence, we first tried to clone a GGPPS gene in order to use it as a probe to clone an entire BCA biosynthetic gene cluster.…”

“…Cloning of Polyprenyl Diphosphate Synthase Genes by PCR Three sets of primers were designed, based on the conserved amino acid sequences of polyprenyl diphosphate synthase genes, which had been cloned from terpentecin [22], furaquinocin A [14,15], and BE-40644 [23] producers:…”