Eubacterial Diterpene Cyclase Genes Essential for Production of the Isoprenoid Antibiotic Terpentecin

Abstract

). Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes. We constructed two mutants, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivated by gene disruptions. The mutants produced no TP, confirming that these cyclase genes are essential for the production of TP. The two cyclase genes were also expressed in Streptomyces lividans together with the GGDP synthase gene under the control of the ermE* constitutive promoter. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced in Escherichia coli and purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is the first report of a eubacterial DC.Isoprenoids are the largest single family of compounds found in nature, with over 22,000 known examples (12), and can be classified into several groups based on the number of C 5 units derived from isopentenyl diphosphate (IPP), such as monoterpenes (C 10 ), sesquiterpenes (C 15 ), diterpenes (C 20 ), and triterpenes (C 30 ), etc. (12). These compounds are biosynthesized from the corresponding prenyl diphosphate. Geranyl diphosphate gives rise to monoterpenes, farnesyl diphosphate gives rise to sesquiterpenes, and geranylgeranyl diphosphate (GGDP) gives rise to diterpenes (9, 29) (Fig. 1). In many cases, these prenyl diphosphates undergo a range of cyclizations to produce the parent skeletons of each class, followed by a variety of modifications to give many thousands of different isoprenoid metabolites (9, 29).A variety of isoprenoid synthases (cyclases), most of which are from plants and fungi, have been purified and extensively studied (12). As for the genes encoding isoprenoid cyclases, more than 30 eukaryotic isoprenoid synthases have been cloned as cDNAs (9, 29). On the other hand, there have been few reports about eubacterial isoprenoid cyclases and genes because the vast majority of isoprenoids are produced by eukaryotes. Pentalenene synthase, a sesquiterpene cyclase from a Streptomyces strain (10), and squalene-hopene cyclases, triterpene cyclases from bacteria, are the only examples (22,32,34,35,42,46). There are no reports, to the best of our knowledge, about eubacterial monoterpene cyclases and diterpene cyclases (DCs).We have been studying the biosynthesis of isoprenoid antibiotics produced by actinomycetes. Although actinomycetes produce approximately 70% of all natural compounds, a very limited number of isoprenoid compounds are known to be produced by them (30). The gene cluster containing the mevalonate pathway genes used to synthesize IPP had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpene antibiotic terpentecin (TP) producer (15). The GGDP synthase gene encoding the enzyme catalyz...

Studies on Biosynthetic Genes and Enzymes of Isoprenoids Produced by Actinomycetes

“…As described above, we showed that gene clusters concerned with the biosyntheses of TP [75] and BE-40644 [78], a diterpene antibiotic and a sesquiterpene antibiotic, respectively, were located in the adjacent MV pathway gene clusters. Next, to examine whether an MV pathway gene cluster and isoprenoid biosynthetic genes are always clustered in all the actinomycete strains possessing both pathways, an MV pathway gene cluster was cloned from Streptomyces sp.…”

“…To verify the enzymatic functions of Cyc1 and Cyc2, these ORF products were overproduced as His-tagged fusion proteins in E. coli and were used for an enzyme assay. Incubation of both the recombinant proteins with GGDP (1) resulted in the formation of TTE (3), confirming that the Cyc1 and Cyc2 products were the diterpene cyclases essential for the formation of TP (4) [75].…”

“…Considering that TP was previously reported to be synthesized from GGDP after successive cyclization, hydroxylation, and epoxidation, these ORFs were believed to encode TP biosynthetic genes. To confirm this probability, gene disruption experiments were employed [75]. We constructed two types of mutants in which cyc1 or cyc2 was inactivated by a gene replacement technique and confirmed that either of the mutants did not produce TP, showing that both cyc1 and cyc2 were essential for the production of TP.…”

“…5A). Each of the ORFs had a significant similarity to efflux proteins responsible for antibiotic resistance (ORF8), an unknown protein found in the genomic DNA of Streptomyces coelicolor A3(2) (ORF9), P450 (ORF10), diterpene cyclase from eukaryote (Cyc1), a pentalenene synthase of Streptomyces strain (Cyc2), P450 (ORF13), and ferredoxin (ORF14), respectively [75]. Considering that TP was previously reported to be synthesized from GGDP after successive cyclization, hydroxylation, and epoxidation, these ORFs were believed to encode TP biosynthetic genes.…”

“…As described above, the MV pathway gene cluster was located in the region adjacent to the TP biosynthetic gene cluster [75], and it supplied more than 50% of the IPP required for TP biosynthesis. These facts led us to investigate whether MV pathway genes and isoprenoid biosynthetic genes are always clustered in strains possessing both the MV and MEP pathways.…”