Studies on Biosynthetic Genes and Enzymes of Isoprenoids Produced by Actinomycetes

Abstract

Most Streptomyces strains are equipped with only the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the formation of isopentenyl diphosphate, a common precursor of isoprenoids. In addition to this pathway, some Streptomyces strains possess the mevalonate (MV) pathway via which isoprenoid antibiotics are produced. We have recently cloned and analyzed the MV pathway gene clusters and their flanking regions from terpentecin, BE-40644, and furaquinocin A producers. All these clusters contained genes coding for mevalonate kinase, mevalonate diphosphate decarboxylase, phosphomevalonate kinase, type 2 IPP isomerase, HMGCoA reductase, and HMG-CoA synthase. The order of each of the open reading frames (ORFs) is also the same, and the respective homologous ORFs show more than 70% amino acid identity with each other. In contrast to these conservative gene organizations, the biosynthetic genes of terpentecin, BE-40644, and furaquinocin A were located just upstream and/or downstream of the MV pathway gene cluster. These facts suggested that all the actinomycete strains possessing both the MV and MEP pathways produce isoprenoid compounds and the biosynthetic genes of one of these isoprenoids usually exist adjacent to the MV pathway gene cluster. Therefore, when the presence of the MV cluster is detected by molecular genetic techniques, isoprenoids may be produced by the cultivation of these actinomycete strains. During the course of these studies, we identified diterpene cyclases possessing unique primary structures that differ from those of eukaryotes and catalyze unique reactions.Keywords isoprenoid, mevalonate pathway, biosynthesis, cyclase, Actinomycetes IntroductionActinomycete strains usually produce a number of secondary metabolites that often possess pharmaceutical activities. Therefore, for a long time, the strains have been used for many screenings to find novel, medically useful compounds. Consequently, more than 60 percent of the known antibiotics, including not only antibacterial antibiotics but also bioactive microbial compounds, have been reported to be produced by actinomycetes [1]. Presently, such pharmaceutically important, novel compounds can be found in the culture broth of actinomycete strains by screening rare actinomycetes, developing new screening strategies, employing sensitive assay methods for the detection of low concentrations of antibiotics, etc. However, sometimes, "semi-new antibiotics"-derivatives of known antibiotics-were isolated; discovering novel antibiotics every year became very difficult.In the past two decades, recombinant DNA technology has come into play in this field. Gene clusters encoding many natural products have been cloned and characterized. Moreover, whole-genome sequencing has uncovered hundreds of candidates for secondary metabolic pathways. Among them, the biosynthetic machinery of nonribosomal peptide and polyketide natural products has been extensively investigated [2ϳ7]. Recent progress in the application of combinatorial biosynthesis methods to these bi...

An alternative menaquinone biosynthetic pathway operating in microorganisms: an attractive target for drug discovery to pathogenic Helicobacter and Chlamydia strains

“…7 HOW IT STARTED I have been studying the enzymes and genes responsible for the biosyntheses of isoprenoids produced by actinomycetes. 8 Approxi-mately 6 years ago, I tried to clone a gene cluster participating in the biosynthesis of KS-505a (Figure 2), which is produced by Streptomyces argenteolus strain A-2 and has a unique cyclized tetraterpene moiety and an aromatic moiety derived from OSB. 9 Antibiotic biosynthetic genes cloned from actinomycetes are usually clustered in a genomic DNA region, 8 and OSB is synthesized by the MenF, -D and -C enzymes in the known MK biosynthetic pathway.…”

“…8 Approxi-mately 6 years ago, I tried to clone a gene cluster participating in the biosynthesis of KS-505a (Figure 2), which is produced by Streptomyces argenteolus strain A-2 and has a unique cyclized tetraterpene moiety and an aromatic moiety derived from OSB. 9 Antibiotic biosynthetic genes cloned from actinomycetes are usually clustered in a genomic DNA region, 8 and OSB is synthesized by the MenF, -D and -C enzymes in the known MK biosynthetic pathway. 1,2 Therefore, I first tried to clone menF, -D and -C orthologs from the KS505a producer, as actinomycetes are known to synthesize MK and the length of the prenyl side chain of MK has been used for taxonomic studies of actinomycetes.…”

Unique Biosynthesis of Sesquarterpenes (C35Terpenes)

“…31) It has been reported that many terpene cyclases have broad substrate specificities, and that other classes of terpenes have been created in vitro. [2][3][4][5][6][7][8][9][10][11][12][13][14][15] A combination of studies identifying enzymatic products biosynthesized from unusual substrates and the analysis of genome sequences should lead not only to the discovery of additional functions of these enzymes in the organism, but also to the identification of novel natural terpenoids that have functions in their producers.…”

“…1. Since a number of genes responsible for the biosynthesis of terpenoids form clusters in microbial genomes, 5) we searched for ts among the genes around the HepPP synthase gene (hepS and hepT), as well as a putative TC gene (sqhC), in the B. subtilis genome, but we were not able to find any candidate. 29) Hence, we investigated 2,514 gene-disrupted strains of B. subtilis, provided by the National BioResource Project (NIG, Japan) to isolate ts.…”

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] These compounds including hormones, vitamins, drugs, flavorings, and pigments, are categorized by number of C 5 isoprene units, as follows: hemi-(C 5 ), 1 unit; mono-(C 10 ), 2 units; sesqui-(C 15 ), 3 units; di-(C 20 ), 4 units; sester-(C 25 ), 5 units; tri-(C 30 ), 6 units; and tetraterpenes (C 40 ), 8 units. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] In contrast, little is known about C 35 terpenes. To the best of my knowledge, only 19 cyclic and eight linear C 35 terpenes have been identified to date, [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33]…”