Nucleotide sequences and expression of genes from Streptomyces purpurascens that cause the production of new anthracyclines in Streptomyces galilaeus

Abstract

Six open reading frames, rdmA to rdmF, in a 6,077-bp segment of Streptomyces purpurascens DNA which caused the production of hybrid anthracyclines were identified. The minimal fragment that produced anthracyclines modified at the 10th position contained rdmB to rdmD; rdmE is the gene for aklavinone-11-hydroxylase. RdmC is similar to a putative open reading frame in the daunorubicin biosynthetic cluster of Streptomyces peucetius and is likely to participate in the removal of the side chain at the 10th position.Anthracyclines are a group of substances that contain the 7,8,9,10-tetrahydro-5,12-naphthacene quinone structure (31); they are usually antibiotics produced by actinomycetes or their synthetic analogs. Some of them are important pharmaceutical agents which are used in cancer therapy. The anthracycline chromophore, aglycone, is synthesized by the polyketide pathway in producing organisms (6). The polyketide synthases of several streptomycetes have been found to have a high degree of conservation (17,18), which has enabled the biosynthetic genes of additional polyketide antibiotics to be cloned (22).Hybrid antibiotics are molecules which combine structural features of two or more antibiotics that are not normally produced by the same organism and are often produced by transferring genes from one antibiotic producer to another strain that produces a structurally related antibiotic by recombinant DNA techniques (14, 16).Previously, Niemi et al. (25,27) described hybridizing a segment of Streptomyces purpurascens DNA near the locus with an actI (polyketide synthase) probe from Streptomyces coelicolor (22), which caused the production of glycosides of several modified aglycones ( Fig. 1) in Streptomyces galilaeus, a producer of aklavinone glycosides. The genes that caused hybrid production were located on a 6.1-kb BamHI-Sau3AI fragment, EB3 (including 9 bp from the polylinker of EMBL4 [8] with an EcoRI site).We have sequenced this fragment and expressed the aglycone-modifying enzyme activities it encodes by using a strong, constitutive promoter. Expression and sequence data indicate interesting similarities with daunorubicin and doxorubicin biosynthesis in Streptomyces peucetius.Sequence analysis. A random subcloning strategy was used (28). M13mp18 (37) clones were sequenced with Taq polymerase (Promega Corporation, Madison, Wis.); with most clones, 7-deazaguanidine nucleotide mixes were used. Clone preselection by single-nucleotide tracking (26), clone inversion, and subcloning of selected restriction fragments were also used to completely sequence both strands of DNA. Assembly and analysis of the nucleotide sequence were performed with the Genetics Computer Group package (10). ATG and GTG start codons (30) were accepted, and the codon usage table for CODONPREFERENCE was prepared by using previously published data (36).On the basis of codon bias (1), six presumed protein-coding open reading frames (ORFs), rdmA to rdmF, were observed (Fig. 2).The beginning of rdmA is uncertain because although an apparent ribosom...

Divergent evolution of an atypical S -adenosyl- l -methionine–dependent monooxygenase involved in anthracycline biosynthesis

“…On the other hand, RdmB from the β-rhodomycin pathway in Streptomyces purpurascens is a very unusual enzyme that is evolutionarily closely related to DnrK with a sequence identity of 52% (24). Determination of the crystal structure of RdmB confirmed that the enzyme binds SAM (25) and that the ternary complex is indeed very similar to that of DnrK with a root-meansquare deviation of 1.14 Å for 335 equivalent Cα atoms (26).…”

“…Determination of the crystal structure of RdmB confirmed that the enzyme binds SAM (25) and that the ternary complex is indeed very similar to that of DnrK with a root-meansquare deviation of 1.14 Å for 335 equivalent Cα atoms (26). Despite these features, RdmB completely lacks methyltransferase activity (24,27,28); it is an anthracycline 10-hydroxylase, which requires SAM, molecular oxygen, and a thiol reducing agent for activity (26). Contrary to DnrK, RdmB is able to use both monoglycosylated and triglycosylated anthracyclines as substrates.…”

Crystal Structure of Aclacinomycin Methylesterase with Bound Product Analogues

“…Enzymes encoded by this gene cluster include RdmA, an aklanonic acid methylester cyclase, and aklavinone-11-hydroxylase RdmE, which converts aklavinone to ⑀-rhodomycinone (11). Genetic evidence suggests that rdmB and rdmC code for two enzymes that catalyze the steps leading from ⑀-rhodomycinone glycoside to ␤-rhodomycinone glycoside (16). Both in vivo and in vitro experiments have shown that these two enzymes can also catalyze the conversion of aclacinomycin to 10-hydroxy-10-decarboxymethylaklavin (14).…”

“…In Streptomyces purpurascens, the rdm operon contains several genes that code for tailoring enzymes of rhodomycin biosynthesis (5,16). Enzymes encoded by this gene cluster include RdmA, an aklanonic acid methylester cyclase, and aklavinone-11-hydroxylase RdmE, which converts aklavinone to ⑀-rhodomycinone (11).…”

DNA Sequencing and Transcriptional Analysis of the Kasugamycin Biosynthetic Gene Cluster from Streptomyces kasugaensis M338-M1

“…The deduced KasR protein (399 amino acids, M r of 42,562, pI of 6.06) showed significant similarity to hexose C-3 dehydrases which are involved in 3-deoxygenation of deoxysugar moieties of some secondary metabolites. Homology analysis for KasR revealed similarity with LanQ [21], an NDP-hexose 3,4-dehydratase involved in landomycin biosynthesis in Streptomyces cyanogenus (38% identity); RdmI [22], a hexose C-3 dehydratase involved in rhodomycin biosynthesis in Streptomyces purpurascens (37% identity); UrdQ [23], an NDP-hexose 3,4-dehydratase involved in the formation of the L-rhodinose moiety of urdamycin in Streptomyces fradiae (37% identity); AscC [24], and a CDP-4-keto-6-deoxy-D-glucose-3-dehydrase involved in ascarylose biosynthesis in Yersinia pseudotuberculosis (36% identity). These enzymes are dependent on pyridoxamine 5Ј-phosphate (PMP) and contain an ironsulfur cluster.…”