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At least 19 recordsLinked to original sources
PubMed2025

Multichassis Expression of Cyanobacterial and Other Bacterial Biosynthetic Gene Clusters.

Heterologous expression of biosynthetic gene clusters (BGCs) is a powerful strategy for natural product (NP) discovery, yet achieving consistent expression across microbial hosts remains challenging. Here, we developed cross-phyla vector systems enabling the expression of BGCs from cyanobacteria and other bacterial origins in Gram-negative Escherichia coli, Gram-positive Bacillus subtilis, and two model cyanobacterial strains including unicellular Synechocystis PCC 6803 and filamentous Anabaena sp. PCC 7120. Following validation using constitutive and inducible expression of the enhanced yellow fluorescent protein (eYFP), we applied these vectors to express the shinorine and violacein BGCs in all four hosts. Promoter tuning, substrate feeding, BGC refactoring, and inducible control enhanced NP production and mitigated host toxicity. Notably, we demonstrated that B. subtilis can serve as a chassis for cyanobacterial NP BGC expression. Our results provide versatile expression platforms for probing BGC function and accelerating natural product discovery from diverse cyanobacterial and other bacterial lineages.

Multigene Family↗
PubMed2004

A gene cluster for biosynthesis of kanamycin from Streptomyces kanamyceticus: comparison with gentamicin biosynthetic gene cluster.

Gene clusters for the biosynthesis of kanamycin (Km) and gentamicin (Gm) were isolated from the genomic libraries of Streptomyces kanamyceticus and Micromonospora echinospora, respectively. The sequencing of the 47 kb-region of S. kanamyceticus genomic DNA revealed 40 putative open reading frames (ORFs) encoding Km biosynthetic proteins, regulatory proteins, and resistance and transport proteins. Similarly, the sequencing of 32.6 kb genomic DNA of M. echinospora revealed a Gm biosynthetic gene cluster flanked by resistant genes. Biosynthetic pathways for the formation of Km were proposed by the comparative study of biosynthetic genes. Out of 12 putative Km biosynthetic genes, kanA was expressed in Escherichia coli and determined its function as a 2-deoxy-scyllo-inosose synthase. Furthermore, the acetylations of aminoglycoside-aminocyclitols (AmAcs) by Km acetyltransferase (KanM) were also demonstrated. The acetylated derivatives completely lost their antibacterial activities against Bacillus subtilis. The comparative genetic studies of Gm, Km, tobramycin (Tm), and butirosin (Bn) reveal their similar biosynthetic routes and provide a framework for the further biosynthetic studies.

Base Sequence↗
PubMed2005

Cloning and characterization of the goadsporin biosynthetic gene cluster from Streptomyces sp. TP-A0584.

The biosynthetic gene cluster of goadsporin, a polypeptide antibiotic containing thiazole and oxazole rings, was cloned from Streptomyces sp. TP-A0584. The cluster contains a structural gene, godA, and nine god (goadsporin) genes involved in post-translational modification, immunity and transcriptional regulation. Although the gene organization is similar to typical bacteriocin biosynthetic gene clusters, each goadsporin biosynthetic gene shows low homology to these genes. Goadsporin biosynthesis is initiated by the translation of godA, and the subsequent cyclization, dehydration and acetylation are probably catalysed by godD, godE, godF, godG and godH gene products. godI shows high similarity to the 54 kDa subunit of the signal recognition particle and plays an important role in goadsporin immunity. Furthermore, four goadsporin analogues were produced by site-directed mutagenesis of godA, suggesting that this biosynthesis machinery is used for the heterocyclization of peptides.

Cloning, Molecular↗
PubMed2006

The small MbtH-like protein encoded by an internal gene of the balhimycin biosynthetic gene cluster is not required for glycopeptide production.

The balhimycin biosynthetic gene cluster of the glycopeptide producer Amycolatopsis balhimycina includes a gene (orf1) with unknown function. orf1 shows high similarity to the mbtH gene from Mycobacterium tuberculosis. In almost all nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters, we could identify a small mbtH-like gene whose function in peptide biosynthesis is not known. The mbtH-like gene is always colocalized with the NRPS genes; however, it does not have a specific position in the gene cluster. In all glycopeptide biosynthetic gene clusters the orf1-like gene is always located downstream of the gene encoding the last module of the NRPS. We inactivated the orf1 gene in A. balhimycina by generating a deletion mutant. The balhimycin production is not affected in the orf1-deletion mutant and is indistinguishable from that of the wild type. For the first time, we show that the inactivation of an mbtH-like gene does not impair the biosynthesis of a nonribosomal peptide.

Actinomycetales↗
PubMed2004

Isolation and characterization of the tobramycin biosynthetic gene cluster from Streptomyces tenebrarius.

The biosynthetic gene cluster for tobramycin, a 2-deoxystreptamine-containing aminoglycoside antibiotic, was isolated from Streptomyces tenebrarius ATCC 17920. A genomic library of S. tenebrarius was constructed, and a cosmid, pST51, was isolated by the probes based on the core regions of 2-deoxy-scyllo-inosose (DOI) synthase, and L-glutamine:DOI aminotransferase and L-glutamine:scyllo-inosose aminotransferase. Sequencing of 33.9 kb revealed 24 open reading frames (ORFs) including putative tobramycin biosynthetic genes. We demonstrated that one of these ORFs, tbmA, encodes DOI synthase by in vitro enzyme assay of the purified protein. The catalytic residues of TbmA and dehydroquinate synthase were studied by homology modeling. The gene cluster found is likely to be involved in the biosynthesis of tobramycin.

Bacterial Proteins↗
PubMed2002

Cloning and analysis of the simocyclinone biosynthetic gene cluster of Streptomyces antibioticus Tü 6040.

The biosynthetic gene cluster of the aminocoumarin antibiotic simocyclinone D8 was cloned by screening a cosmid library of Streptomyces antibioticusTü 6040 with a heterologous probe from a gene encoding a cytochrome P450 enzyme involved in the biosynthesis of the aminocoumarin antibiotic novobiocin. Sequence analysis of a 39.4-kb region revealed the presence of 38 ORFs. Six of the identified ORFs showed striking similarity to genes from the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin and coumermycin A(1). Simocyclinone also contains an angucyclinone moiety, and 12 of the ORFs showed high sequence similarity to biosynthetic genes of other angucyclinone antibiotics. Possible functions within the biosynthesis of simocyclinone D8 could be assigned to 23 ORFs by comparison with sequences in GenBank. Experimental proof for the function of the identified gene cluster was provided by a gene inactivation experiment, which resulted in the abolishment of the formation of the aminocoumarin moiety of simocyclinone. Feeding of the mutant with the aminocoumarin moiety of novobiocin led to a new, artificial simocyclinone derivative.

Aminocoumarins↗
PubMed2006

The enduracidin biosynthetic gene cluster from Streptomyces fungicidicus.

The biosynthetic gene cluster for the 17 aa peptide antibiotic enduracidin has been cloned and sequenced from Streptomyces fungicidicus ATCC 21013. The 84 kb gene cluster contains 25 ORFs and is located within a 116 kb genetic locus that was fully sequenced. Targeted disruption of non-ribosomal peptide synthetase (NRPS) genes in the cluster abolished enduracidin production and confirmed function. The cluster includes four genes, endA-D, encoding two-, seven-, eight- and one-module NRPSs, respectively, and includes unique modules for the incorporation of citrulline and enduracididine. The NRPS organization generally follows the collinearity principle, and starts with a condensation domain (C domain) similar to those found in other lipopeptide systems for the coupling of an acyl group to the starting amino acid. The sixth module of EndB, corresponding to Thr(8), is missing an adenylation domain (A domain) and this module is presumed to be loaded in trans by the single module protein EndD. The most striking feature of the NRPS organization is the lack of epimerization domains (E domains) in light of the fact that the product has seven d-amino acid residues. Sequence analysis reveals that C domains following modules corresponding to d-amino acids belong to a unique subset of C domains able to catalyse both epimerization and condensation reactions. Other genes directing lipid modification and activation, and formation of the non-proteinogenic amino acids 4-hydroxyphenylglycine and enduracididine are readily identified, as are genes possibly involved in regulation of antibiotic biosynthesis and export. These findings provide the basis to further genetically manipulate and improve lipodepsipeptide antibiotics via combinatorial and chemical methods.

Cloning, Molecular↗
PubMed2002

Molecular cloning and sequence analysis of the clorobiocin biosynthetic gene cluster: new insights into the biosynthesis of aminocoumarin antibiotics.

The biosynthetic gene cluster of the aminocoumarin antibiotic clorobiocin was cloned by screening of a cosmid library of Streptomyces roseochromogenes DS 12.976 with two heterologous probes from the novobiocin biosynthetic gene cluster. Sequence analysis revealed 27 ORFs with striking similarity to the biosynthetic gene clusters of novobiocin and coumermycin A(1). Inactivation of a putative aldolase gene, cloR, by in-frame deletion led to the abolishment of the production of clorobiocin. Feeding of the mutant with 3-dimethylallyl-4-hydroxybenzoic acid (Ring A of clorobiocin) restored clorobiocin production. Here, it is suggested that the formation of Ring A of clorobiocin may proceed via a retro-aldol reaction catalysed by CloR, i.e. by a mechanism different from the previously elucidated benzoic acid biosynthetic pathway in Streptomyces maritimus. A comparison of the gene clusters for clorobiocin, novobiocin and coumermycin A(1) showed that the structural differences between the three antibiotics were reflected remarkably well by differences in the organization of their respective biosynthetic gene clusters.

Amino Acid Sequence↗
PubMed2005

The lipopeptide antibiotic A54145 biosynthetic gene cluster from Streptomyces fradiae.

Ca(2+)-dependent cyclic lipodepsipeptides are an emerging class of antibiotics for the treatment of infections caused by Gram-positive pathogens. These compounds are synthesized by nonribosomal peptide synthetase (NRPS) complexes encoded by large gene clusters. The gene cluster encoding biosynthetic pathway enzymes for the Streptomyces fradiae A54145 NRP was cloned from a cosmid library and characterized. Four NRPS-encoding genes, responsible for subunits of the synthetase, as well as genes for accessory functions such as acylation, methylation and hydroxylation, were identified by sequence analysis in a 127 kb region of DNA that appears to be located subterminally in the bacterial chromosome. Deduced epimerase domain-encoding sequences within the NRPS genes indicated a D: -stereochemistry for Glu, Lys and Asn residues, as observed for positionally analogous residues in two related compounds, daptomycin, and the calcium-dependent antibiotic (CDA) produced by Streptomyces roseosporus and Streptomyces coelicolor, respectively. A comparison of the structure and the biosynthetic gene cluster of A54145 with those of the related peptides showed many similarities. This information may contribute to the design of experiments to address both fundamental and applied questions in lipopeptide biosynthesis, engineering and drug development.

Amino Acid Sequence↗
PubMed2000

Identification of the coumermycin A(1) biosynthetic gene cluster of Streptomyces rishiriensis DSM 40489.

The biosynthetic gene cluster of the aminocoumarin antibiotic coumermycin A(1) was cloned by screening of a cosmid library of Streptomyces rishiriensis DSM 40489 with heterologous probes from a dTDP-glucose 4,6-dehydratase gene, involved in deoxysugar biosynthesis, and from the aminocoumarin resistance gyrase gene gyrB(r). Sequence analysis of a 30.8-kb region upstream of gyrB(r) revealed the presence of 28 complete open reading frames (ORFs). Fifteen of the identified ORFs showed, on average, 84% identity to corresponding ORFs in the biosynthetic gene cluster of novobiocin, another aminocoumarin antibiotic. Possible functions of 17 ORFs in the biosynthesis of coumermycin A(1) could be assigned by comparison with sequences in GenBank. Experimental proof for the function of the identified gene cluster was provided by an insertional gene inactivation experiment, which resulted in an abolishment of coumermycin A(1) production.

Aminocoumarins↗
PubMed2000

Isolation and characterization of the epothilone biosynthetic gene cluster from Sorangium cellulosum.

The epothilone biosynthetic gene cluster was isolated from Sorangium cellulosum strain SMP44. The gene cluster contains seven genes and spans approx. 56kb. The genes encoding the PKS, epoA, epoC, epoD, epoE, and epoF, are divided into nine modules. The EpoB protein is a non-ribosomal peptide synthetase (NRPS) that catalyzes formation of the thiazole found in the epothilones. EpoK is a P450 enzyme responsible for the epoxidation of epothilones C and D to epothilones A and B, respectively. EpoK was expressed in Escherichia coli, and the purified protein was shown to convert epothilone D to epothilone B in vitro.

Amino Acid Sequence↗
PubMed2002

Biosynthetic gene cluster of simocyclinone, a natural multihybrid antibiotic.

The entire simocyclinone biosynthetic cluster (sim gene cluster) from the producer Streptomyces antibioticus Tü6040 was identified on six overlapping cosmids (1N1, 5J10, 2L16, 2P6, 4G22, and 1K3). In total, 80.7 kb of DNA from these cosmids was sequenced, and the analysis revealed 49 complete open reading frames (ORFs). These ORFs include genes responsible for the formation and attachment of four different moieties originating from at least three different pools of primary metabolites. Also in the sim gene cluster, four ORFs were detected that resemble putative regulatory and export functions. Based on the putative function of the gene products, a model for simocyclinone D8 biosynthesis was proposed. Biosynthetic mutants were generated by insertional gene inactivation experiments, and culture extracts of these mutants were analyzed by high-performance liquid chromatography. Production of simocyclinone D8 was clearly detectable in the wild-type strain but was not detectable in the mutant strains. This indicated that indeed the sim gene cluster had been cloned.

Bacterial Proteins↗
PubMed2003

Characterization of the biosynthetic gene cluster of rebeccamycin from Lechevalieria aerocolonigenes ATCC 39243.

The biosynthetic gene cluster for rebeccamycin, an indolocarbazole antibiotic, from Lechevalieria aerocolonigenes ATCC 39243 has 11 ORFs. To clarify their functions, mutants with rebG, rebD, rebC, rebP, rebM, rebR, rebH, rebT, or orfD2 disrupted were constructed, and the gene products were examined. rebP disruptants produced 11,11'-dichlorochromopyrrolic acid, found to be a biosynthetic intermediate by a bioconversion experiment. Other genes encoded N-glycosyltransferase (rebG), monooxygenase (rebC), methyltransferase (rebM), a transcriptional activator (rebR), and halogenase (rebH). rebT disruptants produced rebeccamycin as much as the wild strain, so rebT was probably not involved in rebeccamycin production. Biosynthetic genes of staurosporine, an another indolocarbazole antibiotic, were cloned from Streptomyces sp. TP-A0274. staO, staD, and staP were similar to rebO, rebD, and rebP, respectively, all of which are responsible for indolocarbazole biosynthesis, But a rebC homolog, encoding a putative enzyme oxidizing the C-7 site of pyrrole rings, was not found in the staurosporine biosynthetic gene cluster. These results suggest that indolocarbazole is constructed by oxidative decarboxylation of chromopyrrolic acid (11,11'-dichlorochromopyrrolic acid in rebeccamycin) generated from two molecules of tryptophan by coupling and that the oxidation state at the C-7 position depends on the additional enzyme(s) encoded by the biosynthetic genes.

Aminoglycosides↗
PubMed2005

Construction of a bacterial artificial chromosome library for a myxobacterium of the genus Cystobacter and characterization of an antibiotic biosynthetic gene cluster.

A bacterial artificial chromosome (BAC) library was constructed to isolate the biosynthetic gene cluster for the polyketide/peptide hybrid-type antibiotic cystothiazole A from the myxobacterium Cystobacter fuscus strain AJ-13278. Sequence analysis of a 63.9 kb contiguous region that encompasses the biosynthetic gene cluster (cta) led to the identification of a polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) hybrid gene cluster 32.1 kb in size, which consists of six open reading frames (ORFs), ctaB to ctaG, as well as downstream genes ctaJ and ctaK (1.0 and 0.9 kb, respectively) responsible for the final biosynthetic steps. The genes ctaB, ctaE, and ctaF encode PKSs, the genes ctaC and ctaG encode NRPSs, and ctaD encodes an NRPS-PKS hybrid enzyme. Disruption of ctaD impaired cystothiazole A production. Additionally, two downstream genes, ctaJ and ctaK, which encode a nitrilase and an O-methyltransferase, respectively, must be responsible for the final methyl ester formation in the cystothiazole A biosynthesis.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2003

Co-expression of 15 contiguous genes delineates a fumonisin biosynthetic gene cluster in Gibberella moniliformis.

Fumonisins are mycotoxins produced by the maize pathogen Gibberella moniliformis and are associated with cancer in rodents. In this study, we determined the nucleotide sequence of a 75-kb region of G. moniliformis DNA and identified 18 heretofore undescribed genes flanking a cluster of five previously identified fumonisin biosynthetic (FUM) genes. Ten of the newly identified genes downstream of the cluster were coregulated with FUM genes and exhibited patterns of expression that were correlated with fumonisin production. BLASTX analyses indicated that the predicted functions of proteins encoded by the 10 genes were consistent with activities expected for fumonisin biosynthesis or self-protection. These data indicate that the 10 newly identified genes and the previously identified FUM genes constitute a fumonisin biosynthetic gene cluster. Disruption of two of the new genes, encoding longevity assurance factors, had no apparent effect on fumonisin production, but disruption of a third, encoding an ABC transporter, had a subtle effect on ratios of fumonisins produced.

Amino Acid Sequence↗
PubMed2000

Identification of the novobiocin biosynthetic gene cluster of Streptomyces spheroides NCIB 11891.

The novobiocin biosynthetic gene cluster from Streptomyces spheroides NCIB 11891 was cloned by using homologous deoxynucleoside diphosphate (dNDP)-glucose 4,6-dehydratase gene fragments as probes. Double-stranded sequencing of 25.6 kb revealed the presence of 23 putative open reading frames (ORFs), including the gene for novobiocin resistance, gyrB(r), and at least 11 further ORFs to which a possible role in novobiocin biosynthesis could be assigned. An insertional inactivation experiment with a dNDP-glucose 4, 6-dehydratase fragment resulted in abolishment of novobiocin production, since biosynthesis of the deoxysugar moiety of novobiocin was blocked. Heterologous expression of a key enzyme of novobiocin biosynthesis, i.e., novobiocic acid synthetase, in Streptomyces lividans TK24 further confirmed the involvement of the analyzed genes in the biosynthesis of the antibiotic.

Amide Synthases↗
PubMed2005

Identification and analysis of the chivosazol biosynthetic gene cluster from the myxobacterial model strain Sorangium cellulosum So ce56.

Myxobacteria belonging to the genus Sorangium are known to produce a variety of biologically active secondary metabolites. Chivosazol is a macrocyclic antibiotic active against yeast, filamentous fungi and especially against mammalian cells. The compound specifically destroys the actin skeleton of eucaryotic cells and does not show activity against bacteria. Chivosazol contains an oxazole ring and a glycosidically bound 6-deoxyglucose (except for chivosazol F). In this paper we describe the biosynthetic gene cluster that directs chivosazol biosynthesis in the model strain Sorangium cellulosum So ce56. This biosynthetic gene cluster spans 92 kbp on the chromosome and contains four polyketide synthase genes and one hybrid polyketide synthase/nonribosomal peptide synthetase gene. An additional gene encoding a protein with similarity to different methyltransferases and presumably involved in post-polyketide modification was identified downstream of the core biosynthetic gene cluster. The chivosazol biosynthetic gene locus belongs to the recently identified and rapidly growing class of trans-acyltransferase polyketide synthases, which do not contain acyltransferase domains integrated into the multimodular megasynthetases.

Genes, Bacterial↗
PubMed2006

The tallysomycin biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 unveiling new insights into the biosynthesis of the bleomycin family of antitumor antibiotics.

The tallysomycins (TLMs) belong to the bleomycin (BLM) family of antitumor antibiotics. The BLM biosynthetic gene cluster has been cloned and characterized previously from Streptomyces verticillus ATCC 15003, but engineering BLM biosynthesis for novel analogs has been hampered by the lack of a genetic system for S. verticillus. We now report the cloning and sequencing of the TLM biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 and the development of a genetic system for S. hindustanus, demonstrating the feasibility to manipulate TLM biosynthesis in S. hindustanus by gene inactivation and mutant complementation. Sequence analysis of the cloned 80.2 kb region revealed 40 open reading frames (ORFs), 30 of which were assigned to the TLM biosynthetic gene cluster. The TLM gene cluster consists of nonribosomal peptide synthetase (NRPS) genes encoding nine NRPS modules, a polyketide synthase (PKS) gene encoding one PKS module, genes encoding seven enzymes for deoxysugar biosynthesis and attachment, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The involvement of the cloned gene cluster in TLM biosynthesis was confirmed by inactivating the tlmE glycosyltransferase gene to generate a TLM non-producing mutant and by restoring TLM production to the DeltatlmE::ermE mutant strain upon expressing a functional copy of tlmE. The TLM gene cluster is highly homologous to the BLM cluster, with 25 of the 30 ORFs identified within the two clusters exhibiting striking similarities. The structural similarities and differences between TLM and BLM were reflected remarkably well by the genes and their organization in their respective biosynthetic gene clusters.

Amino Acid Sequence↗