Nestor Zaburannyi

Targeting DnaN for tuberculosis therapy using novel griselimycins

New for old—TB drug development Tuberculosis (TB) is a global health threat for which there is only lengthy drug treatment. Patients need to consume multiple tablets over several months and frequently fail to complete their treatment. Consequently, drug-resistant strains of the pathogen have emerged, which add to the threat. Kling et al. revisited a natural product called griselimycin, extracted from the same organism that produced the prototype anti-TB drug, streptomycin. Unmodified griselimycin has poor pharmacological properties. However, one synthetic derivative had improved oral uptake and penetrated cells of the immune system that harbor the TB mycobacterium. In combination with other drugs, the griselimycin derivative showed high potency in mice with TB. Science, this issue p. 1106 A griselimycin-derived drug that blocks the DNA polymerase sliding clamp is a potent anti-tuberculosis lead. The discovery of Streptomyces-produced streptomycin founded the age of tuberculosis therapy. Despite the subsequent development of a curative regimen for this disease, tuberculosis remains a worldwide problem, and the emergence of multidrug-resistant Mycobacterium tuberculosis has prioritized the need for new drugs. Here we show that new optimized derivatives from Streptomyces-derived griselimycin are highly active against M. tuberculosis, both in vitro and in vivo, by inhibiting the DNA polymerase sliding clamp DnaN. We discovered that resistance to griselimycins, occurring at very low frequency, is associated with amplification of a chromosomal segment containing dnaN, as well as the ori site. Our results demonstrate that griselimycins have high translational potential for tuberculosis treatment, validate DnaN as an antimicrobial target, and capture the process of antibiotic pressure-induced gene amplification.

Genetic factors that influence moenomycin production in streptomycetes

Moenomycin, a natural phosphoglycolipid product that has a long history of use in animal nutrition, is currently considered an attractive starting point for the development of novel antibiotics. We recently reconstituted the biosynthesis of this natural product in a heterologous host, Streptomyces lividans TK24, but production levels were too low to be useful. We have examined several other streptomycetes strains as hosts and have also explored the overexpression of two pleiotropic regulatory genes, afsS and relA, on moenomycin production. A moenomycin-resistant derivative of S. albus J1074 was found to give the highest titers of moenomycin, and production was improved by overexpressing relA. Partial duplication of the moe cluster 1 in S. ghanaensis also increased average moenomycin production. The results reported here suggest that rational manipulation of global regulators combined with increased moe gene dosage could be a useful technique for improvement of moenomycin biosynthesis.

Genome Analysis of the Fruiting Body-Forming Myxobacterium Chondromyces crocatus Reveals High Potential for Natural Product Biosynthesis

ABSTRACT Here, we report the complete genome sequence of the type strain of the myxobacterial genus Chondromyces, Chondromyces crocatus Cm c5. It presents one of the largest prokaryotic genomes featuring a single circular chromosome and no plasmids. Analysis revealed an enlarged set of tRNA genes, along with reduced pressure on preferred codon usage compared to that of other bacterial genomes. The large coding capacity and the plethora of encoded secondary metabolite biosynthetic gene clusters are in line with the capability of Cm c5 to produce an arsenal of antibacterial, antifungal, and cytotoxic compounds. Known pathways of the ajudazol, chondramide, chondrochloren, crocacin, crocapeptin, and thuggacin compound families are complemented by many more natural compound biosynthetic gene clusters in the chromosome. Whole-genome comparison of the fruiting-body-forming type strain (Cm c5, DSM 14714) to an accustomed laboratory strain which has lost this ability (nonfruiting phenotype, Cm c5 fr−) revealed genetic changes in three loci. In addition to the low synteny found with the closest sequenced representative of the same family, Sorangium cellulosum, extensive genetic information duplication and broad application of eukaryotic-type signal transduction systems are hallmarks of this 11.3-Mbp prokaryotic genome.

Biosynthesis of Crocacin Involves an Unusual Hydrolytic Release Domain Showing Similarity to Condensation Domains

The crocacins are potent antifungal and cytotoxic natural compounds from myxobacteria of the genus Chondromyces. Although total synthesis approaches have been reported, the molecular and biochemical basis guiding the formation of the linear crocacin scaffold has remained unknown. Along with the identification and functional analysis of the crocacin biosynthetic gene cluster from Chondromyces crocatus Cm c5, we here present the identification and biochemical characterization of an unusual chain termination domain homologous to condensation domains responsible for hydrolytic release of the product from the assembly line. In particular, gene inactivation studies and in vitro experiments using the heterologously produced domain CroK-C2 confirm this surprising role giving rise to the linear carboxylic acid. Additionally, we determined the kinetic parameters of CroK-C2 by monitoring hydrolytic cleavage of the substrate mimic N-acetylcysteaminyl-crocacin B using an innovative high-performance liquid chromatography mass spectrometry-based assay.

Evaluation of heterologous promoters for genetic analysis of Actinoplanes teichomyceticus--Producer of teicoplanin, drug of last defense.

Actinoplanes teichomyceticus is the only known producer of the valuable glycopeptide antibiotic teicoplanin. Random mutagenesis and selection were extensively applied to teicoplanin producers, while the gene engineering methods were not used, because of the paucity of genetic tools for A. teichomyceticus. Particularly, availability of promoters of different strength that are functional in Actinoplanes would be very useful for overexpression of beneficial genes. Here we report the use of a glucuronidase reporter system (gusA) for studying transcriptional activity in A. teichomyceticus and describe the behavior of a set of heterologous promoters in this strain. We reveal several elements that exceed in their strength the well-established Streptomyces promoter ermEp, underscoring the utility of the gusA reporter for Actinoplanes sp. Remarkable overproduction of teicoplanin was achieved by constructing strains carrying additional copies of the regulatory gene tcp28 under the control of one of the two most active promoters, moeE5p and actp, discovered in this study.

Isolation, Structure Elucidation, and (Bio)Synthesis of Haprolid, a Cell-Type-Specific Myxobacterial Cytotoxin

Myxobacteria are well-established sources for novel natural products exhibiting intriguing bioactivities. We here report on haprolid (1) isolated from Byssovorax cruenta Har1. The compound exhibits an unprecedented macrolactone comprising four modified amino acids and a polyketide fragment. As configurational assignment proved difficult, a bioinformatic analysis of the biosynthetic gene cluster was chosen to predict the configuration of each stereocenter. In-depth analysis of the corresponding biosynthetic proteins established a hybrid polyketide synthase/nonribosomal peptide synthetase origin of haprolid and allowed for stereochemical assignments. A subsequent total synthesis yielded haprolid and corroborated all predictions made. Intriguingly, haprolid showed cytotoxicity against several cell lines in the nanomolar range whereas other cells were almost unaffected by treatment with the compound.

Thioholgamides: Thioamide-Containing Cytotoxic RiPP Natural Products

Thioviridamide is a structurally unique ribosomally synthesized and post-translationally modified peptide that contains several thioamide bonds and is active against a number of cancer cell lines. In the search for naturally occurring thioviridamide analogs, we employed genome mining that led to the identification of several related gene clusters. Chemical screening followed by cultivation and isolation yielded thioholgamides A and B, two new additions to the thioviridamide family with several amino acid substitutions, a different N-capping moiety, and with one less thioamide bond. Thioholgamides display improved cytotoxicity in the submicromolar range against a range of cell lines and an IC50 of 30 nM for thioholgamide A against HCT-116 cells. Herein, we report the isolation and structural elucidation of thioholgamides A and B, a proposed biosynthetic cluster for their production, and their bioactivities against a larger panel of microorganisms and cancer cell lines.

Mutations improving production and secretion of extracellular lipase by Burkholderia glumae PG1

Burkholderia glumae is a Gram-negative phytopathogenic bacterium known as the causative agent of rice panicle blight. Strain B. glumae PG1 is used for the production of a biotechnologically relevant lipase, which is secreted into the culture supernatant via a type II secretion pathway. We have comparatively analyzed the genome sequences of B. glumae PG1 wild type and a lipase overproducing strain obtained by classical strain mutagenesis. Among a total number of 72 single nucleotide polymorphisms (SNPs) identified in the genome of the production strain, two were localized in front of the lipAB operon and were analyzed in detail. Both mutations contribute to a 100-fold overproduction of extracellular lipase in B. glumae PG1 by affecting transcription of the lipAB operon and efficiency of lipase secretion. We analyzed each of the two SNPs separately and observed a stronger influence of the promoter mutation than of the signal peptide modification but also a cumulative effect of both mutations. Furthermore, fusion of the mutated LipA signal peptide resulted in a 2-fold increase in secretion of the heterologous reporter alkaline phosphatase from Escherichia coli.

A highly unusual polyketide synthase directs dawenol polyene biosynthesis in Stigmatella aurantiaca

Enormous progress in the field of polyketide biosynthesis has led to the establishment of rules for general text book biosynthetic logic and consequently to the assumption that biosynthetic genes can be easily correlated with the corresponding natural products. However, non-textbook examples of polyketide assembly continue to be discovered suggesting the gene to product and product to gene predictions need improvement, especially as they are increasingly used in the post-genomic era. Here, we analyzed the genomic blueprint of a myxobacterial multi-producer of secondary metabolites, Stigmatella aurantiaca DW4/3-1, for its biosynthetic potential by genome-mining. In addition to the five polyketide synthase and/or nonribosomal peptide synthetase gene clusters of known function we identified a further 13 genomic regions exemplifying the enormous genetic potential for the production of additional chemical diversity by this strain. We show by gene inactivation and heterologous expression of the newly identified biosynthetic pathway for dawenol that the biosynthesis of this known polyene does not follow text book biosynthetic logic. Intriguingly, a genomic locus encoding an unusual polyketide synthase exhibiting similarity to gene loci involved in the formation of polyunsaturated fatty acids and secondary lipids was identified.

Draft Genome Sequence and Annotation of the Obligate Bacterial Endosymbiont Caedibacter taeniospiralis, Causative Agent of the Killer Phenotype in Paramecium tetraurelia

ABSTRACT Caedibacter taeniospiralis is an obligate endosymbiont living in the cytoplasm of Paramecium tetraurelia. C. taeniospiralis causes the so-called killer trait, eliminating intraspecific competitors of its host when released into the medium by the concerted action of the unusual protein structure R-body (refractile body) in addition to an as-yet-unknown toxin.