An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current ...level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.
There is a constant need for new and improved drugs to combat infectious diseases, cancer, and other major life-threatening conditions. The recent development of genomics-guided approaches for novel ...natural product discovery has stimulated renewed interest in the search for natural product-based drugs. Genome sequence analysis of Streptomyces ambofaciens ATCC23877 has revealed numerous secondary metabolite biosynthetic gene clusters, including a giant type I modular polyketide synthase (PKS) gene cluster, which is composed of 25 genes (nine of which encode PKSs) and spans almost 150 kb, making it one of the largest polyketide biosynthetic gene clusters described to date. The metabolic product(s) of this gene cluster are unknown, and transcriptional analyses showed that it is not expressed under laboratory growth conditions. The constitutive expression of a regulatory gene within the cluster, encoding a protein that is similar to Large ATP binding of the LuxR (LAL) family proteins, triggered the expression of the biosynthetic genes. This led to the identification of four 51-membered glycosylated macrolides, named stambomycins A-D as metabolic products of the gene cluster. The structures of these compounds imply several interesting biosynthetic features, including incorporation of unusual extender units into the polyketide chain and in trans hydroxylation of the growing polyketide chain to provide the hydroxyl group for macrolide formation. Interestingly, the stambomycins possess promising antiproliferative activity against human cancer cell lines. Database searches identify genes encoding LAL regulators within numerous cryptic biosynthetic gene clusters in actinomycete genomes, suggesting that constitutive expression of such pathway-specific activators represents a powerful approach for novel bioactive natural product discovery.
Abstract
Bacteria of the genus
Streptomyces
are prolific producers of specialized metabolites, including antibiotics. The linear chromosome includes a central region harboring core genes, as well as ...extremities enriched in specialized metabolite biosynthetic gene clusters. Here, we show that chromosome structure in
Streptomyces ambofaciens
correlates with genetic compartmentalization during exponential phase. Conserved, large and highly transcribed genes form boundaries that segment the central part of the chromosome into domains, whereas the terminal ends tend to be transcriptionally quiescent compartments with different structural features. The onset of metabolic differentiation is accompanied by a rearrangement of chromosome architecture, from a rather ‘open’ to a ‘closed’ conformation, in which highly expressed specialized metabolite biosynthetic genes form new boundaries. Thus, our results indicate that the linear chromosome of
S. ambofaciens
is partitioned into structurally distinct entities, suggesting a link between chromosome folding, gene expression and genome evolution.
The modular organization of the type I polyketide synthases (PKSs) would seem propitious for rational engineering of desirable analogous. However, despite decades of efforts, such experiments remain ...largely inefficient. Here, we combine multiple, state-of-the-art approaches to reprogram the stambomycin PKS by deleting seven internal modules. One system produces the target 37-membered mini-stambomycin metabolites - a reduction in chain length of 14 carbons relative to the 51-membered parental compounds - but also substantial quantities of shunt metabolites. Our data also support an unprecedented off-loading mechanism of such stalled intermediates involving the C-terminal thioesterase domain of the PKS. The mini-stambomycin yields are reduced relative to wild type, likely reflecting the poor tolerance of the modules downstream of the modified interfaces to the non-native substrates. Overall, we identify factors contributing to the productivity of engineered whole assembly lines, but our findings also highlight the need for further research to increase production titers.
The naturally occurring diazobenzofluorenes, kinamycins, fluostatins and lomaiviticins, possess highly oxygenated A-rings, via which the last forms a dimeric pharmacophore. However, neither the ...A-ring transformation nor the dimerization mechanisms have been explored thus far. Here we propose a unified biosynthetic logic for the three types of antibiotics and verify one key reaction via detailed genetic and enzymatic experiments. Alp1U and Lom6 from the kinamycin and lomaiviticin biosynthesis, respectively, are shown to catalyse epoxy hydrolysis on a substrate that is obtained by chemical deacetylation of a kinamycin-pathway-derived intermediate. Thus, our study provides the first evidence for the existence of an epoxy intermediate in lomaiviticin biosynthesis. Furthermore, our results suggest that the dimerization in the lomaiviticin biosynthesis proceeds after dehydration of a product generated by Lom6.
Signaling molecule receptors play a central role in quorum sensing and in the coordination onset of specialized metabolite biosynthesis inStreptomycesdue to their dual function in signal detection ...and gene expression control through DNA-binding in the promoter region of their target genes. InStreptomyces ambofaciensthealpbiosynthetic gene cluster includes the signaling molecule receptor AlpZ that negatively regulates through a complex regulatory cascade the expression of key genes involved in the kinamycin antibiotic production until its cognate ligand, a yet unidentified signaling molecule, prompts its release from target promoters. Here we use an original molecular dynamics method to evaluate the DNA-binding properties of AlpZ to its target DNA sequence and the impact the signaling molecule has on the interaction. It is the first time this approach is used to characterize a regulator from the gamma-butyrolactone receptor family. The observed K(D)in the nanomolar range indicates that AlpZ-DNA constitute a particularly stable complex. The signaling molecule ably disturbs this binding while kinamycin has no effect on the activity of AlpZ. Regulator size was determined and found to be considerably large regarding protein sequence, indicating that AlpZ regulates gene expression by binding the DNA as a homodimer, and structural modeling comparison with closely related gamma-butyrolactone receptors supports this conclusion.
The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty ...acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, samT0172 (alpN) and samL0372, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by S. ambofaciens. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of samL0372 to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones.
Streptomycete bacteria are renowned as a prolific source of natural products with diverse biological activities. Production of these metabolites is often subject to transcriptional regulation: the ...biosynthetic genes remain silent until the required environmental and/or physiological signals occur. Consequently, in the laboratory environment, many gene clusters that direct the biosynthesis of natural products with clinical potential are not expressed or at very low level preventing the production/detection of the associated metabolite. Genetic engineering of streptomycetes can unleash the production of many new natural products. This chapter describes the overexpression of pathway-specific activators, the genetic disruption of pathway-specific repressors, and the main strategy used to identify and characterize new natural products from these engineered Streptomyces strains.
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