Tailoring specialized metabolite production in streptomyces.

Streptomycetes are prolific producers of a plethora of medically useful metabolites. These compounds are made by complex secondary (specialized) metabolic pathways, which utilize primary metabolic intermediates as building blocks. In this review we discuss the evolution of specialized metabolites and how expansion of gene families in primary metabolism has lead to the evolution of diversity in these specialized metabolic pathways and how developing a better understanding of expanded primary metabolic pathways can help enhance synthetic biology approaches to industrial pathway engineering.

Construction of a new class of tetracycline lead structures with potent antibacterial activity through biosynthetic engineering.

Antimicrobial resistance and the shortage of novel antibiotics have led to an urgent need for new antibacterial drug leads. Several existing natural product scaffolds (including chelocardins) have not been developed because their suboptimal pharmacological properties could not be addressed at the time. It is demonstrated here that reviving such compounds through the application of biosynthetic engineering can deliver novel drug candidates. Through a rational approach, the carboxamido moiety of tetracyclines (an important structural feature for their bioactivity) was introduced into the chelocardins, which are atypical tetracyclines with an unknown mode of action. A broad-spectrum antibiotic lead was generated with significantly improved activity, including against all Gram-negative pathogens of the ESKAPE panel. Since the lead structure is also amenable to further chemical modification, it is a platform for further development through medicinal chemistry and genetic engineering.

Oxytetracycline hyper-production through targeted genome reduction of Streptomyces rimosus.

Most biosynthetic gene clusters (BGC) encoding the synthesis of important microbial secondary metabolites, such as antibiotics, are either silent or poorly expressed; therefore, to ensure a strong pipeline of novel antibiotics, there is a need to develop rapid and efficient strain development approaches. This study uses comparative genome analysis to instruct rational strain improvement, using Streptomyces rimosus, the producer of the important antibiotic oxytetracycline (OTC) as a model system. Sequencing of the genomes of two industrial strains M4018 and R6-500, developed independently from a common ancestor, identified large DNA rearrangements located at the chromosome end. We evaluated the effect of these genome deletions on the parental S. rimosus Type Strain (ATCC 10970) genome where introduction of a 145 kb deletion close to the OTC BGC in the Type Strain resulted in massive OTC overproduction, achieving titers that were equivalent to M4018 and R6-500. Transcriptome data supported the hypothesis that the reason for such an increase in OTC biosynthesis was due to enhanced transcription of the OTC BGC and not due to enhanced substrate supply. We also observed changes in the expression of other cryptic BGCs; some metabolites, undetectable in ATCC 10970, were now produced at high titers. This study demonstrated for the first time that the main force behind BGC overexpression is genome rearrangement. This new approach demonstrates great potential to activate cryptic gene clusters of yet unexplored natural products of medical and industrial value.IMPORTANCEThere is a critical need to develop novel antibiotics to combat antimicrobial resistance. Streptomyces species are very rich source of antibiotics, typically encoding 20-60 biosynthetic gene clusters (BGCs). However, under laboratory conditions, most are either silent or poorly expressed so that their products are only detectable at nanogram quantities, which hampers drug development efforts. To address this subject, we used comparative genome analysis of industrial Streptomyces rimosus strains producing high titers of a broad spectrum antibiotic oxytetracycline (OTC), developed during decades of industrial strain improvement. Interestingly, large-scale chromosomal deletions were observed. Based on this information, we carried out targeted genome deletions in the native strain S. rimosus ATCC 10970, and we show that a targeted deletion in the vicinity of the OTC BGC significantly induced expression of the OTC BGC, as well as some other silent BGCs, thus suggesting that this approach may be a useful way to identify new natural products.

Cardiolipin synthase is required for Streptomyces coelicolor morphogenesis.

The fluid mosaic model has recently been amended to account for the existence of membrane domains enriched in certain phospholipids. In rod-shaped bacteria, the anionic phospholipid cardiolipin is enriched at the cell poles but its role in the morphogenesis of the filamentous bacterium Streptomyces coelicolor is unknown. It was impossible to delete clsA (cardiolipin synthase; SCO1389) unless complemented by a second copy of clsA elsewhere in the chromosome. When placed under the control of an inducible promoter, clsA expression, phospholipid profile and morphogenesis became inducer dependent. TLC analysis of phospholipid showed altered profiles upon depletion of clsA expression. Analysis of cardiolipin by mass spectrometry showed two distinct cardiolipin envelopes that reflected differences in acyl chain length; the level of the larger cardiolipin envelope was reduced in concert with clsA expression. ClsA-EGFP did not localize to specific locations, but cardiolipin itself showed enrichment at hyphal tips, branch points and anucleate regions. Quantitative analysis of hyphal dimensions showed that the mycelial architecture and the erection of aerial hyphae were affected by the expression of clsA. Overexpression of clsA resulted in weakened hyphal tips, misshaped aerial hyphae and anucleate spores and demonstrates that cardiolipin synthesis is a requirement for morphogenesis in Streptomyces.

Identification of the chelocardin biosynthetic gene cluster from Amycolatopsis sulphurea: a platform for producing novel tetracycline antibiotics.

Tetracyclines (TCs) are medically important antibiotics from the polyketide family of natural products. Chelocardin (CHD), produced by Amycolatopsis sulphurea, is a broad-spectrum tetracyclic antibiotic with potent bacteriolytic activity against a number of Gram-positive and Gram-negative multi-resistant pathogens. CHD has an unknown mode of action that is different from TCs. It has some structural features that define it as 'atypical' and, notably, is active against tetracycline-resistant pathogens. Identification and characterization of the chelocardin biosynthetic gene cluster from A. sulphurea revealed 18 putative open reading frames including a type II polyketide synthase. Compared to typical TCs, the chd cluster contains a number of features that relate to its classification as 'atypical': an additional gene for a putative two-component cyclase/aromatase that may be responsible for the different aromatization pattern, a gene for a putative aminotransferase for C-4 with the opposite stereochemistry to TCs and a gene for a putative C-9 methylase that is a unique feature of this biosynthetic cluster within the TCs. Collectively, these enzymes deliver a molecule with different aromatization of ring C that results in an unusual planar structure of the TC backbone. This is a likely contributor to its different mode of action. In addition CHD biosynthesis is primed with acetate, unlike the TCs, which are primed with malonamate, and offers a biosynthetic engineering platform that represents a unique opportunity for efficient generation of novel tetracyclic backbones using combinatorial biosynthesis.

Multitargeted anti-infective drugs: resilience to resistance in the antimicrobial resistance era.

The standard drug discovery paradigm of single molecule - single biological target - single biological effect is perhaps particularly unsuitable for anti-infective drug discovery. This is due to the rapid evolution of resistance likely to be observed with single target drugs. Multitargeted anti-infective drugs are likely to be superior due to their lower susceptibility to target-related resistance mechanisms. Strathclyde minor groove binders are a class of compounds which have been developed by adopting the multitargeted anti-infective drugs paradigm, and their effectiveness against a wide range of pathogenic organisms is discussed. The renaming of this class to Strathclyde nucleic acid binders is also presented due to their likely targets including both DNA and RNA.

Insights into the Spectrum of Activity and Mechanism of Action of MGB-BP-3.

MGB-BP-3 is a potential first-in-class antibiotic, a Strathclyde Minor Groove Binder (S-MGB), that has successfully completed Phase IIa clinical trials for the treatment of Clostridioides difficile associated disease. Its precise mechanism of action and the origin of limited activity against Gram-negative pathogens are relatively unknown. Herein, treatment with MGB-BP-3 alone significantly inhibited the bacterial growth of the Gram-positive, but not Gram-negative, bacteria as expected. Synergy assays revealed that inefficient intracellular accumulation, through both permeation and efflux, is the likely reason for lack of Gram-negative activity. MGB-BP-3 has strong interactions with its intracellular target, DNA, in both Gram-negative and Gram-positive bacteria, revealed through ultraviolet-visible (UV-vis) thermal melting and fluorescence intercalator displacement assays. MGB-BP-3 was confirmed to bind to dsDNA as a dimer using nano-electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Type II bacterial topoisomerase inhibition assays revealed that MGB-BP-3 was able to interfere with the supercoiling action of gyrase and the relaxation and decatenation actions of topoisomerase IV of both Staphylococcus aureus and Escherichia coli. However, no evidence of stabilization of the cleavage complexes was observed, such as for fluoroquinolones, confirmed by a lack of induction of DSBs and the SOS response in E. coli reporter strains. These results highlight additional mechanisms of action of MGB-BP-3, including interference of the action of type II bacterial topoisomerases. While MGB-BP-3's lack of Gram-negative activity was confirmed, and an understanding of this presented, the recognition that MGB-BP-3 can target DNA of Gram-negative organisms will enable further iterations of design to achieve a Gram-negative active S-MGB.

Bilateral symmetry of linear streptomycete chromosomes.

Here, we characterize an uncommon set of telomeres from Streptomyces rimosus ATCC 10970, the parental strain of a lineage of one of the earliest-discovered antibiotic producers. Following the closure of its genome sequence, we compared unusual telomeres from this organism with the other five classes of replicon ends found amongst streptomycetes. Closed replicons of streptomycete chromosomes were organized with respect to their phylogeny and physical orientation, which demonstrated that different telomeres were not associated with particular clades and are likely shared amongst different strains by plasmid-driven horizontal gene transfer. Furthermore, we identified a ~50 kb origin island with conserved synteny that is located at the core of all streptomycete chromosomes and forms an axis around which symmetrical chromosome inversions can take place. Despite this chromosomal bilateral symmetry, a bias in parS sites to the right of oriC is maintained across the family Streptomycetaceae and suggests that the formation of ParB/parS nucleoprotein complexes on the right replichore is a conserved feature in streptomycetes. Consequently, our studies reveal novel features of linear bacterial replicons that, through their manipulation, may lead to improvements in growth and productivity of this important industrial group of bacteria.

ActDES - a curated Actinobacterial Database for Evolutionary Studies.

Actinobacteria is a large and diverse phylum of bacteria that contains medically and ecologically relevant organisms. Many members are valuable sources of bioactive natural products and chemical precursors that are exploited in the clinic and made using the enzyme pathways encoded in their complex genomes. Whilst the number of sequenced genomes has increased rapidly in the last 20 years, the large size, complexity and high G+C content of many actinobacterial genomes means that the sequences remain incomplete and consist of large numbers of contigs with poor annotation, which hinders large-scale comparative genomic and evolutionary studies. To enable greater understanding and exploitation of actinobacterial genomes, specialized genomic databases must be linked to high-quality genome sequences. Here, we provide a curated database of 612 high-quality actinobacterial genomes from 80 genera, chosen to represent a broad phylogenetic group with equivalent genome re-annotation. Utilizing this database will provide researchers with a framework for evolutionary and metabolic studies, to enable a foundation for genome and metabolic engineering, to facilitate discovery of novel bioactive therapeutics and studies on gene family evolution. This article contains data hosted by Microreact.

Genetics of Streptomyces rimosus, the oxytetracycline producer.

From a genetic standpoint, Streptomyces rimosus is arguably the best-characterized industrial streptomycete as the producer of oxytetracycline and other tetracycline antibiotics. Although resistance to these antibiotics has reduced their clinical use in recent years, tetracyclines have an increasing role in the treatment of emerging infections and noninfective diseases. Procedures for in vivo and in vitro genetic manipulations in S. rimosus have been developed since the 1950s and applied to study the genetic instability of S. rimosus strains and for the molecular cloning and characterization of genes involved in oxytetracycline biosynthesis. Recent advances in the methodology of genome sequencing bring the realistic prospect of obtaining the genome sequence of S. rimosus in the near term.

Differential transcription of expanded gene families in central carbon metabolism of Streptomyces coelicolor A3(2).

BACKGROUND: Streptomycete bacteria are prolific producers of specialized metabolites, many of which have clinically relevant bioactivity. A striking feature of their genomes is the expansion of gene families that encode the same enzymatic function. Genes that undergo expansion events, either by horizontal gene transfer or duplication, can have a range of fates: genes can be lost, or they can undergo neo-functionalization or sub-functionalization. To test whether expanded gene families in Streptomyces exhibit differential expression, an RNA-Seq approach was used to examine cultures of wild-type Streptomyces coelicolor grown with either glucose or tween as the sole carbon source. RESULTS: RNA-Seq analysis showed that two-thirds of genes within expanded gene families show transcriptional differences when strains were grown on tween compared to glucose. In addition, expression of specialized metabolite gene clusters (actinorhodin, isorenieratane, coelichelin and a cryptic NRPS) was also influenced by carbon source. CONCLUSIONS: Expression of genes encoding the same enzymatic function had transcriptional differences when grown on different carbon sources. This transcriptional divergence enables partitioning to function under different physiological conditions. These approaches can inform metabolic engineering of industrial Streptomyces strains and may help develop cultivation conditions to activate the so-called silent biosynthetic gene clusters.

Emergence of an Australian-like pstS-null vancomycin resistant Enterococcus faecium clone in Scotland.

Multi-locus sequencing typing (MLST) is widely used to monitor the phylogeny of microbial outbreaks. However, several strains of vancomycin-resistant Enterococcus faecium (VREfm) with a missing MLST locus (pstS) have recently emerged in Australia, with a few cases also reported in England. Here, we identified similarly distinct strains circulating in two neighbouring hospitals in Scotland. Whole genome sequencing of five VREfm strains isolated from these hospitals identified four pstS-null strains in both hospitals, while the fifth was multi-locus sequence type (ST) 262, which is the first documented in the UK. All five Scottish isolates had an insertion in the tetM gene, which is associated with increased susceptibility to tetracyclines, providing no other tetracycline-resistant gene is present. Such an insertion, which encompasses a dfrG gene and two currently uncharacterised genes, was additionally identified in all tested vanA-type pstS-null VREfm strains (5 English and 68 Australian). Phylogenetic comparison with other VREfm genomes indicates that the four pstS-null Scottish isolates sequenced in this study are more closely related to pstS-null strains from Australia rather than the English pstS-null isolates. Given how rapidly such pstS-null strains have expanded in Australia, the emergence of this clone in Scotland raises concerns for a potential outbreak.

The association between biocide tolerance and the presence or absence of qac genes among hospital-acquired and community-acquired MRSA isolates.

OBJECTIVES: The MBCs of three commonly used hospital biocides [containing quaternary ammonium compounds (QACs), chlorhexidine gluconate and triclosan] were determined for clinical isolates of Staphylococcus aureus, which were also screened for genes encoding Qac efflux pumps. METHODS: MBCs were determined by broth microdilution for 94 clinical isolates of S. aureus, including 38 hospital-acquired methicillin-resistant S. aureus (HA-MRSA), 25 community-associated methicillin-resistant S. aureus (CA-MRSA), 25 methicillin-susceptible S. aureus (MSSA) and 6 with intermediate resistance to vancomycin (VISA). All isolates were screened by PCR for the presence of qacA, B, C, G, H and J. RESULTS: Biocides had MBCs 10-1000-fold lower than the concentration recommended for use by the manufacturer. HA-MRSA isolates developed significantly enhanced tolerance to QACs following repeat exposure to subinhibitory concentrations. Ten HA-MRSA and four VISA isolates carried qacA. Two HA-MRSA isolates, one MSSA isolate and one VISA isolate carried qacC. One VISA isolate carried qacA and qacC. The CA-MRSA isolates did not carry qac genes. qacG, H and J were not detected in any HA-MRSA. Isolates with qac genes had significantly (P < 0.0001) higher MBCs for biocides containing QACs and chlorhexidine gluconate. These biocides induced expression of qac genes when assayed with a luciferase reporter. CONCLUSIONS: Biocides commonly used in the hospital environment should be effective against clinical isolates of S. aureus if used at concentrations recommended by the manufacturer. However, isolates have the potential to develop increased tolerance to these agents and the expression of Qac efflux pumps results in isolates with a selective advantage when challenged with biocides containing QACs and chlorhexidine gluconate.

Efficacy of common hospital biocides with biofilms of multi-drug resistant clinical isolates.

The hospital environment is particularly susceptible to contamination by bacterial pathogens that grow on surfaces in biofilms. The effects of hospital biocides on two nosocomial pathogens, meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, growing as free-floating (planktonic) and adherent biofilm populations (sessile) were examined. Clinical isolates of MRSA and P. aeruginosa were grown as biofilms on discs of materials found in the hospital environment (stainless steel, glass, polyethylene and Teflon) and treated with three commonly used hospital biocides containing benzalkonium chloride (1 % w/v), chlorhexidine gluconate (4 % w/v) and triclosan (1 % w/v). Cell viability following biocide treatment was determined using an XTT assay and the LIVE/DEAD BacLight Bacterial Viability kit. The minimum bactericidal concentration (MBC) of all biocides for planktonic populations of both organisms was considerably less than the concentration recommended for use by the manufacturer. However, when isolates were grown as biofilms, the biocides were ineffective at killing bacteria at the concentrations recommended for use. Following biocide treatment, 0-11 % of cells in MRSA biofilms survived, and up to 80 % of cells in P. aeruginosa biofilms survived. This study suggests that although biocides may be effective against planktonic populations of bacteria, some biocides currently used in hospitals are ineffective against nosocomial pathogens growing as biofilms attached to surfaces and fail to control this reservoir for hospital-acquired infection.

Expanding Primary Metabolism Helps Generate the Metabolic Robustness To Facilitate Antibiotic Biosynthesis in Streptomyces.

The expansion of the genetic repertoire of an organism by gene duplication or horizontal gene transfer (HGT) can aid adaptation. Streptomyces bacteria are prolific producers of bioactive specialized metabolites that have adaptive functions in nature and have found extensive utility in human medicine. While the biosynthesis of these specialized metabolites is directed by dedicated biosynthetic gene clusters, little attention has been focused on how these organisms have evolved robustness in their genomes to facilitate the metabolic plasticity required to provide chemical precursors for biosynthesis during the complex metabolic transitions from vegetative growth to specialized metabolite production and sporulation. Here, we examine genetic redundancy in actinobacteria and show that specialized metabolite-producing bacterial families exhibit gene family expansion in primary metabolism. Focusing on a gene duplication event, we show that the two pyruvate kinases in the genome of Streptomyces coelicolor arose by an ancient duplication event and that each has evolved altered enzymatic kinetics, with Pyk1 having a 20-fold-higher kcat than Pyk2 (4,703 s-1 compared to 215 s-1, respectively), and yet both are constitutively expressed. The pyruvate kinase mutants were also found to be compromised in terms of fitness compared to wild-type Streptomyces These data suggest that expanding gene families can help maintain cell functionality during metabolic perturbation such as nutrient limitation and/or specialized metabolite production.IMPORTANCE The rise of antimicrobial-resistant infections has prompted a resurgence in interest in understanding the production of specialized metabolites, such as antibiotics, by Streptomyces The presence of multiple genes encoding the same enzymatic function is an aspect of Streptomyces biology that has received little attention; however, understanding how the metabolic expansion influences these organisms can help enhance production of clinically useful molecules. Here, we show that expanding the number of pyruvate kinases enables metabolic adaptation, increases strain fitness, and represents an excellent target for metabolic engineering of industrial specialized metabolite-producing bacteria and the activation of cryptic specialized metabolites.

Antimicrobial lexitropsins containing amide, amidine, and alkene linking groups.

The synthesis and properties of 80 short minor groove binders related to distamycin and the thiazotropsins are described. The design of the compounds was principally predicated upon increased affinity arising from hydrophobic interactions between minor groove binders and DNA. The introduction of hydrophobic aromatic head groups, including quinolyl and benzoyl derivatives, and of alkenes as linkers led to several strongly active antibacterial compounds with MIC for Staphylococcus aureus, both methicillin-sensitive and -resistant strains, in the range of 0.1-5 microg mL-1, which is comparable to many established antibacterial agents. Antifungal activity was also found in the range of 20-50 microg mL-1 MIC against Aspergillus niger and Candida albicans, again comparable with established antifungal drugs. A quinoline derivative was found to protect mice against S. aureus infection for a period of up to six days after a single intraperitoneal dose of 40 mg kg-1.

Engineering primary metabolic pathways of industrial micro-organisms.

Metabolic engineering is a powerful tool for the optimisation and the introduction of new cellular processes. This is mostly done by genetic engineering. Since the introduction of this multidisciplinary approach, the success stories keep accumulating. The primary metabolism of industrial micro-organisms has been studied for long time and most biochemical pathways and reaction networks have been elucidated. This large pool of biochemical information, together with data from proteomics, metabolomics and genomics underpins the strategies for design of experiments and choice of targets for manipulation by metabolic engineers. These targets are often located in the primary metabolic pathways, such as glycolysis, pentose phosphate pathway, the TCA cycle and amino acid biosynthesis and mostly at major branch points within these pathways. This paper describes approaches taken for metabolic engineering of these pathways in bacteria, yeast and filamentous fungi.

The structure and mechanism of the type II dehydroquinase from Streptomyces coelicolor.

The structure of the type II DHQase from Streptomyces coelicolor has been solved and refined to high resolution in complexes with a number of ligands, including dehydroshikimate and a rationally designed transition state analogue, 2,3-anhydro-quinic acid. These structures define the active site of the enzyme and the role of key amino acid residues and provide snap shots of the catalytic cycle. The resolution of the flexible lid domain (residues 21-31) shows that the invariant residues Arg23 and Tyr28 close over the active site cleft. The tyrosine acts as the base in the initial proton abstraction, and evidence is provided that the reaction proceeds via an enol intermediate. The active site of the structure of DHQase in complex with the transition state analog also includes molecules of tartrate and glycerol, which provide a basis for further inhibitor design.

In vivo antimalarial activity of the endophytic actinobacteria, Streptomyces SUK 10.

Endophytic bacteria, such as Streptomyces, have the potential to act as a source for novel bioactive molecules with medicinal properties. The present study was aimed at assessing the antimalarial activity of crude extract isolated from various strains of actinobacteria living endophytically in some Malaysian medicinal plants. Using the four day suppression test method on male ICR strain mice, compounds produced from three strains of Streptomyces (SUK8, SUK10, and SUK27) were tested in vivo against Plasmodium berghei PZZ1/100 in an antimalarial screen using crude extracts at four different concentrations. One of these extracts, isolated from Streptomyces SUK10 obtained from the bark of Shorea ovalis tree, showed inhibition of the test organism and was further tested against P. berghei-infected mice for antimalarial activity at different concentrations. There was a positive relationship between the survival of the infected mouse group treated with 50 µg/kg body weight (bw) of ethyl acetate-SUK10 crude extract and the ability to inhibit the parasites growth. The parasite inhibition percentage for this group showed that 50% of the mice survived for more than 90 days after infection with the parasite. The nucleotide sequence and phylogenetic tree suggested that Streptomyces SUK10 may constitute a new species within the Streptomyces genus. As part of the drug discovery process, these promising finding may contribute to the medicinal and pharmaceutical field for malarial treatment.

Draft Genome Sequence of the Oxytetracycline-Producing Bacterium Streptomyces rimosus ATCC 10970.

We report the draft genome of Streptomyces rimosus (ATCC 10970), a soil isolate that produces oxytetracycline, a commercially important and clinically useful antibiotic.