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Polymyxins are used as the last-line therapy against multidrug-resistant bacteria. However, their further clinical development needs to solve problems related to the presence of heterogeneous analogs, but there is still no platform or methods that can regulate the biosynthesis of polymyxin analogs. In this study, we present an approach to swap domains in the polymyxin gene cluster to regulate the production of different analogs. Following adenylation domain swapping, the proportion of polymyxin B1 increased from 41.36 to 52.90%, while that of B1-1 decreased from 18.25 to 3.09%. The ratio of polymyxin B1 and B3 following starter condensation domain swapping changed from 41.36 and 16.99 to 55.03 and 6.39%, respectively. The two domain-swapping strains produced 62.96% of polymyxin B1, 6.70% of B3 and 3.32% of B1-1. This study also revealed the presence of overflow fluxes between acetoin, 2,3-butanediol and polymyxin. To our best knowledge, this is the first report of engineering the polymyxin synthetase gene cluster in situ to regulate the relative proportions of polymyxin analogs. This research paves a way for regulating lipopeptide analogs and will facilitate the development of novel lipopeptide derivatives. [ABSTRACT FROM AUTHOR]

The development of drug-resistant bacteria and the necessity for unique antimicrobial agents, directed to the search of new habitats to screen the production of anti-infective substances. Culture-dependent studies of heterotrophic bacteria from the intertidal macroalgae thriving along the Southern coast of India resulted in the isolation of 148 strains, which were assayed for antibacterial activities against wide spectrum of pathogens including drug-resistant pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE). Two of the most active strains with a zone of inhibition ≥ 30 mm on spot over lawn assay, belonging to the phyla Firmicutes and Gamma-proteobacteria, isolated from a Rhodophycean marine macroalga, Hypnea valentiae, were selected for bioprospecting studies. They were further characterized as Shewanella algae MTCC 12715 and Bacillus amyloliquefaciens MTCC 12716, based on integrated phenotypic and genotypic analysis. The bacterial extracts exhibited significant antibacterial activities against MRSA and VRE with minimum inhibitory concentrations of 6.25–12.5 µg/mL. Time kill kinetic profiles of these bacteria revealed rapid bactericidal activity against both MRSA and E. coli, showing a ≥ 3log10 decline in viable cell count compared to the initial. In BacLight™ live/dead staining technique, the propidium iodide uptake results appropriately attributed that the components in the B. amyloliquefaciens extract might compromise the integrity of the cytoplasmic membrane of the pathogenic bacteria. Type-1 pks gene (MH157093) of S. algae and hybrid nrps/pks gene (MH157092) of B. amyloliquefaciens could be amplified. Antibacterial activity study combined with the results of amplified genes coding for polyketide synthase and nonribosomal peptide synthetase showed that these marine symbiotic bacteria had a promising broad-spectrum activity, and therefore, could be used against the emerging dilemma of antibiotic-resistant bacterial infections. [ABSTRACT FROM AUTHOR]

Marine sediments host diverse actinomycetes that serve as a source of new natural products to combat infectious diseases and cancer. Here, we report the biodiversity, bioactivities against ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) and ovarian cancer, and metabolites variation among culturable actinomycetes isolated from the marine sediments of Visayan Sea, Philippines. We identified 15 Streptomyces species based on a 16S rRNA gene sequence analysis. The crude extracts of 10 Streptomyces species have inhibited the growth of ESKAPE pathogens with minimum inhibitory concentration (MIC) values ranging from 0.312 mg/mL to 20 mg/mL depending on the strain and pathogens targeted. Additionally, ten crude extracts have antiproliferative activity against A2780 human ovarian carcinoma at 2 mg/mL. To highlight, we observed that four phylogenetically identical Streptomyces albogriseolus strains demonstrated variation in antibiotic and anticancer activities. These strains harbored type I and II polyketide synthase (PKS) and non-ribosomal synthetase (NRPS) genes in their genomes, implying that their bioactivity is independent of the polymerase chain reaction (PCR)-detected bio-synthetic gene clusters (BGCs) in this study. Metabolite profiling revealed that the taxonomically identical strains produced core and strain-specific metabolites. Thus, the chemical diversity among these strains influences the variation observed in their biological activities. This study expanded our knowledge on the potential of marine-derived Streptomyces residing from the unexplored regions of the Visayan Sea as a source of small molecules against ESKAPE pathogens and cancer. It also highlights that Streptomyces species strains produce unique strain-specific secondary metabolites; thus, offering new chemical space for natural product discovery.

It has been suggested that microorganisms present in honey are a potential source of antimicrobial compounds. This study aimed to isolate and characterize bacteria from 46 Greek honey samples of diverse botanical and geographical origin and to determine whether these bacteria demonstrate antibacterial activity against five important nosocomial and foodborne pathogens. In total, 2014 bacterial isolates were obtained and screened for antibacterial activity. Overall, 16% of the isolates inhibited the growth of Staphylococcus aureus, 11.2% inhibited the growth of Pseudomonas aeruginosa and Acinetobacter baumannii, 10.2% inhibited the growth of Salmonella Typhimurium and 12.4% of the isolates affected the growth of Citrobacter freundii. In total, 316 isolates that inhibited the growth of more than two of the tested pathogens were grouped by restriction fragment length polymorphisms (RFLP) analysis of the 16S rRNA gene amplicon. Fifty of them were identified by 16S rRNA gene sequencing. The majority, 62% of the isolates, belonged to the genus Bacillus. Only 10% of the isolates were identified as Gram-negative bacteria. Furthermore, in several bacterial isolates, genes encoding polyketide synthases and nonribosomal peptide synthetases that catalyze the biosynthesis of secondary metabolites which might contribute to the exerted antimicrobial activity, were detected. This study demonstrates that honey microbiota exerts antimicrobial activity and is a putative source of secondary metabolites against important nosocomial and food pathogens that warrants further investigation.

Fusarium pseudograminearum-induced crown rot causes significant reduction to wheat production worldwide. To date, efforts to develop effective resistance to this disease have been hampered by the quantitative nature of resistance trait and a lack of understanding of the molecular pathogenesis. Non-ribosomal peptides have important roles in development, pathogenicity, and toxins in many plant pathogens, while less is known in F. pseudograminearum. In this work, we studied the expression and function of a nonribosomal peptide gene FpNPS9 in F. pseudograminearum. We determined the expression of FpNPS9 which was significantly up regulated during the infection of wheat. A deletion mutant Δfpnps9 produced in this study displayed a normal growth and conidiation phenotype, however, hyphae polar growth was obviously affected. Deoxynivalenol production in this mutant was significantly reduced and the infection of wheat coleoptiles and wheat spikelet was attenuated. The Δfpnps9 showed serious defects on the extension of infectious hyphae in plant and inhibition of roots elongation compared with the wild type. The complementation assay using a FpNPS9-GFP fusion construct fully restored the defects of the mutant. GFP signal was detected in the germinating conidia and infectious hyphae in coleoptiles of the infected plants. Interestingly, the signal was not observed when it was grown on culture medium, suggesting that the expression of FpNPS9 was regulated by an unknown host factor. This observation was supported by the result of qRT-PCR. In summary, we provided new knowledge on FpNPS9 expression in F. pseudograminearum and its function in F. pseudograminearum pathogenicity in wheat. [ABSTRACT FROM AUTHOR]

Lipopeptides are important non-ribosomal peptide synthetases (NRPSs) products with broad therapeutic potential in biotechnology and biopharmaceutical applications. Fatty acyl modifications in N-terminal of lipopeptides have attracted wide interest in the engineering processes of altered fatty acyl selectivity. In this study, we focused on the starter condensation domain of antibiotic A54145 (lptC1) and its indiscriminate selectivity of fatty acyl substrates, which results in multi-component products. Using in silico analysis, five site-directed mutations at protein–protein interface were identified with altered activity and selectivity towards wild type lptC1. The variants Y149W and A330T exhibited changed substrate selectivity to prefer longer branched chain fatty acyl substrate, while T16A and A350D showed improved selectivity for shorter linear chain fatty acyl substrates. Subsequently, molecular dynamics (MD) simulations were performed to analyze the impact of these residues on the changes of catalytic activity and conformation. Through in silico analysis, the altered binding free energy were coincident with the corresponding activity performance of the variants, and surface forces indicated that other factors or processes may influence the activity and selectivity. Moreover, the MD results revealed even altered active center conformations, implying the importance of these interface residues affected on distant active center thus reflected to catalysis activity. Based on the biochemistry and computational results, our work provides detailed insights from molecular and dynamics aspects into the role of C1's interface residues during complex NRPS biosynthesis machinery, prompting further rational engineering for lipopeptide catalysis. [ABSTRACT FROM AUTHOR]

The impact of the global secondary metabolite regulators LaeA and VeA on echinocandin B production and morphological development was evaluated in the industrial production strain Aspergillus pachycristatus NRRL 11440. Other representative secondary metabolites were examined as well to determine if the velvet complex functions as in A. nidulans and other species of fungi. Genetic methods used for gene manipulations in A. nidulans were applied to A. pachycristatus. Separate deletions of genes Apc.laeA and Apc.veA resulted in similar yet differing phenotypes in strain NRRL 11440. Disruption of Apc.laeA and Apc.veA significantly reduced, but did not eliminate, the production of echinocandin B. Similar to what has been observed in A. nidulans, the production of sterigmatocystin was nearly eliminated in both mutants. Quantitative reverse transcription PCR analyses confirmed that selected genes of both the echinocandin B and sterigmatocystin gene clusters were down-regulated in both mutant types. The two mutants differed with respect to growth of aerial hyphae, pigmentation, development of conidiophores, conidial germination rate, and ascospore maturation. Further functional annotation of key regulatory genes in A. pachycristatus and related Aspergillus species will improve our understanding of regulation of echinocandin production and co-produced metabolites. [ABSTRACT FROM AUTHOR]

The increasing threat posed by multiresistant bacterial pathogens necessitates the discovery of novel antibacterials with unprecedented modes of action. ADEP1, a natural compound produced by Streptomyces hawaiiensis NRRL 15010, is the prototype for a new class of acyldepsipeptide (ADEP) antibiotics. ADEP antibiotics deregulate the proteolytic core ClpP of the bacterial caseinolytic protease, thereby exhibiting potent antibacterial activity against Gram-positive bacteria, including multiresistant pathogens. ADEP1 and derivatives, here collectively called ADEP, have been previously investigated for their antibiotic potency against different species, structure-activity relationship, and mechanism of action; however, knowledge on the biosynthesis of the natural compound and producer self-resistance have remained elusive. In this study, we identified and analyzed the ADEP biosynthetic gene cluster in S. hawaiiensis NRRL 15010, which comprises two NRPSs, genes necessary for the biosynthesis of (4S,2R)-4-methylproline, and a type II polyketide synthase (PKS) for the assembly of highly reduced polyenes. While no resistance factor could be identified within the gene cluster itself, we discovered an additional clpP homologous gene (named clpPADEP) located further downstream of the biosynthetic genes, separated from the biosynthetic gene cluster by several transposable elements. Heterologous expression of ClpPADEP in three ADEP-sensitive Streptomyces species proved its role in conferring ADEP resistance, thereby revealing a novel type of antibiotic resistance determinant. [ABSTRACT FROM AUTHOR]

Cyclization of linear dipeptidyl precursors derived from nonribosomal peptide synthetases (NRPSs) into 2,5‐diketopiperazines (DKPs) is a crucial step in the biosynthesis of a large number of bioactive natural products. However, the mechanism of DKP formation in fungi has remained unclear, despite extensive studies of their biosyntheses. Here we show that DKP formation en route to the fungal virulence factor gliotoxin requires a seemingly extraneous couplet of condensation (C) and thiolation (T) domains in the NRPS GliP. In vivo truncation of GliP to remove the CT couplet or just the T domain abrogated production of gliotoxin and all other gli pathway metabolites. Point mutation of conserved active sites in the C and T domains diminished cyclization activity of GliP in vitro and abolished gliotoxin biosynthesis in vivo. Verified NRPSs of other fungal DKPs terminate with similar CT domain couplets, suggesting a conserved strategy for DKP biosynthesis by fungal NRPSs. [ABSTRACT FROM AUTHOR]

• Wildtype and engineered carboxylic acid reductases expand the bioorganic synthesis toolbox. • Protein engineering unravels key catalytic residues in carboxylic acid reductases. • Protein engineering consolidates mechanistic understanding as well as structure-function relationship of carboxylic acid reductases. • Diverse protein engineering strategies are use to engineer multi-domain and multi-functional carboxylic acid reductases. • Protein engineering research of carboxylic acid reductases will continue to flourish, supported by solid groundwork. Carboxylic acids (CAs) are widespread in Nature. A prominent example is fatty acids, a major constituent of lipids. CAs are potentially economical precursors for bio-based products such as bio-aldehydes and bio-alcohols. However, carboxylate reduction is a challenging chemical transformation due to the thermodynamic stability of carboxylate. Carboxylic acid reductases (CARs), found in bacteria and fungi, offer a good solution to this challenge. These enzymes catalyse the NADPH- and ATP-dependent reduction of aliphatic and aromatic CAs. This review summarised all the protein engineering work that has been done on these versatile biocatalysts to date. The intricate catalytic mechanism and structure of CARs prompted us to first examine their domain architecture to facilitate the subsequent discussion of various protein engineering strategies. This then led to a survey of assays to detect aldehyde formation and to monitor aldenylation activity. Strategies for NADPH and ATP regeneration were also incorporated, as they are deemed vital to developing preparative-scale biocatalytic process and high-throughput screening systems. The objectives of the review are to consolidate CAR engineering research, stimulate interest, discussion or debate, and advance the field of bioreduction. [ABSTRACT FROM AUTHOR]

Filamentous fungi produce a vast number of bioactive secondary metabolites (SMs), some of which have found applications in the pharmaceutical industry including as antibiotics and immunosuppressants. As more and more species are whole genome sequenced the number of predicted clusters of genes for SM biosynthesis is ever increasing – holding a promise of novel useful bioactive SMs. To be able to fully utilize the potential of novel SMs, it is necessary to link the SM and the genes responsible for producing it. This can be challenging, but many strategies and tools have been developed for this purpose. Here we provide an overview of the methods used to establish the link between SM and biosynthetic gene cluster (BGC) and vice versa, along with the challenges and advantages of each of the methods. Part I of the review, associating BCG with SM, is divided into gene manipulations native strain and heterologous expression strategies, depending on the fungal species. Part II, associating SM with BGC, is divided into three main approaches: (1) homology search (2) retro-biosynthesis and (3) comparative genomics. [ABSTRACT FROM AUTHOR]

Recent papers have reported dipeptides containing D-amino acids to have novel effects that cannot be observed with LL-dipeptides, and such dipeptides are expected to be novel functional compounds for pharmaceuticals and food additives. Although the functions of D-amino acid-containing dipeptides are gaining more attention, there are few reports on the synthetic enzymes that can accept D-amino acids as substrates, and synthetic methods for D-amino acid-containing dipeptides have not yet been constructed. Previously, we developed a chemoenzymatic system for amide synthesis that comprised enzymatic activation and a subsequent nucleophilic substitution reaction. In this study, we demonstrated the application of the system for D-amino acid-containing-dipeptide synthesis. We chose six adenylation domains as targets according to our newly constructed hypothesis, i.e., an adenylation domain located upstream from the epimerization domain may activate D-amino acid as well as L-amino acid. We successfully synthesized over 40 kinds of D-amino acid-containing dipeptides, including LD-, DL-, and DD-dipeptides, using only two adenylation domains, TycA-A from tyrocidine synthetase and BacB2-A from bacitracin synthetase. Furthermore, this study offered the possibility that the epimerization domain could be a clue to the activity of the adenylation domains toward D-amino acid. This paper provides additional information regarding D-amino acid-containing-dipeptide synthesis through the combination of enzymatic adenylation and chemical nucleophilic reaction, and this system will be a useful tool for dipeptide synthesis. [ABSTRACT FROM AUTHOR]

Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis.

Nostocyclopeptides (Ncps) constitute a small class of nonribosomal peptides, exclusively produced by cyanobacteria of the genus Nostoc. The peptides inhibit the organic anion transporters, OATP1B3 and OATP1B1, and prevent the transport of the toxic microcystins and nodularin into hepatocytes. So far, only three structural analogues, Ncp-A1, Ncp-A2 and Ncp-M1, and their linear forms were identified in Nostoc strains as naturally produced cyanometabolites. In the current work, the whole genome sequence of the new Ncps producer, N. edaphicum CCNP1411 from the Baltic Sea, has been determined. The genome consists of the circular chromosome (7,733,505 bps) and five circular plasmids (from 44.5 kb to 264.8 kb). The nostocyclopeptide biosynthetic gene cluster (located between positions 7,609,981–7,643,289 bps of the chromosome) has been identified and characterized in silico. The LC-MS/MS analyzes of N. edaphicum CCNP1411 cell extracts prepared in aqueous methanol revealed several products of the genes. Besides the known peptides, Ncp-A1 and Ncp-A2, six other compounds putatively characterized as new noctocyclopeptide analogues were detected. This includes Ncp-E1 and E2 and their linear forms (Ncp-E1-L and E2-L), a cyclic Ncp-E3 and a linear Ncp-E4-L. Regardless of the extraction conditions, the cell contents of the linear nostocyclopeptides were found to be higher than the cyclic ones, suggesting a slow rate of the macrocyclization process.

The formation of the trapping device induced by nematodes has been assumed as an indicator for a switch from saprophytic to predacious lifestyles for nematode-trapping fungi. However, fungal nematocidal activity is not completely synonymous with fungal trap formation. We found that the predominant nematode-trapping fungus Arthrobotrys oligospora harbored a rare NRPS ( Ao415 ) gene cluster that was mainly distributed in nematode-trapping fungi. The gene Ao415 putatively encodes a protein with a unique domain organization, distinct from other NRPSs in other fungi. Mutation of the two key biosynthetic genes Ao415 and Ao414 combined with nontarget metabolic analysis revealed that the Ao415 gene cluster was responsible for the biosynthesis of a hydroxamate siderophore, desferriferrichrome ( 1 ). Lack of desferriferrichrome ( 1 ) and its hydroxamate precursor ( 3 ) could lead to significantly increased Fe 3+ content, which induced fungal trap formation without a nematode inducer. Furthermore, the addition of Fe 3+ strongly improved fungal trap formation but deleteriously caused broken traps. The addition of 1 significantly attenuated trap formation but enhanced fungal nematicidal activity. Our findings indicate that iron is a key factor for trap formation and provide a new insight into the underlying mechanism of siderophores in nematode-trapping fungi.

The protein Ebony from Drosophila melanogaster plays a central role in the regulation of histamine and dopamine in various tissues through condensation of these amines with β-alanine. Ebony is a rare example of a nonribosomal peptide synthetase (NRPS) from a higher eukaryote and contains a C-terminal sequence that does not correspond to any previously characterized NRPS domain. We have structurally characterized this C-terminal domain and have discovered that it adopts the aryl-alkylamine-N-acetyl transferase (AANAT) fold, which is unprecedented in NRPS biology. Through analysis of ligand-bound structures, activity assays, and binding measurements, we have determined how this atypical condensation domain is able to provide selectivity for both the carrier protein-bound amino acid and the amine substrates, a situation that remains unclear for standard condensation domains identified to date from NRPS assembly lines. These results demonstrate that the C terminus of Ebony encodes a eukaryotic example of an alternative type of NRPS condensation domain; they also illustrate how the catalytic components of such assembly lines are significantly more diverse than a minimal set of conserved functional domains. [ABSTRACT FROM AUTHOR]

Natural products from the human microbiota may mediate host health and disease. However, discovery of the biosynthetic gene clusters that generate these metabolites has far outpaced identification of the molecules themselves. Here, we used an isolation-independent approach to access the probable products of a nonribosomal peptide synthetase-encoding gene cluster from Ruminococcus bromii , an abundant gut commensal bacterium. By combining bioinformatics with in vitro biochemical characterization of biosynthetic enzymes, we predicted that this pathway likely generates an N -acylated dipeptide aldehyde (ruminopeptin). We then used chemical synthesis to access putative ruminopeptin scaffolds. Several of these compounds inhibited Staphylococcus aureus endoproteinase GluC (SspA/V8 protease). Homologs of this protease are found in gut commensals and opportunistic pathogens as well as human gut metagenomes. Overall, this work reveals the utility of isolation-independent approaches for rapidly accessing bioactive compounds and highlights a potential role for gut microbial natural products in targeting gut microbial proteases. [ABSTRACT FROM AUTHOR]

Abstract: Oxyvinylglycines are a family of nonproteinogenic amino acids featuring an essential vinyl ether conferring mechanism‐based inhibition of pyridoxal phosphate enzymes. The gene clusters for a few oxyvinylglycines are known, yet the biosynthetic origin of the vinyl ether is elusive. The in vitro biosynthesis of methoxyvinylglycine or l‐2‐amino‐4‐methoxy‐trans‐3‐butenoic acid (AMB) is reported. It is shown that AMB is made from glutamate as an alanyl‐AMB dipeptide and the rationale is provided for the N‐term Ala. Using a chemical capture method, the order and timing of the modifications on non‐ribosomal peptide synthetase (NRPS)‐bound substrates was determined, including a cryptic hydroxylation of the Glu β‐carbon. Eliminating this hydroxy group likely generates a key α,β‐dehydroamino acid intermediate that facilitates decarboxylation. This work sheds light on vinyl ether biosynthesis and uncovers new NRPS chemistry. [ABSTRACT FROM AUTHOR]

An ATP regeneration system is advantageous for industrial processes that are coupled with ATP-dependent enzymes. For ATP regeneration from AMP, a few methods have been reported; however, these methods employ multiple enzymes. To establish an ATP regeneration system using a single enzyme, we focused on class III polyphosphate kinase 2 (class III PPK2) that can synthesize ATP from AMP and polyphosphate. We constructed an ATP regeneration system from AMP using Deipr_1912, a class III PPK2 from Deinococcus proteolyticus NBRC 101906 T , coupled with aminoacyl proline (Xaa-Pro) synthesis catalyzed by the adenylation domain of tyrocidine synthetase A (TycA-A). Using this system, 0.87 mM of l -Trp- l -Pro was successfully synthesized from AMP after 72 h. Farther, addition of inorganic pyrophosphatase from Escherichia coli to the coupling reaction increased the reaction rate by 14-fold to yield 6.2 mM l -Trp- l -Pro. When the coupling reaction was applied to whole-cell reactions in E. coli BL21(DE3) pepQ − putA − , ATP was successfully regenerated from AMP by Deipr_1912, and 6.7 mM of l -Trp- l -Pro was produced after 24 h with the supplementation of 10 mM AMP. In addition, by altering the substrate amino acid of TycA-A, not only l -Trp- l -Pro, but also various other l -Xaa- l -Pro (Xaa = Val, Leu, Met, or Tyr) were produced using the whole-cell reaction incorporating ATP regeneration. Therefore, a production method for Xaa-Pro employing the adenylation domain of a nonribosomal peptide synthetase was established by introducing an ATP regeneration system that utilizes class III PPK2 with pyrophosphatase. [ABSTRACT FROM AUTHOR]

Abstract: Bacterial resistance to existing drugs is becoming a serious public health issue, urging extensive search for new antibiotics. Teixobactin, a cyclic depsipeptide discovered in a screen of uncultured bacteria, shows potent activity against all the tested Gram‐positive bacteria. Remarkably, no teixobactin‐resistant bacterial strain has been obtained despite extensive efforts, highlighting the great potential of teixobactin as a lead compound in the fight against antimicrobial resistance (AMR). This review summarizes recent progresses in the understanding of many aspects of teixobactin, including chemical structure, biological activity, biosynthetic pathway, and mode of action. We also discuss the different synthetic strategies in producing teixobactin and its analogues, and the structure–activity relationship (SAR) studies. [ABSTRACT FROM AUTHOR]

We used a temperature differential assay with the opportunistic fungal pathogen Cryptococcus neoformans as a simple screening platform to detect small molecules with antifungal activity in natural product extracts. By screening of a collection extracts from two different strains of the coprophilous fungus, Amphichorda felina, we detected strong, temperature-dependent antifungal activity using a two-plate agar zone of inhibition assay at 25 and 37 °C. Bioassay-guided fractionation of the crude extract followed by liquid chromatography–mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) identified cyclosporin C (CsC) as the main component of the crude extract responsible for growth inhibition of C. neoformans at 37 °C. The presence of CsC was confirmed by comparison with a commercial standard. We sequenced the genome of A. felina to identify and annotate the CsC biosynthetic gene cluster. The only previously characterized gene cluster for the biosynthesis of similar compounds is that of the related immunosuppressant drug cyclosporine A (CsA). The CsA and CsC gene clusters share a high degree of synteny and sequence similarity. Amino acid changes in the adenylation domain of the CsC nonribosomal peptide synthase’s sixth module may be responsible for the substitution of l-threonine compared to l-α-aminobutyric acid in the CsA peptide core. This screening strategy promises to yield additional antifungal natural products with a focused spectrum of antimicrobial activity. [ABSTRACT FROM AUTHOR]

Magnetotactic bacteria (MTB) produce intracellular organelles called magnetosomes which are magnetic nanoparticles composed of magnetite (Fe3O4) or greigite (Fe3S4) enveloped by a lipid bilayer. The synthesis of a magnetosome is through a genetically controlled process in which the bacterium has control over the composition, direction of crystal growth, and the size and shape of the mineral crystal. As a result of this control, magnetosomes have narrow and uniform size ranges, relatively specific magnetic and crystalline properties, and an enveloping biological membrane. These features are not observed in magnetic particles produced abiotically and thus magnetosomes are of great interest in biotechnology. Most currently described MTB have been isolated from saline or brackish environments and the availability of their genomes has contributed to a better understanding and culturing of these fastidious microorganisms. Moreover, genome sequences have allowed researchers to study genes related to magnetosome production for the synthesis of magnetic particles for use in future commercial and medical applications. Here, we review the current information on the biology of MTB and apply, for the first time, a genome mining strategy on these microorganisms to search for secondary metabolite synthesis genes. More specifically, we discovered that the genome of the cultured MTB Magnetovibrio blakemorei, among other MTB, contains several metabolic pathways for the synthesis of secondary metabolites and other compounds, thereby raising the possibility of the co-production of new bioactive molecules along with magnetosomes by this species.

ABSTRACT Bacteria possess an amazing capacity to synthesize a diverse range of structurally complex, bioactive natural products known as specialized (or secondary) metabolites. Many of these specialized metabolites are used as clinical therapeutics, while others have important ecological roles in microbial communities. The biosynthetic gene clusters (BGCs) that generate these metabolites can be identified in bacterial genome sequences using their highly conserved genetic features. We analyzed an unprecedented 1,566 bacterial genomes from Bacillus species and identified nearly 20,000 BGCs. By comparing these BGCs to one another as well as a curated set of known specialized metabolite BGCs, we discovered that the majority of Bacillus natural products are comprised of a small set of highly conserved, well-distributed, known natural product compounds. Most of these metabolites have important roles influencing the physiology and development of Bacillus species. We identified, in addition to these characterized compounds, many unique, weakly conserved BGCs scattered across the genus that are predicted to encode unknown natural products. Many of these “singleton” BGCs appear to have been acquired via horizontal gene transfer. Based on this large-scale characterization of metabolite production in the Bacilli, we go on to connect the alkylpyrones, natural products that are highly conserved but previously biologically uncharacterized, to a role in Bacillus physiology: inhibiting spore development. IMPORTANCE Bacilli are capable of producing a diverse array of specialized metabolites, many of which have gained attention for their roles as signals that affect bacterial physiology and development. Up to this point, however, the Bacillus genus’s metabolic capacity has been underexplored. We undertook a deep genomic analysis of 1,566 Bacillus genomes to understand the full spectrum of metabolites that this bacterial group can make. We discovered that the majority of the specialized metabolites produced by Bacillus species are highly conserved, known compounds with important signaling roles in the physiology and development of this bacterium. Additionally, there is significant unique biosynthetic machinery distributed across the genus that might lead to new, unknown metabolites with diverse biological functions. Inspired by the findings of our genomic analysis, we speculate that the highly conserved alkylpyrones might have an important biological activity within this genus. We go on to validate this prediction by demonstrating that these natural products are developmental signals in Bacillus and act by inhibiting sporulation.

The cyclic depsipeptides ohmyungsamycin (OMS) A (1) and B (2), isolated from the marine-derived Streptomyces sp. SNJ042, contain two non-proteinogenic amino acid residues, β-hydroxy-l-phenylalanine (β-hydroxy-l-Phe) and 4-methoxy-l-tryptophan (4-methoxy-l-Trp). Draft genome sequencing of Streptomyces sp. SNJ042 revealed the OMS biosynthetic gene cluster consisting of a nonribosomal peptide synthetase (NRPS) gene and three genes for amino acid modification. By gene inactivation and analysis of the accumulated products, we found that OhmL, encoding a P450 gene, is an l-Phe β-hydroxylase. Furthermore, OhmK, encoding a Trp 2,3-dioxygenase homolog, and OhmJ, encoding an O-methyltransferase, are suggested to be involved in hydroxylation and O-methylation reactions, respectively, in the biosynthesis of 4-methoxy-l-Trp. In addition, the antiproliferative and antituberculosis activities of the OMS derivatives dehydroxy-OMS A (4) and demethoxy-OMS A (6) obtained from the mutant strains were evaluated in vitro. Interestingly, dehydroxy-OMS A (4) displayed significantly improved antituberculosis activity and decreased cytotoxicity compared to wild-type OMS A.

Nonribosomal peptide synthetases (NRPSs) are incredible macromolecular machines that produce a wide range of biologically- and therapeutically-relevant molecules. During synthesis, peptide elongation is performed by the condensation (C) domain, as it catalyzes amide bond formation between the nascent peptide and the amino acid it adds to the chain. Since their discovery more than two decades ago, C domains have been subject to extensive biochemical, bioinformatic, mutagenic, and structural analyses. They are composed of two lobes, each with homology to chloramphenicol acetyltransferase, have two binding sites for their two peptidyl carrier protein-bound ligands, and have an active site with conserved motif HHxxxDG located between the two lobes. This review discusses some of the important insights into the structure, catalytic mechanism, specificity, and gatekeeping functions of C domains revealed since their discovery. In addition, C domains are the archetypal members of the C domain superfamily, which includes several other members that also function as NRPS domains. The other family members can replace the C domain in NRP synthesis, can work in concert with a C domain, or can fulfill diverse and novel functions. These domains include the epimerization (E) domain, the heterocyclization (Cy) domain, the ester-bond forming C domain, the fungal NRPS terminal C domain (C T ), the β-lactam ring forming C domain, and the X domain. We also discuss structural and function insight into C, E, Cy, C T and X domains, to present a holistic overview of historical and current knowledge of the C domain superfamily. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. [ABSTRACT FROM AUTHOR]

The model white-rot basidiomycete, Ceriporiopsis (Gelatoporia) subvermispora B, encodes putative natural product biosynthesis genes. Among them is the gene for the seven-domain nonribosomal peptide synthetase CsNPS2. It is a member of the as-yet-uncharacterized fungal type VI siderophore synthetase family, which is highly conserved and widely distributed among the basidiomycetes. These enzymes include only one adenylation (A) domain, i.e., one complete peptide synthetase module, and two thiolation/condensation (T-C) didomain partial modules which together constitute an AT1C1T2C2T3C3 domain setup. The full-length CsNPS2 enzyme (274.5 kDa) was heterologously produced as a polyhistidine fusion in Aspergillus niger as a soluble and active protein. N5-acetyl-N5-hydroxy-L-ornithine (L-AHO) and N5- cis-anhydromevalonyl-N5-hydroxy-L-ornithine (L-AMHO) were accepted as the substrates, based on results of an in vitro substrate-dependent [32P]ATP-pyrophosphate radioisotope exchange assay. Full-length holo-CsNPS2 catalyzed amide bond formation between three L-AHO molecules to release the linear L-AHO trimer, called basidioferrin, as the product in vitro, which was verified by liquid chromatography-high-resolution electrospray ionization-mass spectrometry analysis. Phylogenetic analyses suggested that type VI family siderophore synthetases are widespread in mushrooms and evolved in a common ancestor of basidiomycetes. IMPORTANCE The basidiomycete nonribosomal peptide synthetase CsNPS2 represents a member of a widely distributed but previously uninvestigated class (type VI) of fungal siderophore synthetases. Genes orthologous to CsNPS2 are highly conserved across various phylogenetic clades of the basidiomycetes. Hence, our work serves as a broadly applicable model for siderophore biosynthesis and iron metabolism in higher fungi. Also, our results on the amino acid substrate preference of CsNPS2 support a further understanding of the substrate selectivity of fungal adenylation domains. Methodologically, this report highlights the Aspergillus niger/SMXpress-based system as a suitable platform to heterologously express multimodular basidiomycete biosynthesis enzymes in the >250-kDa range in soluble and active form. [ABSTRACT FROM AUTHOR]

Although bacterial bioactive metabolites have been one of the most prolific sources of lead structures for the development of smallmolecule therapeutics, very little is known about the environmental factors associated with changes in secondary metabolism across natural environments. Large-scale sequencing of environmental microbiomes has the potential to shed light on the richness of bacterial biosynthetic diversity hidden in the environment, how it varies from one environment to the next, and what environmental factors correlate with changes in biosynthetic diversity. In this study, the sequencing of PCR amplicons generated using primers targeting either ketosynthase domains from polyketide biosynthesis or adenylation domains from nonribosomal peptide biosynthesis was used to assess biosynthetic domain composition and richness in soils collected across the Australian continent. Using environmental variables collected at each soil site, we looked for environmental factors that correlated with either high overall domain richness or changes in the domain composition. Among the environmental variables we measured, changes in biosynthetic domain composition correlate most closely with changes in latitude and to a lesser extent changes in pH. Although it is unclear at this time the exact mix of factors that may drive the relationship between biosynthetic domain composition and latitude, from a practical perspective the identification of a latitudinal basis for differences in soil metagenome biosynthetic domain compositions should help guide future natural product discovery efforts. [ABSTRACT FROM AUTHOR]

Streptomyces lavendulae FRI-5 produces the blue pigment indigoidine and other secondary metabolites ( d -cycloserine and nucleoside antibiotics). The production of these useful compounds is controlled by a signaling cascade mediated by the γ-butyrolactone autoregulator IM-2. Previously we revealed that the far regulatory island includes the IM-2 receptor, the IM-2 biosynthetic enzyme, and several transcriptional regulators, and that it contributes to the regulation of indigoidine production in response to the signaling molecule. Here, we found that the vicinity of the far regulatory island includes the putative gene cluster for the biosynthesis of indigoidine and unidentified compounds, and demonstrated that the expression of the gene cluster is under the control of the IM-2 regulatory system. Heterologous expression of lbpA , encoding a plausible nonribosomal peptide synthetase, in the versatile model host Streptomyces avermitilis SUKA22 led to indigoidine production, which was enhanced dramatically by feeding of the indigoidine precursor l -glutamine. These results confirmed that LbpA is an indigoidine biosynthetic enzyme in the IM-2 signaling cascade. [ABSTRACT FROM AUTHOR]

Genomic mining revealed one major nonribosomal peptide synthetase (NRPS) phylogenetic cluster in 12 marine sponge species, one ascidian, an actinobacterial isolate and seawater. Phylogenetic analysis predicts its taxonomic affiliation to the actinomycetes and hydroxy-phenyl-glycine as a likely substrate. Additionally, a phylogenetically distinct NRPS gene cluster was discovered in the microbial metagenome of the sponge Aplysina aerophoba, which shows highest similarities to NRPS genes that were previously assigned, by ways of single cell genomics, to a Chloroflexi sponge symbiont. Genomic mining studies such as the one presented here for NRPS genes, contribute to on-going efforts to characterize the genomic potential of sponge-associated microbiota for secondary metabolite biosynthesis.