Accraspiroketides A-B, Phenylnaphthacenoid-Derived Polyketides with Unprecedented [6 + 6+6 + 6] + [5 + 5] Spiro-Architecture.

Two novel polyketides, accraspiroketides A (1) and B (2), which feature unprecedented [6 + 6+6 + 6] + [5 + 5] spiro chemical architectures, were isolated from Streptomyces sp. MA37 ΔaccJ mutant strain. Compounds 1-2 exhibit excellent activity against Gram-positive bacteria (MIC = 1.5-6.3 µg/mL). Notably, 1 and 2 have superior activity against clinically isolated Enterococcus faecium K60-39 (MIC = 4.0 µg/mL and 4.7 µg/mL, respectively) than ampicillin (MIC = 25 µg/mL).

Chemoenzymatic Synthesis of Indole-Containing Acyloin Derivatives.

Indole-containing acyloins are either key intermediates of many antimicrobial/antiviral natural products or building blocks in the synthesis of biologically active molecules. As such, access to structurally diverse indole-containing acyloins has attracted considerable attention. In this report, we present a pilot study of using biotransformation to provide acyloins that contain various indole substituents. The biotransformation system contains the tryptophan synthase standalone ß-subunit variant, PfTrpB6, generated from directed evolution in the literature; a commercially available L-amino acid oxidase (LAAO); and the thiamine-diphosphate (ThDP)-dependent enzyme NzsH, encoded in the biosynthetic gene cluster (nzs) of the bacterial carbazole alkaloid natural product named neocarazostatin A. The utilization of the first two enzymes, the PfTrpB variant and LAAO, is designed to provide structurally diverse indole 3-pyruvate derivatives as donor substrates for NzsH-catalysed biotransformation to provide acyloin derivatives. Our results demonstrate that NzsH displays a considerable substrate profile toward donor substrates for production of acyloins with different indole ring systems, suggesting that NzsH could be further explored as a potential biocatalyst via directed evolution to improve the catalytic efficiency in the future.

Correction: Antischistosomal, antionchocercal and antitrypanosomal potentials of some Ghanaian traditional medicines and their constituents.

[This corrects the article DOI: 10.1371/journal.pntd.0008919.].

Deletion of the accramycin pathway-specific regulatory gene accJ activates the production of unrelated polyketide metabolites.

The manipulation of regulatory genes has been employed to awaken cryptic metabolites in Streptomyces. Of particular interest in recent years is the effect of disruption of a pathway-specific gene to other biosynthetic pathways. Herein, we report the inactivation of the accramycin pathway-specific regulatory gene, accJ in Streptomyces sp. MA37 resulted in the production of unrelated polyketide metabolites. Through detailed mass spectrometric and spectroscopic analyses, and comparison with literature data, their structures were deduced as 3-methoxy-2-methyl-4H-pyran-4-one (1), zanthopyranone (2), propioveratrone (3), and TW94a (4). To the best of our knowledge, this is the first report of the isolation of 1-3 from bacteria. Compounds 1, 2, and 4 showed weak to moderate activity against Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium. Propioveratrone (3) displayed better inhibitory activity (MIC = 6.3 µg/mL) than ampicillin against multi-drug resistant E. faecium K60-39 clinical isolate (MIC = 25 µg/mL), suggesting a promising structural template for the drug development targeting Enterococcus isolates.

A ribosomally synthesised and post-translationally modified peptide containing a ß-enamino acid and a macrocyclic motif.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are structurally complex natural products with diverse bioactivities. Here we report discovery of a RiPP, kintamdin, for which the structure is determined through spectroscopy, spectrometry and genomic analysis to feature a bis-thioether macrocyclic ring and a ß-enamino acid residue. Biosynthetic investigation demonstrated that its pathway relies on four dedicated proteins: phosphotransferase KinD, Lyase KinC, kinase homolog KinH and flavoprotein KinI, which share low homologues to enzymes known in other RiPP biosynthesis. During the posttranslational modifications, KinCD is responsible for the formation of the characteristic dehydroamino acid residues including the ß-enamino acid residue, followed by oxidative decarboxylation on the C-terminal Cys and subsequent cyclization to provide the bis-thioether ring moiety mediated by coordinated action of KinH and KinI. Finally, conserved genomic investigation allows further identification of two kintamdin-like peptides among the kin-like BGCs, suggesting the occurrence of RiPPs from actinobacteria.

In vitro effects and mechanisms of action of Bidens pilosa in Trypanosoma brucei.

Background and aim: African trypanosomiasis poses serious health and economic concerns to humans and livestock in several sub-Saharan African countries. The aim of the present study was to identify the antitrypanosomal compounds from B. pilosa (whole plant) through a bioactivity-guided isolation and investigate the in vitro effects and mechanisms of action against Trypanosoma brucei (T. brucei). Experimental procedure: Crude extracts and fractions were prepared from air-dried pulverized plant material of B. pilosa using the modified Kupchan method of solvent partitioning. The antitrypanosomal activities of the fractions were determined through cell viability analysis. Effects of fractions on cell death and cell cycle of T. brucei were determined using flow cytometry, while fluorescence microscopy was used to investigate alterations in cell morphology and distribution. Results and conclusion: The solvent partitioning dichloromethane (BPFD) and methanol (BPFM) fractions of B. pilosa exhibited significant activities against T. brucei with respective half-maximal inhibitory concentrations (IC50s) of 3.29 µg/ml and 5.86 µg/ml and resulted in the formation of clumpy subpopulation of T. brucei cells. Butyl (compound 1) and propyl (compound 2) esters of tryptophan were identified as the major antitrypanosomal compounds of B. pilosa. Compounds 1 and 2 exhibited significant antitrypanosomal effects with respective IC50 values of 0.66 and 1.46 µg/ml. At the IC50 values, both compounds significantly inhibited the cell cycle of T. brucei at the G0-G1 phase while causing an increase in G2-M phase. The results suggest that tryptophan esters may possess useful chemotherapeutic properties for the control of African trypanosomiasis.

Monodispersed AgNPs Synthesized from the Nanofactories of Theobroma cacao (Cocoa) Leaves and Pod Husk and Their Antimicrobial Activity.

Silver nanoparticles (AgNPs) have been synthesized from the more chemically rich and diverse cocoa pod; the synthesis of silver nanoparticles from cocoa leaves, which are less rich and have low diversity in bioactive molecules, is yet to be achieved. In this work, AgNPs produced using the extracts of the cocoa leaf (CL) and cocoa pods (CP) have been investigated and their antimicrobial activity against E. coli was evaluated. UV-visible absorption spectroscopy was used to examine the reduction of silver ions in solution and the surface plasmon resonance of AgNPs. Scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), dynamic light scattering (DLS), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) were used to further characterize the nanoparticles. The crystalline nature of AgNPs was confirmed by XRD, and the purity and presence of elemental silver were determined by EDX. CL-AgNPs were observed to have a surface plasmon resonance of 425 nm, while CP-AgNPs had a surface plasmon resonance of 440 nm. CL-AgNPs had a significantly higher purity than CP-AgNPs. With a shorter nucleation time, the intensity of the UV-Vis spectrum was always higher in the case of CL-AgNPs, indicating a larger population of bioactive molecules available for CL-AgNPs synthesis. FTIR confirmed the presence of phenolic compounds in the leaf and pod extract, implying that water-soluble polyphenolic and flavonoid chemicals are responsible for nanoparticle reduction, capping, and stability. AgNPs generated from CL and CP extracts are polydispersed, with particle sizes of 10-110 nm and 20-680 nm, respectively, according to DLS. The corresponding zeta potentials measured are -2.7 mV for CL-AgNPs and -0.93 mV for CP-AgNPs. The zeta potential values suggest that the particles have long-term stability. Furthermore, CL-AgNPs outperformed CP-AgNPs in terms of antibacterial activity against Escherichia coli. CL-AgNPs were found to have a maximal inhibitory zone of 21 mm.

Aminoacyl chain translocation catalysed by a type II thioesterase domain in an unusual non-ribosomal peptide synthetase.

Non-Ribosomal Peptide Synthetases (NRPSs) assemble a diverse range of natural products with important applications in both medicine and agriculture. They consist of several multienzyme subunits that must interact with each other in a highly controlled manner to facilitate efficient chain transfer, thus ensuring biosynthetic fidelity. Several mechanisms for chain transfer are known for NRPSs, promoting structural diversity. Herein, we report the first biochemically characterized example of a type II thioesterase (TEII) domain capable of catalysing aminoacyl chain transfer between thiolation (T) domains on two separate NRPS subunits responsible for installation of a dehydrobutyrine moiety. Biochemical dissection of this process reveals the central role of the TEII-catalysed chain translocation event and expands the enzymatic scope of TEII domains beyond canonical (amino)acyl chain hydrolysis. The apparent co-evolution of the TEII domain with the NRPS subunits highlights a unique feature of this enzymatic cassette, which will undoubtedly find utility in biosynthetic engineering efforts.

Digyalipopeptide A, an antiparasitic cyclic peptide from the Ghanaian Bacillus sp. strain DE2B.

During the continued isolation of different bacteria from highly diverse, low human activity environments in Ghana and the subsequent characterization and biological activity studies of their secondary metabolites, we found both Gram-positive and Gram-negative Bacillus strains to be ubiquitous and widespread. One of such strains, the Ghanaian novel Bacillus sp. strain DE2B was isolated from rhizosphere soils collected from the Digya National Park in Ghana. Chromatographic purifications of the fermented culture extract of the strain DE2B, led to the isolation of a cyclic lipopeptide, digyalipopeptide A (1). Using 1D and 2D NMR data, mass spectrometry sequence tagging, advanced Marfey's analysis, and the GNPS molecular networking we solved the full structure of digyalipopeptide A (1). We found that compound 1 is a member of a somewhat homologous series of peptides produced as a mixture by the strain containing the same amino acid sequence in the cyclic peptide backbone but differing only by the length of aliphatic fatty acid side chains. When tested against Trypanosoma brucei subsp. brucei strain GUTat 3.1 and Leishmania donovani (Laveran and Mesnil) Ross (D10), digyalipopeptide A (1) gave IC50 values of 12.89 µM (suramin IC50 0.96 µM) and 4.85 µM (amphotericin B IC50 4.87 µM), respectively. Furthermore, digyalipopeptide A (1) produced IC50 values of 10.07 µM (ampicillin IC50 0.18 µM) and 10.01 µM (ampicillin IC50 1.53 µM) for Staphylococcus aureus and Shigella sonnei, respectively. The selectivity and toxicity profile of compound 1 was investigated using normal cell lines, macrophages RAW 264.7. When tested against normal macrophages, compound 1 gave an IC50 value of 71.32 µM. Selectivity indices (SI) were obtained by calculating the ratio of the IC50 in RAW 264.7 to the IC50 in the respective microbe and neglected parasite. In the presence of RAW 264.7 cell lines, compound 1 was particularly selective towards Leishmania donovani (Laveran and Mesnil) Ross (D10) with an SI value of 14.71. The bioactivity studies conducted confirm the role of these cyclic lipopeptides as defense chemicals in their natural environment and their ability to be biologically active across different species.

Chemical Derivatization and Characterization of Novel Antitrypanosomals for African Trypanosomiasis.

The search for novel antitrypanosomals and the investigation into their mode of action remain crucial due to the toxicity and resistance of commercially available antitrypanosomal drugs. In this study, two novel antitrypanosomals, tortodofuordioxamide (compound 2) and tortodofuorpyramide (compound 3), were chemically derived from the natural N-alkylamide tortozanthoxylamide (compound 1) through structural modification. The chemical structures of these compounds were confirmed through spectrometric and spectroscopic analysis, and their in vitro efficacy and possible mechanisms of action were, subsequently, investigated in Trypanosoma brucei (T. brucei), one of the causative species of African trypanosomiasis (AT). The novel compounds 2 and 3 displayed significant antitrypanosomal potencies in terms of half-maximal effective concentrations (EC50) and selectivity indices (SI) (compound 1, EC50 = 7.3 µM, SI = 29.5; compound 2, EC50 = 3.2 µM, SI = 91.3; compound 3, EC50 = 4.5 µM, SI = 69.9). Microscopic analysis indicated that at the EC50 values, the compounds resulted in the coiling and clumping of parasite subpopulations without significantly affecting the normal ratio of nuclei to kinetoplasts. In contrast to the animal antitrypanosomal drug diminazene, compounds 1, 2 and 3 exhibited antioxidant absorbance properties comparable to the standard antioxidant Trolox (Trolox, 0.11 A; diminazene, 0.50 A; compound 1, 0.10 A; compound 2, 0.09 A; compound 3, 0.11 A). The analysis of growth kinetics suggested that the compounds exhibited a relatively gradual but consistent growth inhibition of T. brucei at different concentrations. The results suggest that further pharmacological optimization of compounds 2 and 3 may facilitate their development into novel AT chemotherapy.

Dermacozine N, the First Natural Linear Pentacyclic Oxazinophenazine with UV-Vis Absorption Maxima in the Near Infrared Region, along with Dermacozines O and P Isolated from the Mariana Trench Sediment Strain Dermacoccus abyssi MT 1.1T.

Three dermacozines, dermacozines N-P (1-3), were isolated from the piezotolerant Actinomycete strain Dermacoccus abyssi MT 1.1T, which was isolated from a Mariana Trench sediment in 2006. Herein, we report the elucidation of their structures using a combination of 1D/2D NMR, LC-HRESI-MSn, UV-Visible, and IR spectroscopy. Further confirmation of the structures was achieved through the analysis of data from density functional theory (DFT)-UV-Visible spectral calculations and statistical analysis such as two tailed t-test, linear regression-, and multiple linear regression analysis applied to either solely experimental or to experimental and calculated 13C-NMR chemical shift data. Dermacozine N (1) bears a novel linear pentacyclic phenoxazine framework that has never been reported as a natural product. Dermacozine O (2) is a constitutional isomer of the known dermacozine F while dermacozine P (3) is 8-benzoyl-6-carbamoylphenazine-1-carboxylic acid. Dermacozine N (1) is unique among phenoxazines due to its near infrared (NIR) absorption maxima, which would make this compound an excellent candidate for research in biosensing chemistry, photodynamic therapy (PDT), opto-electronic applications, and metabolic mapping at the cellular level. Furthermore, dermacozine N (1) possesses weak cytotoxic activity against melanoma (A2058) and hepatocellular carcinoma cells (HepG2) with IC50 values of 51 and 38 µM, respectively.

Genomic scanning enabling discovery of a new antibacterial bicyclic carbamate-containing alkaloid.

Non-ribosomal peptides are a group of structurally diverse natural products with various important therapeutic and agrochemical applications. Bacterial pyrrolizidine alkaloids (PAs), containing a scaffold of two fused five-membered ring system with a nitrogen atom at the bridgehead, have been found to originate from a multidomain non-ribosomal peptide synthetase to generate indolizidine intermediates, followed by multistep oxidation, catalysed by single Bayer-Villiger (BV) enzymes, to yield PA scaffolds. Although bacterial PAs are rare in natural product inventory, bioinformatics analysis suggested that the biosynthetic gene clusters (BGCs) that are likely to be responsible for the production of PA-like metabolites are widely distributed in bacterial genomes. However, most of the strains containing PA-like BGCs are not deposited in the public domain, therefore preventing further assessment of the chemical spaces of this group of bioactive metabolites. Here, we report a genomic scanning strategy to assess the potential of PA metabolites production in our culture collection without prior knowledge of genome information. Among the strains tested, we found fifteen contain the key BV enzymes that are likely to be involved in the last step of PA ring formation. Subsequently one-strain-many-compound (OSMAC) method, supported by a combination of HR-MS, NMR, SMART 2.0 technology, and GNPS analysis, allowed identification and characterization of a new [5 + 7] heterobicyclic carbamate, legoncarbamate, together with five known PAs, bohemamine derivatives, from Streptomyces sp. CT37, a Ghanaian soil isolate. The absolute stereochemistry of legoncarbamate was determined by comparison of measured and calculated ECD spectra. Legoncarbamate displays antibacterial activity against E. coli ATCC 25922 with an MIC value of 3.1 µg/mL. Finally, a biosynthetic model of legoncarbamate and other bohemamines was proposed based on the knowledge we have gained so far.

Isolation and Antitrypanosomal Characterization of Furoquinoline and Oxylipin from Zanthoxylum zanthoxyloides.

In the absence of vaccines, there is a need for alternative sources of effective chemotherapy for African trypanosomiasis (AT). The increasing rate of resistance and toxicity of commercially available antitrypanosomal drugs also necessitates an investigation into the mode of action of new antitrypanosomals for AT. In this study, furoquinoline 4, 7, 8-trimethoxyfuro (2, 3-b) quinoline (compound 1) and oxylipin 9-oxo-10, 12-octadecadienoic acid (compound 2) were isolated from the plant species Zanthoxylum zanthoxyloides (Lam) Zepern and Timler (root), and their in vitro efficacy and mechanisms of action investigated in Trypanosomabrucei (T. brucei), the species responsible for AT. Both compounds resulted in a selectively significant growth inhibition of T. brucei (compound 1, half-maximal effective concentration EC50 = 1.7 µM, selectivity indices SI = 74.9; compound 2, EC50 = 1.2 µM, SI = 107.3). With regards to effect on the cell cycle phases of T. brucei, only compound 1 significantly arrested the second growth-mitotic (G2-M) phase progression even though G2-M and DNA replication (S) phase arrest resulted in the overall reduction of T. brucei cells in G0-G1 for both compounds. Moreover, both compounds resulted in the aggregation and distortion of the elongated slender morphology of T. brucei. Analysis of antioxidant potential revealed that at their minimum and maximum concentrations, the compounds exhibited significant oxidative activities in T. brucei (compound 1, 22.7 µM Trolox equivalent (TE), 221.2 µM TE; compound 2, 15.0 µM TE, 297.7 µM TE). Analysis of growth kinetics also showed that compound 1 exhibited a relatively consistent growth inhibition of T. brucei at different concentrations as compared to compound 2. The results suggest that compounds 1 and 2 are promising antitrypanosomals with the potential for further development into novel AT chemotherapy.

Antischistosomal, antionchocercal and antitrypanosomal potentials of some Ghanaian traditional medicines and their constituents.

BACKGROUND: Ghana is endemic for some neglected tropical diseases (NTDs) including schistosomiasis, onchocerciasis and lymphatic filariasis. The major intervention for these diseases is mass drug administration of a few repeatedly recycled drugs which is a cause for major concern due to reduced efficacy of the drugs and the emergence of drug resistance. Evidently, new treatments are needed urgently. Medicinal plants, on the other hand, have a reputable history as important sources of potent therapeutic agents in the treatment of various diseases among African populations, Ghana inclusively, and provide very useful starting points for the discovery of much-needed new or alternative drugs. METHODOLOGY/PRINCIPAL FINDINGS: In this study, extracts of fifteen traditional medicines used for treating various NTDs in local communities were screened in vitro for efficacy against schistosomiasis, onchocerciasis and African trypanosomiasis. Two extracts, NTD-B4-DCM and NTD-B7-DCM, prepared from traditional medicines used to treat schistosomiasis, displayed the highest activity (IC50 = 30.5 µg/mL and 30.8 µg/mL, respectively) against Schistosoma mansoni adult worms. NTD-B2-DCM, also obtained from an antischistosomal remedy, was the most active against female and male adult Onchocera ochengi worms (IC50 = 76.2 µg/mL and 76.7 µg/mL, respectively). Antitrypanosomal assay of the extracts against Trypanosoma brucei brucei gave the most promising results (IC50 = 5.63 µg/mL to 18.71 µg/mL). Incidentally, NTD-B4-DCM and NTD-B2-DCM, also exhibited the greatest antitrypanosomal activities (IC50 = 5.63 µg/mL and 7.12 µg/mL, respectively). Following the favourable outcome of the antitrypanosomal screening, this assay was selected for bioactivity-guided fractionation. NTD-B4-DCM, the most active extract, was fractionated and subsequent isolation of bioactive constituents led to an eupatoriochromene-rich oil (42.6%) which was 1.3-fold (IC50 <0.0977 µg/mL) more active than the standard antitrypanosomal drug, diminazene aceturate (IC50 = 0.13 µg/mL). CONCLUSION/SIGNIFICANCE: These findings justify the use of traditional medicines and demonstrate their prospects towards NTDs drug discovery.

Discovery of New Antibacterial Accramycins from a Genetic Variant of the Soil Bacterium, Streptomyces sp. MA37.

Continued mining of natural products from the strain Streptomyces sp. MA37 in our laboratory led to the discovery of a minor specialized metabolite (SM) called accramycin A. Owing to its low yield (0.2 mg/L) in the wild type strain, we investigated the roles of regulatory genes in the corresponding biosynthetic gene cluster (acc BGC) through gene inactivation with the aim of improving the titer of this compound. One of the resulting mutants (∆accJ) dramatically upregulated the production of accramycin A 1 by 330-fold (66 mg/L). Furthermore, ten new metabolites, accramycins B-K 2-11, were discovered, together with two known compounds, naphthacemycin B112 and fasamycin C 13 from the mutant extract. This suggested that accJ, annotated as multiple antibiotic resistance regulator (MarR), is a negative regulator gene in the accramycin biosynthesis. Compounds 1-13 inhibited the Gram-positive pathogens (Staphylococcus aureus, Enterococcus faecalis) and clinical isolates Enterococcus faecium (K59-68 and K60-39) and Staphylococcus haemolyticus with minimal inhibitory concentration (MIC) values in the range of 1.5-12.5 µg/mL. Remarkably, compounds 1-13 displayed superior activity against K60-39 (MIC = 3.1-6.3 µg/mL) compared to ampicillin (MIC = 25 µg/mL), and offered promising potential for the development of accramycin-based antibiotics that target multidrug-resistant Enterococcus clinical isolates. Our results highlight the importance of identifying the roles of regulatory genes in natural product discovery.

Identification of 5-Fluoro-5-Deoxy-Ribulose as a Shunt Fluorometabolite in Streptomyces sp. MA37.

A fluorometabolite, 5-fluoro-5-deoxy-D-ribulose (5-FDRul), from the culture broth of the soil bacterium Streptomyces sp. MA37, was identified through a combination of genetic manipulation, chemo-enzymatic synthesis and NMR comparison. Although 5-FDRul has been chemically synthesized before, it was not an intermediate or a shunt product in previous studies of fluorometalism in S. cattleya. Our study of MA37 demonstrates that 5-FDRul is a naturally occurring fluorometabolite, rendering it a new addition to this rare collection of natural products. The genetic inactivation of key biosynthetic genes involved in the fluorometabolisms in MA37 resulted in the increased accumulation of unidentified fluorometabolites as observed from 19F-NMR spectral comparison among the wild type (WT) of MA37 and the mutated variants, providing evidence of the presence of other new biosynthetic enzymes involved in the fluorometabolite pathway in MA37.

Targeted Isolation of Indole Alkaloids from Streptomyces sp. CT37.

Four compounds (1-4) were isolated from the extracts of Streptomyces sp. CT37 using bioassay in conjunction with mass spectrometric molecular networking (MN) driven isolation. Their complete structures were established by high-resolution electrospray ionization mass spectrometry (HR-ESIMS), and 1D and 2D nuclear magnetic resonance (NMR) data. Legonimide 1 was identified as a new alkaloid containing a rare linear imide motif in its structure, while compounds 2-4 were already known and their structures were elucidated as 1H-indole-3-carbaldehyde, actinopolymorphol B, (2R,3R)-1-phenylbutane-2,3-diol, respectively. The biosynthetic pathways of 1-4 were proposed based on the reported biogenesis of indole alkaloids in literature. Bioactivity tests for 1 and 2 revealed moderate growth inhibition activity against Candida albicans ATCC 10231 with MIC95 values of 21.54 µg/mL and 11.47 µg/mL, respectively.

An unusual metal-bound 4-fluorothreonine transaldolase from Streptomyces sp. MA37 catalyses promiscuous transaldol reactions.

ß-Hydroxy-α-amino acids (ßH-AAs) are key components of many bioactive molecules as well as exist as specialised metabolites. Among these ßH-AAs, 4-fluorothreonine (4-FT) is the only naturally occurring fluorinated AA discovered thus far. Here we report overexpression and biochemical characterisation of 4-fluorothreonine transaldolase from Streptomyces sp. MA37 (FTaseMA), a homologue of FTase previously identified in the biosynthesis of 4-FT in S. cattleya. FTaseMA displays considerable substrate plasticity to generate 4-FT as well as other ß-hydroxy-α-amino acids with various functionalities at C4 position, giving the prospect of new chemo-enzymatic applications. The enzyme has a hybrid of two catalytic domains, serine hydroxymethyltransferase (S) and aldolase (A). Site-directed mutagenesis allowed the identification of the key residues of FTases, suggesting that the active site of A domain has a historical reminiscent feature in metal-dependent aldolases. Elemental analysis demonstrated that FTaseMA is indeed a Zn2+-dependent enzyme, the first example of pyridoxal phosphate (PLP) enzyme family fused with a metal-binding domain carrying out a distinct catalytic role. Finally, FTaseMA showed divergent evolutionary origin with other PLP dependent enzymes.

Characterization of the promiscuous N-acyl CoA transferase, LgoC, in legonoxamine biosynthesis.

More than 500 siderophores are known to date, but only three were identified to be aryl-containing hydroxamate siderophores, legonoxamines A and B from Streptomyces sp. MA37, and aryl ferrioxamine 2 from Micrococcus luteus KLE1011. Siderophores are produced by microorganisms to scavenge iron from the environment, thereby making this essential metal nutrient available to the microbe. We demonstrate here that LgoC from MA37 is responsible for the key aryl-hydroxamate forming step in legonoxamine biosynthesis. Biochemical characterization established that LgoC displays considerable promiscuity for the acylation between N-hydroxy-cadaverine and SNAC (N-acetylcysteamines) thioester derivatives.

A Co-Culturing Approach Enables Discovery and Biosynthesis of a Bioactive Indole Alkaloid Metabolite.

Whole-genome sequence data of the genus Streptomyces have shown a far greater chemical diversity of metabolites than what have been discovered under typical laboratory fermentation conditions. In our previous natural product discovery efforts on Streptomyces sp. MA37, a bacterium isolated from the rhizosphere soil sample in Legon, Ghana, we discovered a handful of specialised metabolites from this talented strain. However, analysis of the draft genome of MA37 suggested that most of the encoded biosynthetic gene clusters (BGCs) remained cryptic or silent, and only a small fraction of BGCs for the production of specialised metabolites were expressed when cultured in our laboratory conditions. In order to induce the expression of the seemingly silent BGCs, we have carried out a co-culture experiment by growing the MA37 strain with the Gram-negative bacterium Pseudomonas sp. in a co-culture chamber that allows co-fermentation of two microorganisms with no direct contact but allows exchange of nutrients, metabolites, and other chemical cues. This co-culture approach led to the upregulation of several metabolites that were not previously observed in the monocultures of each strain. Moreover, the co-culture induced the expression of the cryptic indole alkaloid BGC in MA37 and led to the characterization of the known indolocarbazole alkaloid, BE-13793C 1. Neither bacterium produced compound 1 when cultured alone. The structure of 1 was elucidated by Nuclear Magnetic Resonance (NMR), mass spectrometry analyses and comparison of experimental with literature data. A putative biosynthetic pathway of 1 was proposed. Furthermore, BE-13793C 1 showed strong anti-proliferative activity against HT-29 (ATCC HTB-38) cells but no toxic effect to normal lung (ATCC CCL-171) cells. To the best of our knowledge, this is the first report for the activity of 1 against HT-29. No significant antimicrobial and anti-trypanosomal activities for 1 were observed. This research provides a solid foundation for the fact that a co-culture approach paves the way for increasing the chemical diversity of strain MA37. Further characterization of other upregulated metabolites in this strain is currently ongoing in our laboratory.