Discovery of the Lipopeptides Albubactins A-H from Streptomyces albidoflavus RKJM0023 via Chemical Elicitation with Rhamnolipids and Synthesis of Albubactin A.

The marine tunicate-derived Streptomyces albidoflavus RKJM0023 was cultured in the presence of a rhamnolipid mixture in an effort to elicit the production of silent natural products. MS/MS-based molecular networking analysis enhanced with nonparametric statistics highlighted the upregulation of a molecular cluster (Kruskal-Wallis p = 1.6 e-6 for 1) in which no MS/MS features had library matches. Targeted isolation of these features resulted in the discovery of nine new N-acylated lipopeptides, albubactins A-H (1-8) each containing a unique glutamine tripeptide and a C-terminal ethyl ester moiety. Three related albubactin acids A-C (9-11) lacking the ethyl ester were also identified. NMR spectroscopy and UPLC-HR-ESI-MS/MS demonstrated that the albubactins were obtained as mixtures that shared a common m/z and differed only in their acylated terminal groups. Due to the complex spectroscopic elucidation with many overlapping shifts, a total synthesis of albubactin A (1) was completed and used to determine the absolute configuration of the new albubactins.

Discovery of Acyl-Surugamide A2 from Marine Streptomyces albidoflavus RKJM-0023-A New Cyclic Nonribosomal Peptide Containing an N-ε-acetyl-L-lysine Residue.

We report the discovery of a novel cyclic nonribosomal peptide (NRP), acyl-surugamide A2, from a marine-derived Streptomyces albidoflavus RKJM-0023 (CP133227). The structure of acyl-surugamide A2 was elucidated using a combination of NMR spectroscopy, MS2 fragmentation analysis, and comparative analysis of the sur biosynthetic gene cluster. Acyl-surugamide A2 contains all eight core amino acids of surugamide A, with a modified N-ε-acetyl-L-lysine residue. Our study highlights the potential of marine Streptomyces strains to produce novel natural products with potential therapeutic applications. The structure of cyclic peptides can be solved using MS2 spectra and analysis of their biosynthetic gene clusters.

Discovery of Levesquamide B through Global Natural Product Social Molecular Networking.

Levesquamide A is an isothiazolinone-containing anti-tubercular natural product isolated from Streptomyces sp. RKND-216. Through the use of Global Natural Product Social Molecular Networking (GNPS), additional members of the levesquamide family were identified (B-G). Levesquamide B is a glycosylated analogue, isolated and structurally elucidated via spectroscopical techniques along with the putative structures of levesquamide C and D. For masses relating to the additional three levesquamides (E-G), their complete structures remain ambiguous.

Synthesis of (+)-(R)-Tiruchanduramine.

The absolute stereochemistry of the marine alkaloid (+)-(R)-tiruchanduramine was established via a convergent total synthesis in six steps and 15.5% overall yield from Fmoc-D-Dab(Boc)-OH.

SynBio-SynChem Approaches to Diversifying the Pacidamycins through the Exploitation of an Observed Pictet-Spengler Reaction.

A nonenzymatic Pictet-Spengler reaction has been postulated to give rise to a subset of naturally occurring uridyl peptide antibiotics (UPAs). Here, using a combination of strain engineering and synthetic chemistry, we demonstrate that Pictet-Spengler chemistry may be employed to generate even greater diversity in the UPAs. We use an engineered strain to afford access to meta-tyrosine containing pacidamycin 4. Pictet-Spengler diversification of this compound using a small series of aryl-aldehydes was achieved with some derivatives affording remarkable diastereomeric control.

Development of a microbe domestication pod (MD Pod) for in situ cultivation of micro-encapsulated marine bacteria.

Microbial marine natural products hold significant potential for the discovery of new bioactive therapeutics such as antibiotics. Unfortunately, this discovery is hindered by the inability to culture the majority of microbes using traditional laboratory approaches. While many new methods have been developed to increase cultivability, a high-throughput in situ incubation chamber capable of simultaneously isolating individual microbes while allowing cellular communication has not previously been reported. Development of such a device would expedite the discovery of new microbial taxa and, thus, facilitate access to their associated natural products. In this study, this concept is achieved by the development of a new device termed by the authors as the microbe domestication (MD) Pod. The MD Pod enables single-cell cultivation by isolating marine bacterial cells in agarose microbeads produced using microfluidics, while allowing potential transmission of chemical signals between cells during in situ incubation in a chamber, or "Pod," that is deployed in the environment. The design of the MD Pod was optimized to ensure the use of biocompatible materials, allow for simple assembly in a field setting, and maintain sterility throughout incubation. The encapsulation process was designed to ensure that the viability of marine sediment bacteria was not adversely impacted by the encapsulation process. The process was validated using representative bacteria isolated from temperate marine sediment samples: Marinomonas polaris, Psychrobacter aquimaris, and Bacillus licheniformis. The overall process appeared to promote metabolic activity of most representative species. Thus, microfluidic encapsulation of marine bacteria and subsequent in situ incubation in the MD Pod is expected to accelerate marine natural products discovery by increasing the cultivability of marine bacteria.

Auyuittuqamides A-D, Cyclic Decapeptides from Sesquicillium microsporum RKAG 186 Isolated from Frobisher Bay Sediment.

Four new cyclic decapeptides, auyuittuqamides A-D (1-4), were obtained from Sesquicillium microsporum RKAG 186 obtained from marine sediment collected from the intertidal zone of Frobisher Bay, Nunavut, Canada. The structures of the compounds were elucidated by NMR spectroscopy and tandem mass spectrometry. The absolute configurations of the amino acids were determined using Marfey's method.

Heck Diversification of Indole-Based Substrates under Aqueous Conditions: From Indoles to Unprotected Halo-tryptophans and Halo-tryptophans in Natural Product Derivatives.

The blending of synthetic chemistry with biosynthetic processes provides a powerful approach to synthesis. Biosynthetic halogenation and synthetic cross-coupling have great potential to be used together, for small molecule generation, access to natural product analogues and as a tool for chemical biology. However, to enable enhanced generality of this approach, further synthetic tools are needed. Though considerable research has been invested in the diversification of phenylalanine and tyrosine, functionalisation of tryptophans thorough cross-coupling has been largely neglected. Tryptophan is a key residue in many biologically active natural products and peptides; in proteins it is key to fluorescence and dominates protein folding. To this end, we have explored the Heck cross-coupling of halo-indoles and halo-tryptophans in water, showing broad reaction scope. We have demonstrated the ability to use this methodology in the functionalisation of a brominated antibiotic (bromo-pacidamycin), as well as a marine sponge metabolite, barettin.

Microencapsulation and in situ incubation methodology for the cultivation of marine bacteria.

Environmental microorganisms are important sources of biotechnology innovations; however, the discovery process is hampered by the inability to culture the overwhelming majority of microbes. To drive the discovery of new biotechnology products from previously unculturable microbes, several methods such as modification of media composition, incubation conditions, single-cell isolation, and in situ incubation, have been employed to improve microbial recovery from environmental samples. To improve microbial recovery, we examined the effect of microencapsulation followed by in situ incubation on the abundance, viability, and diversity of bacteria recovered from marine sediment. Bacteria from marine sediment samples were resuspended or encapsulated in agarose and half of each sample was directly plated on agar and the other half inserted into modified Slyde-A-Lyzer™ dialysis cassettes. The cassettes were incubated in their natural environment (in situ) for a week, after which they were retrieved, and the contents plated. Colony counts indicated that bacterial abundance increased during in situ incubation and that cell density was significantly higher in cassettes containing non-encapsulated sediment bacteria. Assessment of viability indicated that a higher proportion of cells in encapsulated samples were viable at the end of the incubation period, suggesting that agarose encapsulation promoted higher cell viability during in situ incubation. One hundred and 46 isolates were purified from the study (32-38 from each treatment) to assess the effect of the four treatments on cultivable bacterial diversity. In total, 58 operational taxonomic units (OTUs) were identified using a 99% 16S rRNA gene sequence identity threshold. The results indicated that encapsulation recovered greater bacterial diversity from the sediment than simple resuspension (41 vs. 31 OTUs, respectively). While the cultivable bacterial diversity decreased by 43%-48% after in situ incubation, difficult-to-culture (Verrucomicrobia) and obligate marine (Pseudoalteromonas) taxa were only recovered after in situ incubation. These results suggest that agarose encapsulation coupled with in situ incubation in commercially available, low-cost, diffusion chambers facilitates the cultivation and improved recovery of bacteria from marine sediments. This study provides another tool that microbiologists can use to access microbial dark matter for environmental, biotechnology bioprospecting.

Microfabrication of a micron-scale microbial-domestication pod for in situ cultivation of marine bacteria.

Through the hyphenation of microfabrication, microfluidics and microbiology, we report the development of a µMicrobial-Domestication Pod (µMD Pod). This in situ cultivation device facilitates cell signaling from neighbouring species and interactions with environmental stimuli for marine bacterial growth to overcome current barriers faced by standard laboratory cultivation methods.

Draft Genome Sequence of Streptomyces sp. Strain RKCA744, Isolated from the Arauca River, Colombia.

Streptomyces sp. strain RKCA744 was isolated from sediment collected from the Arauca River, Colombia.

Development of N,O-Carboxymethyl Chitosan-Starch Biomaterial Inks for 3D Printed Wound Dressing Applications.

In this paper, a novel hybrid biomaterial ink consisting of two water-soluble polymers is investigated: starch and N,O-carboxymethyl chitosan (NOCC). The biomaterial ink is used to fabricate controlled release biodegradable wound dressing scaffolds via a novel low-temperature solvent (organic)-free 3D printing technique. NOCC is a variant of chitosan with a high degradation rate that can lead to an immediate release of the drugs, and starch, on the other hand, is used to alter degradation and drug release characteristics of the biomaterial. Mupirocin, a topical anti-infective, is incorporated into the biomaterial inks. Different biomaterial inks in terms of NOCC to starch ratio are prepared and characterized. Printability and rheology of the samples are investigated, and the release of mupirocin over time is quantified. The efficacy of the developed 3D printed wound dressings against Staphylococcus aureus is examined through disk diffusion assays. Increasing NOCC accelerated the release of the drug from the scaffold and led to larger zones of inhibition in the early hours of the in vitro tests; this phenomenon is correlated to the enhanced hydrophilicity of NOCC-dominated scaffolds. The drug release and the zone of inhibition are controlled by altering starch to NOCC ratio in the biomaterial ink.

Development of 3D Printed Drug-Eluting Scaffolds for Preventing Piercing Infection.

Herein, novel drug-eluting, bio-absorbable scaffold intended to cover piercing studs is introduced. This "biopierce" will stay in human tissue following piercing, and will slowly release an antimicrobial agent to prevent infection while the wound heals. Nearly 20% of all piercings lead to local infection. Therefore, it is imperative to develop alternative methods of piercing aftercare to prevent infection. Biopierces were made using mupirocin loaded poly-lactic-co-glycolic acid (PLGA) biomaterial ink, and a low-temperature 3D printing technique was used to fabricate the biopierces. Proton nuclear magnetic resonance (1H NMR) spectroscopy was used to confirm the complete removal of the solvent, and liquid chromatography high-resolution mass spectrometry (LC-HRMS) was used to confirm the structural integrity of mupirocin and to quantify the amount of the released drug over time. The efficacy of the biopierces against Staphylococcus aureus, one of the most common piercing-site pathogens, was confirmed over two weeks using in vitro antimicrobial susceptibility testing.

Buchwald Hartwig diversification of unprotected halotryptophans, halotryptophan containing tripeptides and the natural product barettin in aqueous conditions.

Blending synthetic biology and synthetic chemistry represents a powerful approach to diversity complex molecules. To further enable this, compatible synthetic tools are needed. We report the first Buchwald Hartwig amination reactions with unprotected halotryptophans under aqueous conditions and demonstrate this methodology is applicable also to the modification of unprotected tripeptides and the natural product barettin.

Living GenoChemetics by hyphenating synthetic biology and synthetic chemistry in vivo.

Marrying synthetic biology with synthetic chemistry provides a powerful approach toward natural product diversification, combining the best of both worlds: expediency and synthetic capability of biogenic pathways and chemical diversity enabled by organic synthesis. Biosynthetic pathway engineering can be employed to insert a chemically orthogonal tag into a complex natural scaffold affording the possibility of site-selective modification without employing protecting group strategies. Here we show that, by installing a sufficiently reactive handle (e.g., a C-Br bond) and developing compatible mild aqueous chemistries, synchronous biosynthesis of the tagged metabolite and its subsequent chemical modification in living culture can be achieved. This approach can potentially enable many new applications: for example, assay of directed evolution of enzymes catalyzing halo-metabolite biosynthesis in living cells or generating and following the fate of tagged metabolites and biomolecules in living systems. We report synthetic biological access to new-to-nature bromo-metabolites and the concomitant biorthogonal cross-coupling of halo-metabolites in living cultures.Coupling synthetic biology and chemical reactions in cells is a challenging task. The authors engineer bacteria capable of generating bromo-metabolites, develop a mild Suzuki-Miyaura cross-coupling reaction compatible with cell growth and carry out the cross-coupling chemistry in live cell cultures.

Synthetic analogues of cyanobacterial alkaloid cylindrospermopsin and their toxicological activity.

Cylindrospermopsin (CYN) is a naturally occurring alkaloid produced by a variety of cyanobacteria and known to induce oxidative stress-mediated toxicity in eukaryotic cells. Despite extensive research on the mechanism of CYN toxicity, an understanding of the structural features responsible for this toxicity and the mechanism by which it can enter the cell are still not clear. It was established that the presence of both the uracil and guanidine groups is essential in biological activity of CYN whilst not much is known in this regard on the role of tether that separates them and the attached hydroxyl group. Therefore, in the present study we have prepared three synthetic analogues possessing uracil and guanidine groups separated by a variable length tether (4-6 carbons) and containing a hydroxyl function in a position orientation to CYN, together with a tetracyclic analogue of CYN lacking the hydroxyl group at C-7. The toxicity of these compounds was then compared with CYN and guanidinoacetate (GAA; the primary substrate in CYN biosynthesis) in an in vitro model using human neutrophils isolated from healthy subjects. The lowest activity measured by means of reactive oxygen species generation, lipid peroxidation and cell death was observed for GAA and the tetracyclic analogue. The greatest toxicity was found in an analogue with a 6-carbon tether, but all three analogues and CYN caused rapid onset of redox imbalance. These results add to the general understanding of CYN toxicity and preliminary findings suggest that the -OH group at C-7 may be significant for the cellular transport of CYN and/or be involved in its toxic activity inside the cell, a hypothesis which requires further testing.