Exfoliation of 2D Metal-Organic Frameworks: toward Advanced Scalable Materials for Optical Sensing.

Two-dimensional metal-organic frameworks (MOFs) occupy a special place among the large family of functional 2D materials. Even at a monolayer level, 2D MOFs exhibit unique sensing, separation, catalytic, electronic, and conductive properties due to the combination of porosity and organo-inorganic nature. However, lab-to-fab transfer for 2D MOF layers faces the challenge of their scalability, limited by weak interactions between the organic and inorganic building blocks. Here, comparing three top-down approaches to fabricate 2D MOF layers (sonication, freeze-thaw, and mechanical exfoliation), The technological criteria have established for creation of the layers of the thickness up to 1 nm with a record aspect ratio up to 2*10^4:1. The freezing-thaw and mechanical exfoliation are the most optimal approaches; wherein the rate and manufacturability of the mechanical exfoliation rivaling the greatest scalability of 2D MOF layers obtained by freezing-thaw (21300:1 vs 1330:1 aspect ratio), leaving the sonication approach behind (with a record 900:1 aspect ratio) have discovered. The high quality 2D MOF layers with a record aspect ratio demonstrate unique optical sensitivity to solvents of a varied polarity, which opens the way to fabricate scalable and freestanding 2D MOF-based atomically thin chemo-optical sensors by industry-oriented approach.

Effect of Collagen Matrix on Doxorubicin Distribution and Cancer Cells' Response to Treatment in 3D Tumor Model.

The extracellular matrix (ECM) plays an important role in regulation of many aspects of tumor growth and response to therapies. However, the specifics of the interaction of chemotherapeutic agents with cancer cells in the presence of collagen, the major component of ECM, is still poorly investigated. In this study, we explored distribution of doxorubicin (DOX) and its effects on cancer cells' metabolism in the presence of collagen with different structures in 3D models. For this, a combination of second harmonic generation imaging of collagen and multiphoton fluorescence microscopy of DOX, and metabolic cofactor NAD(P)H was used. It was found that collagen slowed down the diffusion of DOX and thus decreased the cellular drug uptake. Besides nuclei, DOX also targeted mitochondria leading to inhibition of oxidative phosphorylation, which was more pronounced in the cells growing in the absence of collagen. As a result, the cells in collagen displayed better viability upon treatment with DOX. Taken together, our data illustrate that tumor collagen contributes to heterogeneous and sub-optimal response to DOX and highlight the challenges in improving drug delivery and efficacy.

Microfluidic synthesis of optically responsive materials for nano- and biophotonics.

Recently, nanomaterials demonstrating optical response under illumination, the so-called optically responsive nanoparticles (NPs), have found their broad application as optical switchers, gas adsorbents, data storage devices, and optical and biological sensors. Unique optical properties of such nanomaterials are strongly related to their chemical composition, geometrical parameters and morphology. Microfluidic approaches for NPs' synthesis allow overcoming the known critical stages in conventional synthesis of NPs due to a high rate of heat/mass transfer and precise regulation of synthesis conditions, which results in reproducible synthesis outcomes with the desired physico-chemical properties. Here, we review the recent advances in microfluidic approach for synthesis of optically responsive nanomaterials (plasmonic, photoluminescent, shape-changeable NPs), highlighting the general background of microfluidics, common considerations in the design of microfluidic chips (MFCs), and theoretical models of the NPs' formation mechanisms. Comparative analysis of microfluidic synthesis with conventional synthesis methods is provided further, along with the recent applications of optically responsive NPs in nano- and biophotonics.

Toxicological Analysis of Hepatocytes Using FLIM Technique: In Vitro versus Ex Vivo Models.

The search for new criteria indicating acute or chronic pathological processes resulting from exposure to toxic agents, testing of drugs for potential hepatotoxicity, and fundamental study of the mechanisms of hepatotoxicity at a molecular level still represents a challenging issue that requires the selection of adequate research models and tools. Microfluidic chips (MFCs) offer a promising in vitro model for express analysis and are easy to implement. However, to obtain comprehensive information, more complex models are needed. A fundamentally new label-free approach for studying liver pathology is fluorescence-lifetime imaging microscopy (FLIM). We obtained FLIM data on both the free and bound forms of NAD(P)H, which is associated with different metabolic pathways. In clinical cases, liver pathology resulting from overdoses is most often as a result of acetaminophen (APAP) or alcohol (ethanol). Therefore, we have studied and compared the metabolic state of hepatocytes in various experimental models of APAP and ethanol hepatotoxicity. We have determined the potential diagnostic criteria including the pathologically altered metabolism of the hepatocytes in the early stages of toxic damage, including pronounced changes in the contribution from the bound form of NAD(P)H. In contrast to the MFCs, the changes in the metabolic state of hepatocytes in the ex vivo models are, to a greater extent, associated with compensatory processes. Thus, MFCs in combination with FLIM can be applied as an effective tool set for the express modeling and diagnosis of hepatotoxicity in clinics.

Polyketide Bioderivatization Using the Promiscuous Acyltransferase KirCII.

During polyketide biosynthesis, acyltransferases (ATs) are the essential gatekeepers which provide the assembly lines with precursors and thus contribute greatly to structural diversity. Previously, we demonstrated that the discrete AT KirCII from the kirromycin antibiotic pathway accesses nonmalonate extender units. Here, we exploit the promiscuity of KirCII to generate new kirromycins with allyl- and propargyl-side chains in vivo, the latter were utilized as educts for further modification by "click" chemistry.

Inversion of Extender Unit Selectivity in the Erythromycin Polyketide Synthase by Acyltransferase Domain Engineering.

Acyltransferase (AT) domains of polyketide synthases (PKSs) select extender units for incorporation into polyketides and dictate large portions of the structures of clinically relevant natural products. Accordingly, there is significant interest in engineering the substrate specificity of PKS ATs in order to site-selectively manipulate polyketide structure. However, previous attempts to engineer ATs have yielded mutant PKSs with relaxed extender unit specificity, rather than an inversion of selectivity from one substrate to another. Here, by directly screening the extender unit selectivity of mutants from active site saturation libraries of an AT from the prototypical PKS, 6-deoxyerythronolide B synthase, a set of single amino acid substitutions was discovered that dramatically impact the selectivity of the PKS with only modest reductions of product yields. One particular substitution (Tyr189Arg) inverted the selectivity of the wild-type PKS from its natural substrate toward a non-natural alkynyl-modified extender unit while maintaining more than twice the activity of the wild-type PKS with its natural substrate. The strategy and mutations described herein form a platform for combinatorial biosynthesis of site-selectively modified polyketide analogues that are modified with non-natural and non-native chemical functionality.

Biological conversion of gaseous alkenes to liquid chemicals.

Industrial gas-to-liquid (GTL) technologies are well developed. They generally employ syngas, require complex infrastructure, and need high capital investment to be economically viable. Alternatively, biological conversion has the potential to be more efficient, and easily deployed to remote areas on relatively small scales for the utilization of otherwise stranded resources. The present study demonstrates a novel biological GTL process in which engineered Escherichia coli converts C2-C4 gaseous alkenes into liquid diols. Diols are versatile industrially important chemicals, used routinely as antifreeze agents, polymer precursors amongst many other applications. Heterologous co-expression of a monooxygenase and an epoxide hydrolase in E. coli allows whole cell conversion of C2-C4 alkenes for the formation of ethylene glycol, 1,2-propanediol, 1,2-butanediol, and 2,3-butanediol at ambient temperature and pressure in one pot. Increasing intracellular NADH supply via addition of formate and a formate dehydrogenase increases ethylene glycol production titers, resulting in an improved productivity of 9mg/L/h and a final titer of 250mg/L. This represents a novel biological method for GTL conversion of alkenes to industrially valuable diols.

Evaluating nonpolar surface area and liquid chromatography/mass spectrometry response: an application for site occupancy measurements for enzyme intermediates in polyketide biosynthesis.

RATIONALE: Site occupancy measurements using liquid chromatography/mass spectrometry (LC/MS) are reported throughout the literature. However, site occupancy quantification suffers from ionization bias between modified and unmodified peptides containing the active site. In this study, we explore the MS signal as a function of nonpolar surface area (NPSA) in order to better understand this bias in electrospray response. The correlation between hydrophobicity and LC/MS response was evaluated and applied to study enzyme intermediates in polyketide synthases. METHODS: Site occupancy methods were developed to study acyltransferase activity. To further evaluate these methods, several standard peptides containing one cysteine residue were modified with alkylation reagents of increasing hydrophobicity to study the MS signal as a function of NPSA. RESULTS: A consistent trend in MS response was observed which is dependent on the NPSA of the analyte. An optimal NPSA zone was observed for the peptides studied. CONCLUSIONS: Nonpolar surface area can be used as metric to determine relative LC/MS response for peptides and evaluate site occupancy measurements.

Promiscuity of a modular polyketide synthase towards natural and non-natural extender units.

Combinatorial biosynthesis approaches that involve modular type I polyketide synthases (PKSs) are proven strategies for the synthesis of polyketides. In general however, such strategies are usually limited in scope and utility due to the restricted substrate specificity of polyketide biosynthetic machinery. Herein, a panel of chemo-enzymatically synthesized acyl-CoA's was used to probe the promiscuity of a polyketide synthase. Promiscuity determinants were dissected, revealing that the KS is remarkably tolerant to a diverse array of extender units, while the AT likely discriminates between extender units that are native to the producing organism. Our data provides a clear blueprint for future enzyme engineering efforts, and sets the stage for harnessing extender unit promiscuity by employing various in vivo polyketide diversification strategies.

Poly specific trans-acyltransferase machinery revealed via engineered acyl-CoA synthetases.

Polyketide synthases construct polyketides with diverse structures and biological activities via the condensation of extender units and acyl thioesters. Although a growing body of evidence suggests that polyketide synthases might be tolerant to non-natural extender units, in vitro and in vivo studies aimed at probing and utilizing polyketide synthase specificity are severely limited to only a small number of extender units, owing to the lack of synthetic routes to a broad variety of acyl-CoA extender units. Here, we report the construction of promiscuous malonyl-CoA synthetase variants that can be used to synthesize a broad range of malonyl-CoA extender units substituted at the C2-position, several of which contain handles for chemoselective ligation and are not found in natural biosynthetic systems. We highlighted utility of these enzymes by probing the acyl-CoA specificity of several trans-acyltransferases, leading to the unprecedented discovery of poly specificity toward non-natural extender units, several of which are not found in naturally occurring biosynthetic pathways. These results reveal that polyketide biosynthetic machinery might be more tolerant to non-natural substrates than previously established, and that mutant synthetases are valuable tools for probing the specificity of biosynthetic machinery. Our data suggest new synthetic biology strategies for harnessing this promiscuity and enabling the regioselective modification of polyketides.

A high-throughput screen for directed evolution of the natural product sulfotransferase LipB.

In this article, the authors describe a colorimetric, high-throughput assay suitable for optimizing the activity of the recently discovered sulfotransferase LipB, by directed evolution. Crucially, LipB uses para-nitrophenol sulfate as donor in the sulfation of the nucleoside antibiotic liposidomycin B-I and other acceptor surrogates. Thus, using a robotic liquid-handling device, crude cell extracts were prepared from an Escherichia coli strain that overproduced LipB in wells of a microplate, and production of para-nitrophenol at 405 nm was monitored spectrophotometrically. Enzyme activity could be detected only in the presence of both LipB substrates and overexpressed LipB. The screen displays a suitable standard deviation for directed evolution and importantly is not limited to the natural desulfo-liposidomycin acceptor. The authors plan to use the screen to identify LipB variants with altered acceptor specificity and promiscuity for use in sulfation of natural products and other small-molecule therapeutics.

Mutant malonyl-CoA synthetases with altered specificity for polyketide synthase extender unit generation.

Tailoring guide: We have used structure-guided saturation mutagenesis followed by colorimetric screening to identify mutant malonyl-CoA synthetases with altered substrate specificity. One particular mutant displayed a 240-fold shift in specificity (see graphic). These mutant enzymes will be useful tools for providing extender units to probe the activity of polyketide synthases.