- Associations of P2RX7 Functional Diplotypes with Localized Aggressive Periodontitis. [Journal Article]
- JCJDR Clin Trans Res 2019 Jul 18; :2380084419863789
- CONCLUSIONS: This study detected an association between P2RX7 functional diplotypes and in vitro immune response of whole blood from subjects with LAP. In addition, we found that inhibition of the activated P2X7 receptor leads to increased P2RX7 mRNA levels, suggesting a feedback loop ( ClinicalTrials.gov NCT01330719).
- Site-specific Incorporation of a Dithiolane Containing Amino Acid into Proteins. [Journal Article]
- BCBioconjug Chem 2019 Jul 18
- We have genetically encoded a dithiolane containing amino acid (dtF) in Escherichia coli (E. coli) using a polyspecific aminoacyl-tRNA synthetase (aaRS)/amber suppressor tRNA pair. To demonstrate the…
We have genetically encoded a dithiolane containing amino acid (dtF) in Escherichia coli (E. coli) using a polyspecific aminoacyl-tRNA synthetase (aaRS)/amber suppressor tRNA pair. To demonstrate the utility of dtF for bioapplications, we synthesized gold nanoparticle (AuNP) constructs with a mutant superfolder green fluorescent protein (sfGFP) [sfGFP-AuNP] as a model for the protein-metal conjugation. The resulting sfGFP-AuNP constructs show directional homogeneity and enhanced chemical durability compared to their cysteine analogs towards excess environmental 1,4-dithiothreitol (DTT).
- Assembling a plug-and-play production line for combinatorial biosynthesis of aromatic polyketides in Escherichia coli. [Journal Article]
- PBPLoS Biol 2019; 17(7):e3000347
- Polyketides are a class of specialised metabolites synthesised by both eukaryotes and prokaryotes. These chemically and structurally diverse molecules are heavily used in the clinic and include front…
Polyketides are a class of specialised metabolites synthesised by both eukaryotes and prokaryotes. These chemically and structurally diverse molecules are heavily used in the clinic and include frontline antimicrobial and anticancer drugs such as erythromycin and doxorubicin. To replenish the clinicians' diminishing arsenal of bioactive molecules, a promising strategy aims at transferring polyketide biosynthetic pathways from their native producers into the biotechnologically desirable host Escherichia coli. This approach has been successful for type I modular polyketide synthases (PKSs); however, despite more than 3 decades of research, the large and important group of type II PKSs has until now been elusive in E. coli. Here, we report on a versatile polyketide biosynthesis pipeline, based on identification of E. coli-compatible type II PKSs. We successfully express 5 ketosynthase (KS) and chain length factor (CLF) pairs-e.g., from Photorhabdus luminescens TT01, Streptomyces resistomycificus, Streptoccocus sp. GMD2S, Pseudoalteromonas luteoviolacea, and Ktedonobacter racemifer-as soluble heterodimeric recombinant proteins in E. coli for the first time. We define the anthraquinone minimal PKS components and utilise this biosynthetic system to synthesise anthraquinones, dianthrones, and benzoisochromanequinones (BIQs). Furthermore, we demonstrate the tolerance and promiscuity of the anthraquinone heterologous biosynthetic pathway in E. coli to act as genetically applicable plug-and-play scaffold, showing it to function successfully when combined with enzymes from phylogenetically distant species, endophytic fungi and plants, which resulted in 2 new-to-nature compounds, neomedicamycin and neochaetomycin. This work enables plug-and-play combinatorial biosynthesis of aromatic polyketides using bacterial type II PKSs in E. coli, providing full access to its many advantages in terms of easy and fast genetic manipulation, accessibility for high-throughput robotics, and convenient biotechnological scale-up. Using the synthetic and systems biology toolbox, this plug-and-play biosynthetic platform can serve as an engine for the production of new and diversified bioactive polyketides in an automated, rapid, and versatile fashion.
- EPR spectroscopy of putative enzyme intermediates in the NO reductase and the auto-nitrosylation reaction of Desulfovibrio vulgaris hybrid cluster protein. [Journal Article]
- FLFEBS Lett 2019 Jul 18
- The hybrid cluster protein Hcp contains a unique 4Fe cluster that is a hybrid of μ-S and μ-O bridges. Escherichia coli Hcp has recently been found to carry NO reductase activity as well as S-nitrosyl…
The hybrid cluster protein Hcp contains a unique 4Fe cluster that is a hybrid of μ-S and μ-O bridges. Escherichia coli Hcp has recently been found to carry NO reductase activity as well as S-nitrosylation activity in NO-based signaling. In other species, the physiological activity has not been established. No reaction mechanism of any Hcp has been proposed. Here, we show that D. vulgaris (Hildenborough) Hcp has nitric oxide reductase activity with benzyl viologen as electron donor. With EPR spectroscopy we identify three unexpected putative reaction intermediates: both in reduced and oxidized Hcp, dinitrosyliron complexes are formed. Also, the hybrid cluster in reduced Hcp, but not in oxidized Hcp, binds the product N2 O. Possible implications for a reaction mechanism are discussed. This article is protected by copyright. All rights reserved.
- Glycerol Monolaurate-Mediated Attenuation of Metabolic Syndrome is Associated with the Modulation of Gut Microbiota in High Fat diet-fed Mice. [Journal Article]
- MNMol Nutr Food Res 2019 Jul 18; :e1801417
- CONCLUSIONS: Our findings identify the links between gut microbiota and GML-induced metabolic improvements, suggesting that the attenuation of HFD-induced metabolic disorders by higher GML supplementation might occur through targeting gut microbiota. This article is protected by copyright. All rights reserved.
- A single tool to monitor multiple protein-protein interactions of the Escherichia coli acyl carrier protein. [Journal Article]
- AIACS Infect Dis 2019 Jul 18
- Protein-protein interactions are ubiquitous to all domains of life and have gained recent interest as drug targets. However, many current methods to study protein-protein interactions can be costly a…
Protein-protein interactions are ubiquitous to all domains of life and have gained recent interest as drug targets. However, many current methods to study protein-protein interactions can be costly and are low-throughput. Here, we demonstrate a solvatochromic tool based on the natural post-translational modification of the Escherichia coli acyl carrier protein (EcACP) used to visualize protein-protein interactions between EcACP and thirteen different partner enzymes from several biosynthetic pathways. We use this tool to confirm proposed interactions between EcACP and both catalytic and regulatory proteins. We also show the utility of this method towards detecting allosteric changes to partner enzyme structure and the validation of active site inhibitors. We anticipate the future adaptation of this assay into a high-throughput screen for antibiotic discovery.
- Synthesis of cinnabarinic acid by metabolically engineered Pseudomonas chlororaphis GP72. [Journal Article]
- BBBiotechnol Bioeng 2019 Jul 17
- Cinnabarinic acid is a valuable phenoxazinone that has broad applications in the pharmaceutical, chemical and dyeing industries. However, few studies have investigated the production of cinnabarinic …
Cinnabarinic acid is a valuable phenoxazinone that has broad applications in the pharmaceutical, chemical and dyeing industries. However, few studies have investigated the production of cinnabarinic acid or its derivatives using genetically engineered microorganisms. Herein, an efficient synthetic pathway of cinnabarinic acid was designed and constructed in Pseudomonas chlororaphis GP72 for the first time, which was more straightforward and robust than the known eukaryotic biosynthetic pathways. First, we screened and identified trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) dehydrogenases from Escherichia coli MG1655 (encoded by entA), Streptomyces sp. NRRL12068 (encoded by bomO) and Streptomyces chartreusis NRRL3882 (encoded by calB3) based on the structural similarity of the substrate and product, and the DHHA dehydrogenase encoded by calB3 was selected for the synthesis of cinnabarinic acid due to its high DHHA conversion rate. Subsequently, cinnabarinic acid was synthesized by the expression of the DHHA dehydrogenase CalB3 and the phenoxazinone synthase CotA in the DHHA-producing strain P. chlororaphis GP72, resulting in a cinnabarinic acid titer of 20.3 mg/L at 48 hour. Further fermentation optimization by the addition of Cu2+ , H2 O2 and with adding glycerol increased cinnabarinic acid titer to 136.2 mg/L in shake flasks. The results indicate that P. chlororaphis GP72 may be engineered as a microbial cell factory to produce cinnabarinic acid or its derivatives from renewable bioresources. This article is protected by copyright. All rights reserved.
- Model-based framework for parallel scale down fed-batch cultivations in mini-bioreactors for accelerated phenotyping. [Journal Article]
- BBBiotechnol Bioeng 2019 Jul 17
- Concentration gradients that occur in large industrial-scale bioreactors due to mass transfer limitations have significant effects on process efficiency. Hence, it is desirable to investigate the res…
Concentration gradients that occur in large industrial-scale bioreactors due to mass transfer limitations have significant effects on process efficiency. Hence, it is desirable to investigate the response of strains to such heterogeneities so as to reduce the risk of failure during process scale-up. Although there are various scale-down techniques to study these effects, scale-down strategies are rarely applied in the early developmental phases of a bioprocess as they have not yet been implemented on small scale parallel cultivation devices. In this work, we combine mechanistic growth models with a parallel mini-bioreactor system to create a high-throughput platform for studying the response of Escherichia coli strains to concentration gradients. As a scale-down approach, a model-based glucose pulse feeding scheme is applied and compared to a continuous feed profile, to study the influence of glucose and dissolved oxygen gradients on both cell physiology and the incorporation of non-canonical amino acids into recombinant proinsulin. The results show a significant increase in the incorporation of the non-canonical amino acid norvaline in the soluble intracellular extract and in the recombinant product in cultures with glucose/oxygen oscillations. Interestingly, the amount of norvaline depended on the pulse frequency and was negligible with continuous feeding, confirming observations from large-scale cultivations. Most importantly, the results also show that a larger number of the model parameters are significantly affected by the scale-down scheme, compared to the reference cultivations. In this example, it was possible to describe the effects of oscillations in a single parallel experiment. The platform offers the opportunity to combine strain screening with scale-down studies to select the most robust strains for bioprocess scale-up. This article is protected by copyright. All rights reserved.
- Construction of a novel Escherichia coli expression system: relocation of lpxA from chromosome to a constitutive expression vector. [Journal Article]
- AMAppl Microbiol Biotechnol 2019 Jul 17
- The selective marker in the plasmid-based expression system is usually a gene that encodes an antibiotic-resistant protein; therefore, the antibiotic has to add to maintain the plasmid when growing t…
The selective marker in the plasmid-based expression system is usually a gene that encodes an antibiotic-resistant protein; therefore, the antibiotic has to add to maintain the plasmid when growing the bacteria. This antibiotic addition would lead to increase of production cost and the environment contamination. In this study, a novel Escherichia coli expression system, the lpxA deletion mutant harboring an lpxA-carrying vector, was developed. To develop this system, three plasmids pCas9Cre, pTF-A-UD, and pRSFCmlpxA were constructed. The plasmid pCas9Cre produces enzymes Cas9, λ-Red, and Cre and can be cured by growing at 42 °C; pTF-A-UD contains several DNA fragments required for deleting the chromosomal lpxA and can be cured by adding isopropyl-D-thiogalactopyranoside; pRSFCmlpxA contains the lpxA mutant lpxA123 and CamR. When E. coli were transformed with these three plasmids, the chromosomal lpxA and the CamR in pRSFCmlpxA can be efficiently removed, resulting in an E. coli lpxA mutant harboring pRSFlpxA. The lpxA is essential for the growth of E. coli; its relocation from chromosome to a constitutive expression vector is an ideal strategy to maintain the vector without antibiotic addition. The lpxA123 in pRSFlpxA can complement the deletion of the chromosomal lpxA and provide a strong selective pressure to maintain the plasmid pRSFlpxA. This study provides an experimental evidence that this novel expression system is convenient and efficient to use and can be used to improve L-threonine biosynthesis in the wild type E. coli MG1655 and an L-threonine producing E. coli TWF006.
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- The E. coli MinCDE system in the regulation of protein patterns and gradients. [Review]
- CMCell Mol Life Sci 2019 Jul 17
- Molecular self-organziation, also regarded as pattern formation, is crucial for the correct distribution of cellular content. The processes leading to spatiotemporal patterns often involve a multitud…
Molecular self-organziation, also regarded as pattern formation, is crucial for the correct distribution of cellular content. The processes leading to spatiotemporal patterns often involve a multitude of molecules interacting in complex networks, so that only very few cellular pattern-forming systems can be regarded as well understood. Due to its compositional simplicity, the Escherichia coli MinCDE system has, thus, become a paradigm for protein pattern formation. This biological reaction diffusion system spatiotemporally positions the division machinery in E. coli and is closely related to ParA-type ATPases involved in most aspects of spatiotemporal organization in bacteria. The ATPase MinD and the ATPase-activating protein MinE self-organize on the membrane as a reaction matrix. In vivo, these two proteins typically oscillate from pole-to-pole, while in vitro they can form a variety of distinct patterns. MinC is a passenger protein supposedly operating as a downstream cue of the system, coupling it to the division machinery. The MinCDE system has helped to extract not only the principles underlying intracellular patterns, but also how they are shaped by cellular boundaries. Moreover, it serves as a model to investigate how patterns can confer information through specific and non-specific interactions with other molecules. Here, we review how the three Min proteins self-organize to form patterns, their response to geometric boundaries, and how these patterns can in turn induce patterns of other molecules, focusing primarily on experimental approaches and developments.