scholarly journals In Vivo Assembly of Artificial Metalloenzymes and Application in Whole‐Cell Biocatalysis**

2021 ◽  
Author(s):  
Shreyans Chordia ◽  
Siddarth Narasimhan ◽  
Alessandra Lucini Paioni ◽  
Marc Baldus ◽  
Gerard Roelfes
Keyword(s):  
Whole Cell ◽  
Whole Cell Biocatalysis ◽  
Artificial Metalloenzymes

scholarly journals In Vivo Assembly of Artificial Metalloenzymes and Application in Whole‐Cell Biocatalysis**

2021 ◽  
Author(s):  
Shreyans Chordia ◽  
Siddarth Narasimhan ◽  
Alessandra Lucini Paioni ◽  
Marc Baldus ◽  
Gerard Roelfes
Keyword(s):  
Whole Cell ◽  
Whole Cell Biocatalysis ◽  
Artificial Metalloenzymes

scholarly journals In Vivo Assembly of Artificial Metalloenzymes and Application in Whole‐Cell Biocatalysis

2020 ◽  
Author(s):  
Shreyans Chordia ◽  
Siddarth Narasimhan ◽  
Alessandra Lucini Paioni ◽  
Marc Baldus ◽  
Gerard Roelfes
Keyword(s):  
Directed Evolution ◽  
Cell Fractionation ◽  
Whole Cell ◽  
Whole Cells ◽  
E Coli ◽  
Whole Cell Biocatalysis ◽  
Catalytically Active ◽  
Active Transition ◽  
Artificial Metalloenzymes

Artificial metalloenzymes (ArMs), which are hybrids of catalytically active transition metal complexes and proteins, have emerged as promising approach to the creation of biocatalysts for reactions that have no equivalent in nature. Here we report the assembly and application in catalysis of ArMs in the cytoplasm of E. coli cells based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneously added copper(II)‐phenanthroline (Cu(II)‐phen) complex. The ArMs are spontaneously assembled by addition of Cu(II)‐phen to E. coli cells that express LmrR and it is shown that the ArM containing whole cells are active in the catalysis of the enantioselective vinylogous Friedel‐Crafts alkylation of indoles. The ArM assembly in E. coli is further supported by a combination of cell‐ fractionation and inhibitor experiments and confirmed by in‐cell solid‐state NMR. A mutagenesis study showed that the same trends in catalytic activity and enantioselectivity in response to mutations of LmrR were observed for the ArM containing whole cells and the isolated ArMs. This made it possible to perform a directed evolution study using ArMs in whole cells, which gave rise to a mutant, LmrR_A92E_M8D that showed increased activity and enantioselectivity in the catalyzed vinylogous Friedel‐Crafts alkylation of a variety of indoles. The unique aspect of this whole‐cell ArM system is that no engineering of the microbial host, the protein scaffold or the cofactor is required to achieve ArM assembly and catalysis. This makes this system attractive for applications in whole cell biocatalysis and directed evolution, as demonstrated here. Moreover, our findings represent important step forward towards achieving the challenging goal of a hybrid metabolism by integrating artificial metalloenzymes in biosynthetic pathways.

scholarly journals In Vivo Assembly of Artificial Metalloenzymes and Application in Whole‐Cell Biocatalysis

2020 ◽  
Author(s):  
Shreyans Chordia ◽  
Siddarth Narasimhan ◽  
Alessandra Lucini Paioni ◽  
Marc Baldus ◽  
Gerard Roelfes
Keyword(s):  
Directed Evolution ◽  
Cell Fractionation ◽  
Whole Cell ◽  
Whole Cells ◽  
E Coli ◽  
Whole Cell Biocatalysis ◽  
Catalytically Active ◽  
Active Transition ◽  
Artificial Metalloenzymes

Artificial metalloenzymes (ArMs), which are hybrids of catalytically active transition metal complexes and proteins, have emerged as promising approach to the creation of biocatalysts for reactions that have no equivalent in nature. Here we report the assembly and application in catalysis of ArMs in the cytoplasm of E. coli cells based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneously added copper(II)‐phenanthroline (Cu(II)‐phen) complex. The ArMs are spontaneously assembled by addition of Cu(II)‐phen to E. coli cells that express LmrR and it is shown that the ArM containing whole cells are active in the catalysis of the enantioselective vinylogous Friedel‐Crafts alkylation of indoles. The ArM assembly in E. coli is further supported by a combination of cell‐ fractionation and inhibitor experiments and confirmed by in‐cell solid‐state NMR. A mutagenesis study showed that the same trends in catalytic activity and enantioselectivity in response to mutations of LmrR were observed for the ArM containing whole cells and the isolated ArMs. This made it possible to perform a directed evolution study using ArMs in whole cells, which gave rise to a mutant, LmrR_A92E_M8D that showed increased activity and enantioselectivity in the catalyzed vinylogous Friedel‐Crafts alkylation of a variety of indoles. The unique aspect of this whole‐cell ArM system is that no engineering of the microbial host, the protein scaffold or the cofactor is required to achieve ArM assembly and catalysis. This makes this system attractive for applications in whole cell biocatalysis and directed evolution, as demonstrated here. Moreover, our findings represent important step forward towards achieving the challenging goal of a hybrid metabolism by integrating artificial metalloenzymes in biosynthetic pathways.

Enantiocomplementary decarboxylative hydroxylation combining photocatalysis and whole-cell biocatalysis in a one-pot cascade process

2019 ◽  
Vol 21 (8) ◽  
pp. 1907-1911 ◽  
Author(s):  
Jian Xu ◽  
Mamatjan Arkin ◽  
Yongzhen Peng ◽  
Weihua Xu ◽  
Huilei Yu ◽  
...  
Keyword(s):  
Cascade Process ◽  
One Pot ◽  
Whole Cell ◽  
Whole Cell Biocatalysis

The first demonstration of photochemo-enzymatic whole-cell one-pot enantiocomplementary decarboxylative hydroxylation.

scholarly journals In VitroandIn VivoCharacterization of the Novel Oxabicyclooctane-Linked Bacterial Topoisomerase Inhibitor AM-8722, a Selective, Potent Inhibitor of Bacterial DNA Gyrase

2016 ◽  
Vol 60 (8) ◽  
pp. 4830-4839 ◽  
Author(s):  
Christopher M. Tan ◽  
Charles J. Gill ◽  
Jin Wu ◽  
Nathalie Toussaint ◽  
Jingjun Yin ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Antibacterial Agents ◽  
Cell Activity ◽  
Dna Gyrase ◽  
Topoisomerase Iv ◽  
Bacterial Dna ◽  
Whole Cell ◽  
Content Type

ABSTRACTOxabicyclooctane-linked novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of recently described antibacterial agents with broad-spectrum activity. NBTIs dually inhibit the clinically validated bacterial targets DNA gyrase and topoisomerase IV and have been shown to bind distinctly from known classes of antibacterial agents directed against these targets. Herein we report the molecular, cellular, andin vivocharacterization of AM-8722 as a representative N-alkylated-1,5-naphthyridone left-hand-side-substituted NBTI. Consistent with its mode of action, macromolecular labeling studies revealed a specific effect of AM-8722 to dose dependently inhibit bacterial DNA synthesis. AM-8722 displayed greater intrinsic enzymatic potency than levofloxacin versus both DNA gyrase and topoisomerase IV fromStaphylococcus aureusandEscherichia coliand displayed selectivity against human topoisomerase II. AM-8722 was rapidly bactericidal and exhibited whole-cell activity versus a range of Gram-negative and Gram-positive organisms, with no whole-cell potency shift due to the presence of DNA or human serum. Frequency-of-resistance studies demonstrated an acceptable rate of resistance emergencein vitroat concentrations 16- to 32-fold the MIC. AM-8722 displayed acceptable pharmacokinetic properties and was shown to be efficacious in mouse models of bacterial septicemia. Overall, AM-8722 is a selective and potent NBTI that displays broad-spectrum antimicrobial activityin vitroandin vivo.

Whole‐cell biocatalysis: Advancements toward the biosynthesis of fuels

2021 ◽  
Author(s):  
Tanushree Baldeo Madavi ◽  
Sushma Chauhan ◽  
Anushri Keshri ◽  
Hemasundar Alavilli ◽  
Kwon‐Young Choi ◽  
...  
Keyword(s):  
Whole Cell ◽  
Whole Cell Biocatalysis

scholarly journals ADR1-Mediated Transcriptional Activation Requires the Presence of an Intact TFIID Complex

1998 ◽  
Vol 18 (10) ◽  
pp. 5861-5867 ◽  
Author(s):  
Philip B. Komarnitsky ◽  
Edward R. Klebanow ◽  
P. Anthony Weil ◽  
Clyde L. Denis
Keyword(s):  
Transcriptional Activation ◽  
Yeast Cells ◽  
Binding Studies ◽  
Whole Cell ◽  
Transactivation Domains ◽  
Cell Extracts ◽  
Genes Expression ◽  
Tfiid Complex

ABSTRACT The yeast transcriptional activator ADR1, which is required forADH2 and other genes’ expression, contains four transactivation domains (TADs). While previous studies have shown that these TADs act through GCN5 and ADA2, and presumably TFIIB, other factors are likely to be involved in ADR1 function. In this study, we addressed the question of whether TFIID is also required for ADR1 action. In vitro binding studies indicated that TADI of ADR1 was able to retain TAFII90 from yeast extracts and TADII could retain TBP and TAFII130/145. TADIV, however, was capable of retaining multiple TAFIIs, suggesting that TADIV was binding TFIID from yeast whole-cell extracts. The ability of TADIV truncation derivatives to interact with TFIID correlated with their transcription activation potential in vivo. In addition, the ability of LexA-ADR1-TADIV to activate transcription in vivo was compromised by a mutation in TAFII130/145. ADR1 was found to associate in vivo with TFIID in that immunoprecipitation of either TAFII90 or TBP from yeast whole-cell extracts specifically coimmunoprecipitated ADR1. Most importantly, depletion of TAFII90 from yeast cells dramatically reducedADH2 derepression. These results indicate that ADR1 physically associates with TFIID and that its ability to activate transcription requires an intact TFIID complex.

scholarly journals Compensation of physiological motion enables high-yield whole-cell recording in vivo

2020 ◽  
Author(s):  
William M. Stoy ◽  
Bo Yang ◽  
Ali Kight ◽  
Nathaniel C. Wright ◽  
Peter Y. Borden ◽  
...  
Keyword(s):  
Single Cells ◽  
Strong Dependence ◽  
High Yield ◽  
Patch Clamp Recording ◽  
Gold Standard Method ◽  
Whole Cell ◽  
Whole Cell Recording ◽  
Cell Recording ◽  
Living Mouse

1.1.1AbstractWhole-cell patch-clamp recording in vivo is the gold-standard method for measuring subthreshold electrophysiology from single cells during behavioural tasks, sensory stimulations, and optogenetic manipulation. However, these recordings require a tight, gigaohm resistance, seal between a glass pipette electrode’s aperture and a cell’s membrane. These seals are difficult to form, especially in vivo, in part because of a strong dependence on the distance between the pipette aperture and cell membrane. We elucidate and utilize this dependency to develop an autonomous method for placement and synchronization of pipette’s tip aperture to the membrane of a nearby, moving neuron, which enables high-yield seal formation and subsequent recordings in the deep in the brain of the living mouse, in the thalamus. This synchronization procedure nearly doubles the reported gigaseal yield in the thalamus (>3 mm below the pial surface) from 26% (n=17/64) to 48% (n=32/66). Whole-cell recording yield improved from 10% (n = 9/88) to 24% (n=18/76) when motion compensation was used during the gigaseal formation. As an example of its application, we utilized this system to investigate the role of the sensory environment and ventral posterior medial region (VPM) projection synchrony on intracellular dynamics in the barrel cortex. This method results in substantially greater subcortical whole-cell recording yield than previously reported and thus makes pan-brain whole-cell electrophysiology practical in the living mouse brain.

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