Channeling in native microbial pathways: Implications and challenges for metabolic engineering

Abernathy, Mary H.; He, Lian; Tang, Yinjie J.

doi:10.1016/j.biotechadv.2017.06.004

Cited by 55 publications

(43 citation statements)

References 93 publications

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“…However, the citrate labeling rate was slower than expected with only M + 2 and M + 3 isotopomers detected by LC‐MS at the initial stage. Such enigmatic observations were consistent with confounding labeling patterns due to metabolite channeling, and in turn could introduce errors into 13 C‐based flux analysis.…”

Section: Resultsmentioning

confidence: 56%

“…We demonstrated knocking out the PTS in E. coli loosens the metabolic flux network and changes reaction directions toward competing pathways. Although this work did not directly prove that intracellular enzymes self‐organize to form subtle metabolons, dynamic labeling experiments provide reasonable in vivo channeling tests for systems perturbed at possible channeling flux control nodes . Intuitively, Malate➔Oxaloacetate➔Citrate in the TCA cycle of eukaryote cells have been proved to be highly channeled .…”

Section: Resultsmentioning

confidence: 86%

“…Less experimentally explored, enzymes may dynamically associate via pairwise interactions to form “pearl necklace like” channels or via nonpairwise interactions to form enzyme clusters . Independent of the specific channeling architect, these channels function to direct metabolic fluxes in crowded cytosolic environments . Such protein–protein interactions ( proximity channels ) are linked via noncovalent forces rather than membrane encapsulation or covalent bonds .…”

Section: Introductionmentioning

confidence: 99%

“…Unlike transcription or protein expressions that often show significant variations, channeling might stabilize fluxes under environmental/genetic perturbations . Because small metabolite diffusion is considered sufficiently fast for enzyme kinetics, channeling may increase reaction rates, but it is more likely channeling functions as a regulatory role in controlling metabolic fluxes and protecting reaction intermediates from competing reactions or harsh cytosol environments . Although it has been recognized that enzymes can organize spatially and temporarily to shuttle intermediates without releasing them into the cytosol, it is difficult to validate weak transient interactions between enzymes under in vitro (cell free) conditions, thus proximity channeling in bacteria remains controversial …”

Section: Introductionmentioning

confidence: 99%

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Comparative studies of glycolytic pathways and channeling under in vitro and in vivo modes

et al. 2018

Self Cite

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This study constructed cell‐free glycolytic enzyme systems and compared them to their in vivo functions in Escherichia coli. Under in vitro conditions, flux regulation followed enzyme concentrations and kinetics. In E. coli, only one of the isozymes of phosphofructokinase (PfkA) and fructose‐bisphosphate aldolase (FbaA) facilitate Embden‐Meyerhof‐Parnas (EMP) flux, but under in vitro assays, these isozymes were interchangeable. Additionally, in vitro introduction of the Entner–Doudoroff (ED) pathway improved glycolysis rates, while in vivo overexpression of the ED pathway could not capture significant flux unless its phosphotransferase system (PTS) was knocked out. Lastly, in vivo dynamic 13 C‐experiments revealed that the labeling order of EMP pathway intermediates was not strictly cascade, indicating intracellular metabolites were not well mixed. These enigmatic observations cannot be fully explained by thermodynamics or substrate level regulations. This article supports the long‐time conjecture that EMP enzymes are channeled, and the PTS may be an anchor point to initiate enzyme assemblies. © 2018 American Institute of Chemical Engineers AIChE J, 65: 483–490, 2019

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Section: Resultsmentioning

confidence: 56%

Section: Resultsmentioning

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Comparative studies of glycolytic pathways and channeling under in vitro and in vivo modes

et al. 2018

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“…Now, more and more studies are being conducted on the artificial relocation of pathway enzymes. In addition to the endoplasmic reticulum and vacuole mentioned above, compartments of enzyme complexes can also be peroxisomes, chloroplasts, mitochondria, and the Golgi apparatus [120,121]. Dhurrin (D-glucopyranosyloxy-(S)-p-hydroxymandelonitrile) is a cyanogenic glucoside, and its synthetic pathway consists of two endoplasmic reticulum membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble UDP-glucosyltransferase (UGT85B1) [122,123].…”

Section: Eukaryotic Physical Compartmentsmentioning

confidence: 99%

Enzyme Assembly for Compartmentalized Metabolic Flux Control

Cui

et al. 2020

Metabolites

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Enzyme assembly by ligand binding or physically sequestrating enzymes, substrates, or metabolites into isolated compartments can bring key molecules closer to enhance the flux of a metabolic pathway. The emergence of enzyme assembly has provided both opportunities and challenges for metabolic engineering. At present, with the development of synthetic biology and systems biology, a variety of enzyme assembly strategies have been proposed, from the initial direct enzyme fusion to scaffold-free assembly, as well as artificial scaffolds, such as nucleic acid/protein scaffolds, and even some more complex physical compartments. These assembly strategies have been explored and applied to the synthesis of various important bio-based products, and have achieved different degrees of success. Despite some achievements, enzyme assembly, especially in vivo, still has many problems that have attracted significant attention from researchers. Here, we focus on some selected examples to review recent research on scaffold-free strategies, synthetic artificial scaffolds, and physical compartments for enzyme assembly or pathway sequestration, and we discuss their notable advances. In addition, the potential applications and challenges in the applications are highlighted.

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Polymeric Giant Unilamellar Vesicles with Integrated DNA‐Origami Nanopores: An Efficient Platform for Tuning Bioreaction Dynamics Through Controlled Molecular Diffusion

Cochereau

Maffeis

Santos

et al. 2023

Adv Funct Materials

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Giant unilamellar vesicles (GUVs) are microcompartments serving to confine reactions, allow signaling pathways, or design synthetic cells. Polymer GUVs are composed of copolymer membranes mimicking cell membranes, and present advantages over lipid‐based GUVs, such as higher mechanical stability and chemical versatility. Such microcompartments are essential for understanding reactions/signaling occurring in cells, which are difficult to study by in vivo approaches due to the cell's complexity. However, the lack of control over their production, stability, and membrane diffusion properties is still limiting their use for bio‐related applications. Here, polymer GUVs produced by microfluidics and permeabilized with DNA‐origami nanopores (DoNs) that present a high level of control over these essential properties are introduced. After systematic optimization of conditions, DoN‐GUVs reveal a narrow size distribution, allow for high encapsulation efficiencies, and are stable for weeks, protecting encapsulated biomolecules. The kinetics of diffusion of molecules through the GUV's membrane is tuned by insertion of DoNs with a controlled 3D‐ structure. DNA polymerase I, encapsulated as model for bioreactions, successfully produced DNA duplex strands with spatiotemporal control. DoN‐GUVs loaded with active molecules open new avenues in bioreactions, from the detection of biomolecules, over the tuning of molecular transport rates, to the investigation of cellular processes/signaling.

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Channeling in native microbial pathways: Implications and challenges for metabolic engineering

Cited by 55 publications

References 93 publications

Comparative studies of glycolytic pathways and channeling under in vitro and in vivo modes

Comparative studies of glycolytic pathways and channeling under in vitro and in vivo modes

Enzyme Assembly for Compartmentalized Metabolic Flux Control

Polymeric Giant Unilamellar Vesicles with Integrated DNA‐Origami Nanopores: An Efficient Platform for Tuning Bioreaction Dynamics Through Controlled Molecular Diffusion

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