Programmable Scaffold-Mediated Assembly Regulation Tool for Dynamic Control of a Multienzyme Biocatalyst

ACS Catalysis, Journal Year: 2025, Volume and Issue: unknown, P. 2236 - 2249

Published: Jan. 23, 2025

Surface-displayed whole-cell biocatalysis offers significant advantages over traditional intracellular enzyme-based methods but faces challenges in complex multienzyme processes and low efficiency. We present Scaffold-Mediated Assembly Regulation Tool (SMART), a programmable platform for dynamic control of biocatalysts on Corynebacterium glutamicum surfaces. SMART integrates orthogonal cross-linked scaffolds with the twin Catcher/Tag pairs (SpyCatcher/SpyTag SnoopCatcher/SnoopTag), controllable molecular ratios, high loading capacity, offering user-defined functionality enzyme assembly. optimized through computational modeling experimental validation created bifunctional long tandem repeats optimal loading. The versatility was demonstrated by codisplaying two sequential cascade tunable stoichiometry, significantly improving conversion maltodextrin to trehalose. Furthermore, we integrated Bxb1 recombinase-based state machine (RSM) genetic circuit, allowing temporal regulation scaffold expression within single cell. Finally, system's applicability showcased isomaltulose production from low-cost sucrose. design represents advancement surface-displayed biocatalysis, reactions potential applications various industrially relevant biocatalysts.

Language: Английский

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Protein Design Accelerates the Development and Application of Optogenetic Tools

Computational and Structural Biotechnology Journal, Journal Year: 2025, Volume and Issue: 27, P. 717 - 732

Published: Jan. 1, 2025

Optogenetics has substantially enhanced our understanding of biological processes by enabling high-precision tracking and manipulation individual cells. It relies on photosensitive proteins to monitor control cellular activities, thereby paving the way for significant advancements in complex system research. Photosensitive play a vital role development optogenetics, facilitating establishment cutting-edge methods. Recent breakthroughs protein design have opened up opportunities develop protein-based tools that can precisely manipulate activities. These will significantly accelerate application optogenetic tools. This article emphasizes pivotal tools, offering insights into potential future directions. We begin providing an introduction historical fundamental principles followed exploration operational mechanisms key domains, which includes clarifying conformational changes they undergo response light, such as allosteric modulation dimerization processes. Building this foundation, we reveal enable creation even more sophisticated techniques.

Language: Английский

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Engineering a Self-Assembled Protein Cage for Targeted Dual Functionalization

Nano Letters, Journal Year: 2024, Volume and Issue: 24(30), P. 9237 - 9244

Published: July 17, 2024

Self-assembled protein cages are attractive scaffolds for organizing various proteins of interest (POIs) toward applications in synthetic biology and medical science. However, specifically attaching multiple POIs to a single cage remains challenging, resulting diversity among the functionalized particles. Here, we present engineering self-assembled cage,

Language: Английский

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Assembly of Matryoshka-Type Protein Nanocages for Compartmentalized Oxygen Sensing

Nano Letters, Journal Year: 2025, Volume and Issue: unknown

Published: March 10, 2025

Oxygen permeability is a critical property of protein nanocages (PNCs) that impacts or dictates the functions PNCs. However, it remains challenging to determine experimentally. Here, we report compartmentalized oxygen sensing inside PNCs by assembling matryoshka-type structures through interfacial engineering, namely, one PNC containing another smaller functionalized with small-molecule probes. in lumen outer can be probed conveniently via phosphorescence spectrometry. This method enabled analysis two representative PNCs, MS2 virus-like particles and Thermotoga maritima encapsulin, revealing former permeable, while latter impermeable. study establishes general approach for measuring shells, which provide an experimental basis understanding working mechanisms inspire applications like oxygen-sensitive oxygen-responsive sensing, catalysis, delivery. Also, tunable nested may serve as platforms designing hierarchical devices organelles.

Language: Английский

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Enhanced Targeted Drug Delivery System to Control Avidity and Drug Encapsulation Using E2 Nanocages and SpyTag/SpyCatcher

ACS Biomaterials Science & Engineering, Journal Year: 2025, Volume and Issue: unknown

Published: April 10, 2025

Although antibody-drug conjugates offer advanced targeted anticancer therapy that overcomes the limitations of conventional chemotherapy and therapeutic antibodies, they are restricted in their capacity to carry multiple hydrophobic payloads. Protein nanocages have emerged as versatile platforms for drug delivery, offering advantages like precise molecular assembly, biocompatibility, multivalent targeting. This study presents development engineered E2 functionalized with anti-HER2 single-chain variable fragments (scFv) using SpyTag/SpyCatcher ligation system achieve controlled scFv display valency. The results demonstrate increasing valency enhances HER2 binding affinity via avidity effects, highest showing avidity. Furthermore, cysteine residues were introduced into enable conjugation monomethyl auristatin E (MMAE) through maleimide chemistry, achieving efficient loading. resulting MMAE-conjugated displayed potent, subnanomolar cytotoxicity HER2-positive SKBR3 BT-474 cell lines while sparing HER2-negative MDA-MB-231 cells at concentrations up 1 nM. These underscore critical role enhancing targeting highlight potential protein specific, potent cancer therapy. In this study, we developed an enhanced delivery SpyCatcher/SpyTag regulate encapsulate drugs. modular design pH-sensitive dissociation these establish a foundation next-generation precision medicine strategies.

Language: Английский

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Engineered interaction elements enable enhanced multi-enzyme assembly and cascade biocatalysis for indigo synthesis

Bioresource Technology, Journal Year: 2025, Volume and Issue: 429, P. 132540 - 132540

Published: April 15, 2025

Language: Английский

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Engineering a Mi3_Chemout system for controlled recruitment and release of proteins of interest

Chemical Communications, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

We engineered the Mi3 protein cage with chemically inducible dimerization domains and a photocleavable protein, enabling controlled recruitment release of POIs both in vitro E. coli cells.

Language: Английский

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Programmed protein scaffold for multienzyme assembly empowering the biosynthesis of rare sugars

CHINESE JOURNAL OF CATALYSIS (CHINESE VERSION), Journal Year: 2025, Volume and Issue: 72, P. 95 - 105

Published: May 1, 2025

Language: Английский

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Complete Enzyme Clustering Enhances Coenzyme Q Biosynthesis via Substrate Channeling

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: May 28, 2025

Abstract Metabolons - transient assemblies of sequential metabolic enzymes facilitate the reactions multi-step pathways, yet, how they mechanistically bolster flux remains unknown. Here, we investigate molecular determinants metabolon formation in coenzyme Q (CoQ) biosynthesis using coarse-grained dynamics simulations and biochemical experiments. We show that COQ forms at critical region a phase transition, where both clustering exhibit coordinated sigmoidal responses to changes protein-protein interaction strength. These complete metabolons enable substrate channeling between enzymes, leading crucial enhancement CoQ production efficiency. Selectively disrupting interactions randomly shuffling network demonstrate protein-proximity rather than fine structure clusters is imperative for channeling. Grounded experiment simulation, these findings provide framework understanding organization function across diverse pathways.

Language: Английский

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Biosynthesis of nonnutritive monosaccharide d‐allulose by metabolically engineered Escherichia coli from nutritive disaccharide sucrose

Biotechnology and Bioengineering, Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 11, 2024

Abstract Sucrose is a commonly utilized nutritive sweetener in food and beverages due to its abundance nature low production costs. However, excessive intake of sucrose increases the risk metabolic disorders, including diabetes obesity. Therefore, there growing demand for development nonnutritive sweeteners with almost no calories. d ‐Allulose an ultra‐low‐calorie, rare six‐carbon monosaccharide high sweetness, making it ideal alternative sucrose. In this study, we developed cell factory ‐allulose from using Escherichia coli JM109 (DE3) as chassis host. The genes cscA , cscB cscK alsE a6PP were co‐expressed construction synthesis pathway. Then, introduction ptsG‐F knockout ptsG fruA ptsI ptsH reprogram sugar transport pathways resulted improvement substrate utilization. Next, carbon fluxes Embden‐Meyerhof‐Parnas pentose phosphate regulated by inactivation pfkA zwf achieving increase titer yield 154.2% 161.1%, respectively. Finally, scaled‐up fermentation was performed 5 L fermenter. reached 11.15 g/L, 0.208 g/g on

Language: Английский

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