Abstract
Substrate channeling is a strategy for enhancing flux and yield in enzymatic cascades and is increasingly relevant for applications in biocatalysis, biotechnology, and bioelectrochemical systems. However, efforts to engineer channeling are limited by the lack of high-throughput methods to evaluate and optimize channeling efficiency. Here, we present a fluorescence-based screening assay to rapidly assess substrate channeling in a model system involving fumarase and malate dehydrogenase, two sequential enzymes from the Krebs cycle. By expressing genetic fusions in Escherichia coli, quantifying intermediate (malate) and product (NADH) formation in lysate using orthogonal fluorescent readouts, and comparing product-to-intermediate ratios, we screened a library of linker variants designed to promote electrostatic channeling. A top-performing construct was identified and validated through classical channeling assays. This hit demonstrated increased product yield and current output when immobilized on electrodes with a bilayer architecture, highlighting utility in bioelectrocatalysis. We further showed that the channeling linker could be applied to a de novo designed single-chain fumarase, which preserved channeling capability and exhibited improved thermal stability. These results establish a strategy for engineering and evolving substrate channeling, which can be extended to the screening of other multienzymatic cascades via the design of specific substrate-tailored fluorescent sensors, with broad implications for pathway optimization and enzyme design in synthetic biology, bioprocessing, and energy applications.
Recommended Citation
N. J. Ricks et al., "Fluorescent Biosensor-Guided Engineering of Enzyme Cascades for Electrochemical Applications," ACS Catalysis, vol. 16, no. 5, pp. 4729 - 4748, American Chemical Society, Mar 2026.
The definitive version is available at https://doi.org/10.1021/acscatal.5c08170
Department(s)
Chemistry
Publication Status
Open Access
Keywords and Phrases
bioelectrocatalysis; directed evolution; fluorescent biosensors; fusion protein; protein design; substrate channeling
International Standard Serial Number (ISSN)
2155-5435
Document Type
Article - Journal
Document Version
Citation
File Type
text
Language(s)
English
Rights
© 2026 American Chemical Society, All rights reserved.
Publication Date
06 Mar 2026
Comments
University of Utah, Grant N00014-21-1-2188