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PreprintCited by 2Enzyme Catalysis and ImmobilizationElectrochemical sensors and biosensorsMicrobial Metabolic Engineering and Bioproduction

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Fluorescent Biosensor-Guided Engineering of Enzyme Cascades for Electrochemical Applications

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Ricks, Nathan J., Brachi, Monica, Arun, FNU, Baiarashov, Egor, Minteer, Shelley D., Hammond, Ming C.·2025·ACS Catalysis
DOI 10.1101/2025.11.21.689824

Preprint — published as 10.1021/acscatal.5c08170 · Paper page

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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 E. 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 generalizable and scalable method for engineering and evolving substrate channeling, with broad implications for pathway optimization and enzyme design in synthetic biology, bioprocessing, and energy applications.

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Metadata

Authors
Ricks, N. J., Brachi, M., Arun, F., Baiarashov, E., Minteer, S. D., & Hammond, M. C.
Affiliations
  • University of Utah (US)
  • Missouri University of Science and Technology (US)
Corresponding author
  • Minteer, Shelley D.
  • Hammond, Ming C.
ORCID
Venue
ACS Catalysis
Publisher
openRxiv
Published
2025-11-23
Volume
16
Issue
5
Pages
4729-4748
ISSN
2155-5435 (ISSN-L)
DOI
10.1101/2025.11.21.689824
OpenAlex
W7131368284
Cited by
2
References
1
Citations by year
2026: 2
FWCI
4.18
Type
journal article
Language
en
Open access
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Locations
  • ACS Catalysis (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
Funders
Office of Naval Research (N00014-21-1-2188)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Physical Sciences › Chemistry › Analytical Chemistry › Electrochemical sensors and biosensors
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
Keywords
substrate channeling, enzyme cascade, fluorescent biosensor, fumarase, malate dehydrogenase, TCA cycle, linker engineering, bioelectrocatalysis, enzyme immobilization, NADH production, high-throughput screening, enzyme design

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