Biology subjects

Cowan, A. E.

Publications and source records attributed to Cowan, A. E..

4 recordsLinked to original sources

bioRxiv2026

Expanding the scope of redox-balance growth coupling techniques with a carbon cofeeding strategy

Metabolic engineering to produce molecules not naturally synthesized by the host often requires directed evolution to improve pathway enzyme performance. Growth-coupled selection can dramatically increase directed-evolution throughput, and manipulation of redox balance has proven effective for tying reductase fitness to microbial growth. However, most redox-balance selections require feeding the reductase substrate because of stoichiometric constraints. This is impractical for many biosynthetic pathways either due to practical limitations on cost or complexity of bulk substrate synthesis, or the lack of an ability to transport substrate into cells, for example intracellular acyl-CoA/ACP intermediates. Here we define stoichiometric constraints that make substrate feeding necessary for many acetyl-CoA-derived reduction pathways in NADPH-imbalanced hosts. We overcome these constraints with a dual-feedstock strategy in which glucose provides reducing power while acetate supplies additional acetyl-CoA without directly perturbing redox balance. In an engineered E. coli selection strain, acetate co-feeding enabled growth coupling of acetaldehyde, 3-hydroxybutyrate, and mevalonate production and produced a linear correlation between product formation and growth. We then used this selection to evolve a class II HMG-CoA reductase (HMGR) from Delftia acidovorans toward NADPH utilization, enriching variants with improved NADPH-dependent activity. Finally, propionate co-feeding enabled growth coupling of propionyl-CoA reduction, supporting the generality of carbon co-feeding for selecting enzymes in pathways involving acyl-chain elongation and reduction. HighlightsO_LIStoichiometric limits of redox-balance growth coupling are defined C_LIO_LIAcetate co-feeding supplies acetyl-CoA without perturbing redox balance C_LIO_LICo-feeding enables growth coupling of acetaldehyde, 3-HB, and mevalonate C_LIO_LIGrowth coupling enables evolution of HMGR toward NADPH specificity C_LIO_LIPropionate co-feeding extends growth coupling to additional acyl-CoA substrates C_LI

bioengineering↗

bioRxiv2025

Carbon-Efficient Conversion through Hydrogen and Formate-Driven Metabolism in Escherichia coli

Product yields for biomanufacturing processes are often constrained by the tight coupling of cellular energy generation and carbon metabolism in sugar-based fermentation systems. To overcome this limitation, we engineered Escherichia coli to utilize hydrogen gas (H2) and formate (HCOO-) as alternative sources of energy and reducing equivalents, thereby decoupling energy generation from carbon metabolism. This approach enabled precise suppression of decarboxylative oxidation during acetate growth, with 86.6 {+/-} 1.6% of electrons from hydrogen gas (via soluble hydrogenase from Cupriavidus necator H16) and 98.4 {+/-} 3.6% of electrons from formate (via formate dehydrogenase from Pseudomonas sp. 101) offsetting acetate oxidation. Hydrogen gas supplementation led to a titratable and stoichiometric reduction in CO2 evolution in acetate-fed cultures. Metabolomic analysis suggests that this metabolic decoupling redirects carbon flux through the glyoxylate shunt, partially bypassing two decarboxylative steps in the TCA cycle. We demonstrated the utility of this strategy by applying it to mevalonate biosynthesis, where formate supplementation during glucose fermentation increased titers by 57.6% in our best-performing strain. Flux balance analysis further estimated that 99.0 {+/-} 2.8% of electrons from formate were used to enhance mevalonate production. These findings highlight a broadly applicable strategy for enhancing biomanufacturing efficiency by leveraging external reducing power to optimize feedstock and energy use. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/657096v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@12f77dborg.highwire.dtl.DTLVardef@165d6c1org.highwire.dtl.DTLVardef@f6302forg.highwire.dtl.DTLVardef@d59bb3_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Panich, J. (2025) https://BioRender.com/0tmtp94

synthetic biology↗

bioRxiv2023

Mapping the biochemical landscape of rubisco

Rubisco is the primary CO2 fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineered E. coli where enzyme function is coupled to growth. By assaying >99% of single amino acid mutants across CO2 concentrations, we inferred enzyme velocity and CO2 affinity for thousands of substitutions. We identified many highly conserved positions that tolerate mutation and rare mutations that improve CO2 affinity. These data suggest that non-trivial kinetic improvements are readily accessible and provide a comprehensive sequence-to-function mapping for enzyme engineering efforts.

biochemistry↗

bioRxiv2023

Beyond analytic solution: analysis of FRAP experiments by spatial simulation of the forward problem

Fluorescence redistribution after photobleaching (FRAP) is a commonly used method to understand the dynamic behavior of molecules within cells. Analytic solutions have been developed for specific, well-defined models of dynamic behavior in idealized geometries, but these solutions are inaccurate in complex geometries or when complex binding and diffusion behaviors exist. We demonstrate the use of numerical reaction-diffusion simulation approaches using the easily accessible Virtual Cell (VCell) software, to establish methods for analyzing photobleaching data. We show how multiple simulations employing parameter scans and varying bleaching locations and sizes can help to bracket diffusion coefficients and kinetic rate constants. This approach is applied to problems in membrane surface diffusion, diffusion and binding in cytosolic volumes in complex cell geometries, and analysis of diffusion and binding in intracellular liquid droplets. Statement of SignificanceFluorescence Redistribution After Photobleaching (FRAP) is a widely used experimental method that can reveal important parameters for reaction/diffusion events within cells. However, analytic methods to analyze FRAP experiments are limited to specific geometries and conditions. We demonstrate how spatial numerical simulation methods using the freely available software Virtual Cell can be used to obtain parameter information from FRAP experiments in situations that are not amenable to analytic solutions and that are accessible to most bench biologists.

biophysics↗