Abstract
<jats:title>Abstract</jats:title> <jats:p> Efficient conversion of polymeric feedstocks for sustainable bioprocessing requires robust strategies for enzyme assembly and cell-surface attachment. In nature, cellulosomes achieve highly efficient lignocellulosic polysaccharide deconstruction through scaffoldin-mediated organization of carbohydrate-active enzymes via specific cohesin–dockerin interactions. These modular binding pairs are therefore attractive tools for synthetic biology and engineered whole-cell biocatalysis, yet their performance has been studied mainly <jats:italic>in vitro</jats:italic> or in yeast or Gram-positive bacteria. The factors governing their function on the microbial surfaces - particularly those of Gram-negative bacteria - remain incompletely understood. Here, we investigated the binding efficiency and interaction stability of two thermophilic cohesin–dockerin pairs from <jats:italic>Acetivibrio thermocellus</jats:italic> and <jats:italic>Acetivibrio clariflavus</jats:italic> displayed on the surface of the genome-streamlined strain <jats:italic>Pseudomonas putida</jats:italic> EM371 using an Ag43-based display system from <jats:italic>Escherichia coli</jats:italic> and a dockerin-tagged fluorescent reporter. We show that binding efficiency is strongly affected by the temperature at which the cohesin–dockerin complex is formed. We further demonstrate that the interaction stability of the <jats:italic>A. clariflavus</jats:italic> pair can be substantially improved by targeted amino acid substitutions in the dockerin domain guided by molecular dynamics simulations and free-energy calculations. These results identify key parameters controlling the performance of thermophilic cohesin–dockerin modules on living bacterial cell surfaces and establish a computation-guided strategy for engineering more stable cellulosome-derived assembly interfaces, advancing the development of modular whole-cell platforms for sustainable biotechnology applications. </jats:p> <jats:sec> <jats:title>TOC graphics</jats:title> <jats:fig id="ufig1" position="float" orientation="portrait" fig-type="figure"> <jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="743725v1_ufig1" position="float" orientation="portrait"/> </jats:fig> <jats:p> Cohesin–dockerin pairs provide strong and modular non-covalent interactions for synthetic biology and biotechnology applications. We establish an experimental and computational pipeline to improve their two key properties - binding efficiency and interaction stability - on the surface of <jats:italic>Pseudomonas putida</jats:italic> , enabling more robust cell-surface assembly systems. </jats:p> </jats:sec>