Directed in vitro evolution of bacterial expansin BsEXLX1 for higher cellulose binding and its consequences for plant cell wall-loosening activities

Expansins are cell wall-loosening proteins found in all land plants and many microbial species. Despite homologous structures, bacterial expansins have much weaker cellulose binding and wall-loosening activity than plant expansins. We hypothesized stronger cellulose binding would result in greater wall-loosening activity and used in vitro evolution of Bacillus subtilis BsEXLX1 to test this hypothesis. Mutants with stronger binding generally had greater wall-loosening activity, but the relationship was nonlinear and plateaued at ~ 40% higher than wild-type. Mutant E191K exhibited stronger cellulose binding but failed to induce creep, evidently due to protein mistargeting. These results reveal the complexity of interactions between plant cell walls and wall-modifying proteins, an important consideration when engineering proteins for applications in biofuel production and plant pathogen resistance.