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

Abstract

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.

Expression of Two α-Type Expansins from Ammopiptanthus nanus in Arabidopsis thaliana Enhance Tolerance to Cold and Drought Stresses

“…In the CBM63 domain, the different amino acids of AnEXPA1 (A) and AnEXPA2 (V) were located in the 183rd position, whereas S (AnEXPA1) and P (AnEXPA2) were at the 194th position. Therefore, the different amino acids of these two genes could lead to differences in their protein structure and function [25,27,28].…”

Expansin-mediated developmental and adaptive responses: A matter of cell wall biomechanics?

“…This increased activity can be due to easier access of the cellulase to the cellulose chains, but other effects such as electrostatic or other physical interactions between the adsorbed EXLX1 and cellulases cannot be ruled out (Zhang et al, 2021b ). However, bacterial expansins have much weaker cellulose binding and wall-loosening activity than plant α-expansins (Kerff et al, 2008 ; Kim et al, 2009 ), and recent results suggest that although EXLX1 is homologous with plant expansins, it possibly has distinct effects on plant CWs (Hepler & Cosgrove, 2019 ).…”

“…The CW can undergo several types of deformation that can be measured either in situ (ideally in living plant tissues) or in simplified models, most frequently using onion epidermis peels clamped in a custom-made mechanical testing device (Cosgrove, 1989 ; 2011 ; Durachko & Cosgrove, 2009 ; Durachko et al, 2017 ; Wang et al, 2020 ; Zhang & Cosgrove, 2017 ; Zhang et al, 2019a ). In some cases, slightly more complex systems such as de-frosted Arabidopsis petioles (Park & Cosgrove, 2012a ; Xin et al, 2020 ), cucumber and Arabidopsis hypocotyls (Boron et al, 2015 ; Cosgrove, 1989 ; Marga et al, 2005 ; Park & Cosgrove, 2012b ) or wheat coleoptiles (Hepler & Cosgrove, 2019 ) have been used. The advantage of using onion epidermal peels is that the mechanical properties of isolated CW fragments can be measured, largely neglecting the contribution of neighbouring cells, cell size or shape that might possibly influence the results when using indentation-based (AFM) measurements (Cosgrove, 2018b and references therein).…”

Loosenin-Like Proteins from Phanerochaete carnosa Impact Both Cellulose and Chitin Fiber Networks

“…3 ). Notably, previous mutagenesis of the D2 domain of BsEXLX1 revealed a nonlinear relation between microbial expansin binding to cellulose and filter paper weakening ( 43 ). Based on these results, further assessment of the PcaLOOLs on cellulosic materials focused on the wild-type proteins.…”