Engineering and characterization of carbohydrate‐binding modules for imaging cellulose fibrils biosynthesis in plant protoplasts

Abstract

Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM-based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type-A CBMs from families 3a and 64 with crystalline cellulose-I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose-I using equilibrium binding assays. To compute the adsorption (nk on ) and desorption (k off ) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide-field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts.

Supercharging carbohydrate-binding module alone enhances endocellulase thermostability, binding, and activity on cellulosic biomass

“…The maximum number of binding sites (N max ) on cellulose, binding dissociation constant (K d ), and partition coefficient (N max /K d ) were estimated from the fits and are listed in Table 1 . Recent work from our group has identified that GFP binding to cellulose is insignificant with roughly two orders of magnitude lower available binding sites than CBM tagged GFP 50 , and thus the contribution of GFP binding to cellulose has been assumed to be negligible.…”

Cell-free expression and biochemical characterization of polysaccharide-synthesizing glycosyltransferases

“…Cellulose synthase 8 (CesA8) gene from hybrid aspen (Populus tremula x tremuloides) carrying a C-terminal dodeca-HIS-tag from the previously described vector 15 was moved to the pEU-E01 cell-free wheat germ expression vector using sequence and ligation independent cloning (SLIC) protocol as described previously 21,39 . Similarly, the hyaluronan synthase (Has) gene from…”

“…The first structure of homotrimeric plant cellulose synthase was solved by Purushotham et al 19 . Yet, understanding how these CesAs coordinate and work together as a part of cellulose synthase complex (CSC) is still far from fruition both in vivo and in vitro 20,21 .…”

Supercharging Carbohydrate Binding Module Alone Enhances Endocellulase Thermostability, Binding, and Activity on Cellulosic Biomass

“…Prior work has shown that GFP does not specifically adsorb to bacterial microcrystalline cellulose. 51 Recent work from our group has identified that GFP binding to cellulose is insignificant with roughly 2 orders of magnitude lower available binding sites than CBM-tagged GFP, 52 and thus, the contribution of GFP binding to cellulose has been assumed to be negligible. The charge of each CBM (excluding GFP) at pH 5.5 was estimated with an online charge calculator (http://protcalc.…”