In vivo Functional Characterization of Hydrophilic X2 Modules in the Cellulosomal Scaffolding Protein

Abstract

As part of free cellulases or scaffolding proteins in cellulosomes, the hydrophilic non-catalytic X2 module is widely distributed in cellulolytic Clostridia or other Firmicutes bacteria. Previous biochemical studies suggest that X2 modules might increase the solubility and substrate binding affinity of X2-bearing proteins. However, their in vivo biological functions remain elusive. Here we employed CRISPR-Cas9 editing to genetically modify X2 modules by deleting the conserved motif (NGNT) from the CipC scaffoldin. Both single and double X2 mutants (X2-N: near the N terminus of CipC; X2-C: near the C terminus of CipC) presented similar stoichiometric compositions in isolated cellulosomes as the wildtype strain (WT). These X2 mutants had an elongated adaptation stage during growth on cellulose compared to cellobiose. Compared to WT, the double mutant ΔX2-NC reduced cellulose degradation by 15% and the amount of released soluble sugars by 63%. Since single X2 mutants did not present such obvious physiological changes as ΔX2-NC, there seems to be a functional redundancy between X2 modules in CipC. The in vivo adhesion assay revealed that ΔX2-NC decreased cell attachment to cellulose by 70% but a weaker effect was also overserved in single X2 mutants. These results highlight the in vivo biological role of X2 in increasing cellulose degradation efficiency by enhancing the binding affinity between cells and cellulose, which provides new perspectives for microbial engineering.

Current challenges in designer cellulosome engineering

“…The so-called hydrophilic X-module has been identified next to many catalytic modules of natural DEs and cohesins and/or CBMs of scaffoldins (Adams et al 2004). Although its function is not clear yet, its incorporation into the delineated modules should be considered because the X-module may (i) enhance the catalytic activity, (ii) act as a carbohydrate-binding module and an enzyme/protein module stabilizer, and (iii) assist in disturbing the substrate surface structure, in anchoring the cellulosome complex to the cell surface and in cell growth on cellulose (Charnock et al 2000;Duarte et al 2021;Kataeva et al 2002;Kataeva et al 2003;Pasari et al 2017;Tao et al 2022;Xu et al 2004).…”

“…During recent years, these hurdles have slowed down further progress in the DC research field, leading to a decline of studies focusing on the conversion of natural free lignocellulose-active proteins into cellulosomal docking enzymes (DEs). Yet, cellulosome research has shifted focus to (1) the deep understanding of the cellulosomes overall structure and their natural functionality like deciphering natural cellulosome 4 components and architectures that can be incorporated into DCs (Borne et al 2020;Bule et al 2018;Dorival et al 2022;Phitsuwan et al 2019;Tao et al 2022;Vera et al 2021;Zhivin-Nissan et al 2019), studying the mechanical stability of DCs (Galera-Prat et al 2018a), the cohesin-dockerin interaction specificity and affinity (Carvalho et al 2003;Wojciechowski et al 2018) and the role of linkers in DC scaffoldins (Galera-Prat et al 2018b) and (2) on creative engineering tools to lift the DCs applicability in the bio-economy field like engineering hosts for in vivo DC production/assembly (Liu et al 2019;Tao et al 2020;Tarraran et al 2021).…”