Cell‐surface binding domains from Clostridium cellulovorans can be used for surface display of cellulosomal scaffoldins in Lactococcus lactis

Abstract

Engineering microbial strains combining efficient lignocellulose metabolization and high‐value chemical production is a cutting‐edge strategy towards cost‐sustainable 2nd generation biorefining. Here, protein components of the Clostridium cellulovorans cellulosome were introduced in Lactococcus lactis IL1403, one of the most efficient lactic acid producers but unable to directly ferment cellulose. Cellulosomes are protein complexes with high cellulose depolymerization activity whose synergistic action is supported by scaffolding protein(s) (i.e., scaffoldins). Scaffoldins are involved in bringing enzymes close to each other and often anchor the cellulosome to the cell surface. In this study, three synthetic scaffoldins were engineered by using domains derived from the main scaffoldin CbpA and the Endoglucanase E (EngE) of the C. cellulovorans cellulosome. Special focus was on CbpA X2 and EngE S‐layer homology (SLH) domains possibly involved in cell‐surface anchoring. The recombinant scaffoldins were successfully introduced in and secreted by L. lactis. Among them, only that carrying the three EngE SLH modules was able to bind to the L. lactis surface although these domains lack the conserved TRAE motif thought to mediate binding with secondary cell wall polysaccharides. The synthetic scaffoldins engineered in this study could serve for assembly of secreted or surface‐displayed designer cellulosomes in L. lactis.

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

“…Western blotting did not detect the presence of X2 on any of these three strains post incubation with purified X2, indicating that X2 cannot directly bind to cell surfaces ( Figure 4C ). This agrees well with a recent study ( Tarraran et al, 2021 ).…”

“…Although previous studies indicated that the X2 module might directly bind to the cell wall ( Kosugi et al, 2004 ), our in vitro protein-cell wall binding assay indicated that the X2 module protein could not directly bind to the cell wall ( Figure 4C ). This is consistent with a recent study that scaffoldins containing X2 domains derived from CpbA were not able to bind to the L. lactis surface ( Tarraran et al, 2021 ). Based on structural analysis of the X2 modules ( Mosbah et al, 2000 ), the surface of the X2 module is predominantly covered by hydrophilic amino acids and only contains a hydrophobic shallow groove, which could explain why the X2 module could not directly bind to the cell wall.…”

“…This suggests that X2 modules are widely distributed and may serve an important role in the biodegradation of lignocellulosic biomass. Based on the structure and in vitro biochemistry assays of X2 modules, it belongs to the immunoglobulin superfamily and has been predicted to be associated with the localization of the cellulosome, cellulose binding, cell wall binding, or increased enzymatic activities for free cellulase ( Kosugi et al, 2004 ; Chanal et al, 2011 ; Ravachol et al, 2015 ; Pasari et al, 2017 ; Zhang et al, 2018 ; Tarraran et al, 2021 ). However, little is known about its in vivo biological functions and significance.…”

Current Progress in Production of Building-Block Organic Acids by Consolidated Bioprocessing of Lignocellulose

“…Native cellulolytic strategies (NCSs) aim at introducing and/or improving high-value product biosynthetic pathways into natural (hemi)cellulolytic strains (e.g., Clostridium cellulovorans, Clostridium thermocellum, Figure 1c) [5]. The purpose of recombinant cellulolytic strategies (RCSs) is to confer (hemi)cellulolytic ability to microorganisms with valuable product formation properties (e.g., Saccharomyces cerevisiae, Yarrowia lipolytica, lactic acid bacteria) and include heterologous cellulase expression [47,53,54] (Figure 1d). Current progress of NCSs benefits from recent development of efficient gene tools for manipulating a number of (hemi)cellulolytic bacteria (e.g., Caldicellulosiruptor bescii, C. cellulovorans, C. thermocellum, C. cellulovorans, Thermoanaerobacterium saccharolyticum) [55][56][57][58][59], and fungi (e.g., Myceliophthora thermophila) [60].…”

Editorial: Microorganisms for Consolidated 2nd Generation Biorefining

“…Recombinant cellulolytic strategies have been severely hampered by some major issues: (i) the extreme complexity and sophistication of native cellulase systems (Xu et al, 2015;Leis et al, 2017;Bule et al, 2018;Galera-Prat et al, 2020) (together with the high recalcitrance of lignocellulosic substrates) makes it difficult to mimic their efficiency through minimal artificial enzyme mixtures/complexes; (ii) insufficient understanding of the mechanisms promoting cellulase secretion Wu, 2013, 2014;De Paula et al, 2019) as well as species-specific protein secretion mechanisms challenge rational engineering of recombinant cellulolytic strains. Heterologous expression of cellulases has frequently been associated with cell toxicity (Mingardon et al, 2011;Kovács et al, 2013;Tarraran et al, 2021), and/or cellulase proteolysis by the host (Mingardon et al, 2005(Mingardon et al, , 2011, and/or low levels of cellulase activity (Van Rensburg et al, 2012) and/or the activation of the unfolded protein response (Ilmén et al, 2011), and/or metabolic burden (Ding et al, 2018). Metabolic burden refers to perturbation of host metabolism by heterologous protein expression and is generally attributed to energetic costs and competition for gene transcription/protein translation cell machinery associated with production of heterologous cellulases which generally cause a decrease in growth efficiency (Van Rensburg et al, 2012).…”