Plant cell wall architecture guided design of CBM3-GH11 chimeras with enhanced xylanase activity using a tandem repeat left-handed β-3-prism scaffold

Abstract

Effective use of plant biomass as an abundant and renewable feedstock for biofuel production and biorefinery requires efficient enzymatic mobilization of cell wall polymers. Knowledge of plant cell wall composition and architecture has been exploited to develop novel multifunctional enzymes with improved activity against lignocellulose, where a left-handed β-3-prism synthetic scaffold (BeSS) was designed for insertion of multiple protein domains at the prism vertices. This allowed construction of a series of chimeras fusing variable numbers of a GH11 β-endo-1,4-xylanase and the CipA-CBM3 with defined distances and constrained relative orientations between catalytic domains. The cellulose binding and endoxylanase activities of all chimeras were maintained. Activity against lignocellulose substrates revealed a rapid 1.6- to 3-fold increase in total reducing saccharide release and increased levels of all major oligosaccharides as measured by polysaccharide analysis using carbohydrate gel electrophoresis (PACE). A construct with CBM3 and GH11 domains inserted in the same prism vertex showed highest activity, demonstrating interdomain geometry rather than number of catalytic sites is important for optimized chimera design. These results confirm that the BeSS concept is robust and can be successfully applied to the construction of multifunctional chimeras, which expands the possibilities for knowledge-based protein design.

Keywords: Carbohydrate-binding module; Endoxylanase; Lignocellulose hydrolysis; Multifunctional protein; Protein design; Protein engineering; Synthetic biology.

Graphical abstract

Fig. 1

(A) The logo plot of residue frequency for the six positions in the HPR repeats from the 3D structures of proteins with the left-handed β-3-prism structural motif. The plot was generated by WebLogo . (B) Representation of the hexapeptide repeat motif of BeSSv2.1 protein. Prolines (position 1) and glycines (position 6) of the hexapeptides are represented in pink and orange, respectively. Asparagine residues (position 2) are colored in purple, isoleucine (position 3) in green, and the residues at position 3 and 5 are colored in blue and yellow, respectively. (C) Two orthogonal views of the three-dimensional structure of the BeSSv2.1 modeled structure. The four domain insertion positions at the vertices and the amino acid pairs for insertion of the catalytic domains are represented as spheres. (D) The amino acid sequence of the BeSSv2.1 colored according to position in the hexapeptide repeat: 1 (pink), 2 (purple); 3 (blue); 4 (green); 5 (yellow) and 6 (orange). The amino acids encoded by the four unique restriction sites are shown underlined. The GlySer pair at site 1 are encoded by the recognition site for the restriction enzyme BamHI (GGATCC), the ThrGly pair at site 2, the ThrSer pair at site 3 and the GlyThr pair at site 4 are encoded by the recognition sites for restriction enzymes AgeI (ACCGGT), SpeI (ACTAGT) and KpnI (GGTACC), respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

(A) Three surface representation views of the BeSS-CBM3-GH11 chimeras, with the BeSSv2.1 shown in gray. The CBM3 domain (C) at insertion position 1 is shown in orange and the amino acids in the cellulose binding region are highlighted in yellow. The GH11 domains (X) at insertion positions 2, 3 and 4 are colored in blue, green and violet, respectively, with the active site region highlighted in red. (B) Four ribbon representation views of the BeSS-CBM3-GH11 with all sites occupied (CXXX). The distances between the CBM cellulose binding region (yellow spheres) and the active sites of the GH11 domains (red spheres) are indicated by red dashes and black text. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

(A) Reducing sugar release by the GH11 and BeSS-CBM3-GH11 chimeras against wheat arabinoxylan (WAX). In order to maintain the concentration of catalytic domains equal in all experiments, the protein concentration for single domain constructs (GH11 alone, and the CXOO, COXO and COOX chimeras) was 2.4 μM, and for the CXXO and CXXX chimeras was 1.2 μM and 0.8 μM, respectively. (B) Binding affinity of the BeSS-CBM3-GH11 chimeras and the control (COOO) for crystalline cellulose (Avicel PH-101).

Fig. 4

PACE and total reducing sugar release after 10 min (panels on left) and 16 hr digestion (panels on right) of (A) sugarcane bagasse, (B) maize stems and (C) Miscanthus with the GH11 enzyme and the CXOO, COXO, COOX, CXXO and CXXX chimeras. In all panels, the PACE gel show xylose oligomer ladder (X1 to X6, left-hand and right-hand lanes), together with the control (COOO), and the samples from the treated substrates as indicated at the lower edge of the gel. The relative band intensities of selected saccharides X1 (violet), X2 (dark blue), X3 (light blue), XA3X (green), X4 (yellow) and (XA3XX) were analyzed using ImageJ software and presented as histograms on the right-hand side of each PACE gel image. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)