Chaetomella raphigera β-glucosidase D2-BGL has intriguing structural features and a high substrate affinity that renders it an efficient cellulase supplement for lignocellulosic biomass hydrolysis

Abstract

Background: To produce second-generation biofuels, enzymatic catalysis is required to convert cellulose from lignocellulosic biomass into fermentable sugars. β-Glucosidases finalize the process by hydrolyzing cellobiose into glucose, so the efficiency of cellulose hydrolysis largely depends on the quantity and quality of these enzymes used during saccharification. Accordingly, to reduce biofuel production costs, new microbial strains are needed that can produce highly efficient enzymes on a large scale. Results: We heterologously expressed the fungal β-glucosidase D2-BGL from a Taiwanese indigenous fungus Chaetomella raphigera in Pichia pastoris for constitutive production by fermentation. Recombinant D2-BGL presented significantly higher substrate affinity than the commercial β-glucosidase Novozyme 188 (N188; K m = 0.2 vs 2.14 mM for p-nitrophenyl β-d-glucopyranoside and 0.96 vs 2.38 mM for cellobiose). When combined with RUT-C30 cellulases, it hydrolyzed acid-pretreated lignocellulosic biomasses more efficiently than the commercial cellulase mixture CTec3. The extent of conversion from cellulose to glucose was 83% for sugarcane bagasse and 63% for rice straws. Compared to N188, use of D2-BGL halved the time necessary to produce maximal levels of ethanol by a semi-simultaneous saccharification and fermentation process. We upscaled production of recombinant D2-BGL to 33.6 U/mL within 15 days using a 1-ton bioreactor. Crystal structure analysis revealed that D2-BGL belongs to glycoside hydrolase (GH) family 3. Removing the N-glycosylation N68 or O-glycosylation T431 residues by site-directed mutagenesis negatively affected enzyme production in P. pastoris. The F256 substrate-binding residue in D2-BGL is located in a shorter loop surrounding the active site pocket relative to that of Aspergillus β-glucosidases, and this short loop is responsible for its high substrate affinity toward cellobiose. Conclusions: D2-BGL is an efficient supplement for lignocellulosic biomass saccharification, and we upscaled production of this enzyme using a 1-ton bioreactor. Enzyme production could be further improved using optimized fermentation, which could reduce biofuel production costs. Our structure analysis of D2-BGL offers new insights into GH3 β-glucosidases, which will be useful for strain improvements via a structure-based mutagenesis approach.

Improvements of the productivity and saccharification efficiency of the cellulolytic β-glucosidase D2-BGL in Pichia pastoris via directed evolution

“…Among GH3 β-glucosidases, the substrate-binding subsite + 1 is formed by a triad of hydrophobic amino acid residues, with W34, F256 and Y444 being involved in the binding of the non-reducing sugar moiety of cellobiose in D2-BGL [ 20 ]. Substitution of residue Y305 in S. cerevisiae -expressed Aspergillus niger β-glucosidase BGL1 with cysteine (C) or glycine (G) increased both apparent K m ( app K m ) and apparent V max ( app V max ) towards cellobiose and maintained the reaction rate at high cellobiose concentrations [ 17 ].…”

“…In our previous study, we have proposed to classify fungal GH3 β-glucosidases into two clades based on their protein lengths [ 20 ]. D2-BGL and T. reesei BGL belong to the clade II enzymes with fewer than 800 amino acid residues, whereas Aspergillus enzymes belong to clade I with higher residue number.…”

“…2 b). The F256 and Y260 in D2-BGL are on a loop that is one amino acid shorter than the corresponding loop in clade I enzymes, and this short loop is crucial for the high substrate affinity in D2-BGL [ 20 ]. Since clade I and II enzymes share low similarity of protein sequence and structure in the entry region of the active site, we expect that our discovery would provide new possibilities to improve clade II GH3 β-glucosidases via a structure-based mutagenesis approach.…”

“…We previously identified the GH3 β-glucosidase D2-BGL from the fungus Chaetomella raphigera based on its dynamic synergism with T. reesei cellulases to degrade cellulose efficiently [ 19 ]. Functional and structural analyses have since revealed that P. pastoris -expressed D2-BGL exhibits high substrate affinity, and its specific glycosylation patterns are required for enzyme production [ 20 ]. The study further showed that use of D2-BGL as a β-glucosidase supplement in a T. reesei cellulase mixture shortened the time necessary for bioethanol production via a semi-simultaneous saccharification and fermentation process.…”

Identification and characterization of proteins of unknown function (PUFs) in Clostridium thermocellum DSM 1313 strains as potential genetic engineering targets

“…GC (HP 6890, Agilent, CA, USA) equipped with a MS (HP 5973, Agilent, CA, USA) was used to detect esters [ 44 – 46 , 69 ]. 1 μL sample was injected into the GC capillary column (Zebron ZB-5, 30 m × 0.25 mm × 0.25 μm, Phenomenex, CA, USA) with the splitless mode at an injector temperature of 280 °C.…”