Genomics review of holocellulose deconstruction by aspergilli

Abstract

Biomass is constructed of dense recalcitrant polymeric materials: proteins, lignin, and holocellulose, a fraction constituting fibrous cellulose wrapped in hemicellulose-pectin. Bacteria and fungi are abundant in soil and forest floors, actively recycling biomass mainly by extracting sugars from holocellulose degradation. Here we review the genome-wide contents of seven Aspergillus species and unravel hundreds of gene models encoding holocellulose-degrading enzymes. Numerous apparent gene duplications followed functional evolution, grouping similar genes into smaller coherent functional families according to specialized structural features, domain organization, biochemical activity, and genus genome distribution. Aspergilli contain about 37 cellulase gene models, clustered in two mechanistic categories: 27 hydrolyze and 10 oxidize glycosidic bonds. Within the oxidative enzymes, we found two cellobiose dehydrogenases that produce oxygen radicals utilized by eight lytic polysaccharide monooxygenases that oxidize glycosidic linkages, breaking crystalline cellulose chains and making them accessible to hydrolytic enzymes. Among the hydrolases, six cellobiohydrolases with a tunnel-like structural fold embrace single crystalline cellulose chains and cooperate at nonreducing or reducing end termini, splitting off cellobiose. Five endoglucanases group into four structural families and interact randomly and internally with cellulose through an open cleft catalytic domain, and finally, seven extracellular β-glucosidases cleave cellobiose and related oligomers into glucose. Aspergilli contain, on average, 30 hemicellulase and 7 accessory gene models, distributed among 9 distinct functional categories: the backbone-attacking enzymes xylanase, mannosidase, arabinase, and xyloglucanase, the short-side-chain-removing enzymes xylan α-1,2-glucuronidase, arabinofuranosidase, and xylosidase, and the accessory enzymes acetyl xylan and feruloyl esterases.

FIG 1

Canonical holocellulose structure and deconstructive hydrolytic enzyme interactions. The main polymers integrating biomass are lignin (boxes) and holocellulose, which includes hemicellulose (light-colored, loosely branched chains) and cellulose (black linear bundled chains). Sugars: X, xylose; A, arabinose; Gc, glucuronic acid; M, mannose. Open hexagons, ferulic acid; closed circles, acetyl groups. Biomass is the principal carbon sink on earth and recruits numerous enzymes to deconstruct cellulose and hemicellulose to sway the carbon cycle via the central energy metabolism. Enzymes needed to deconstruct holocellulose include the following: cellulases, i.e., cellobiohydrolases and endoglucanases, with and without CBMs, β-glucosidases, copper-dependent lytic polysaccharide monooxygenases (LPMOs), and cellobiose dehydrogenases; and hemicellulases, i.e., xylanases, mannosidases, xyloglucanases, xylan acetyl esterases, feruloyl esterases, arabinanases, glucuronidases, and arabinofuranosidase xylosidases.

FIG 2

GH6 (A to C) and GH7 (D to F) cellobiohydrolases. (B and E) Typical tunnel-shaped catalytic cleft found in cellobiohydrolases. (C and F) Cleft depth. Cellobiohydrolases fold into an enclosed catalytic core shaped by a β-sandwich with two large, antiparallel β-sheets packed onto each other, forming a long cellulose-binding tunnel (226). The cellulosic substrate chain has to travel through the tunnel, where β-1,4-glycosyl bonds of cellobiose molecules (dimers) are hydrolyzed off the ends (GH6 or GH7 enzymes). The three-dimensional structures are for Trichoderma reesei GH6 (CBHII; PDB entry 1QK2) (108) and GH7 (CBHI or Cel7A; PDB entry 4C4C) enzymes (227).

FIG 3

Three-dimensional structures of GH5 (A and B), GH7 (C and D), and GH12 (E and F) endoglucanases. (B, D, and F) Well-defined open clefts in these endoglucanase families. Endoglucanases from the GH5 family show a catalytic module with a typical compact 8-fold β/α barrel architecture, forming an open cleft similar to those of GH7 and GH12 endoglucanases, which share the β-jelly-roll topology with an extended, open substrate-binding groove. Endoglucanases with the open cleft configuration bind randomly to internal portions of a cellulose chain and cleave β-1,4-glycosidic bonds, resulting in shortened fragments. The three-dimensional structures are for the Thermoascus aurantiacus GH5 endoglucanase Cel5A (PDB entry 1GZJ), the Trichoderma reesei GH7 enzyme EGI (PDB entry 1EG1), and the Trichoderma reesei GH12 enzyme EGIII (Egl3 or Cel12A; PDB entry 1H8V) (116, 119, 122).

FIG 4

Lytic polysaccharide monooxygenases (LPMOs). LPMOs, which are classified in the AA9 family (formerly GH61), are bivalent ion-dependent lytic polysaccharide monooxygenases. These proteins cleave cellulose chains with oxidation of various carbons (C-1, C-4, and C-6). The LPMO three-dimensional structures are for Neurospora crassa (PDB entry 4EIR) (129) (A), Hypocrea jecorina (PDB entry 2VTC) (130) (B), Thielavia terrestris (PDB entry 3EJA) (72) (C), and Phanerochaete chrysosporium (PDB entry 4B5Q) (126) (D).

FIG 5

GH74 xyloglucanobiohydrolases. (A) The three-dimensional structure consists of two tandem repeats of a seven-blade β-propeller domain which forms a large cleft and a loop where the substrate is bound. (B and C) Two views of the open cleft. The three-dimensional structure of Geotrichum sp. GH74 endoglucanase is from PDB entry 1SQJ (199, 200).