Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity.

Engineering enhanced cellobiohydrolase activity.

Glycoside Hydrolase Family 7 cellobiohydrolases (GH7 CBHs) catalyze cellulose depolymerization in cellulolytic eukaryotes, making them key discovery and engineering targets. However, there remains a lack of robust structure-activity relationships for these industrially important cellulases. Here, we compare CBHs from Trichoderma reesei (TrCel7A) and Penicillium funiculosum (PfCel7A), which exhibit a multi-modular architecture consisting of catalytic domain (CD), carbohydrate-binding module, and linker.

Distinct roles of N- and O-glycans in cellulase activity and stability.

In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood.

A versatile 2A peptide-based bicistronic protein expressing platform for the industrial cellulase producing fungus, .

Background: The industrial workhorse fungus, , is typically exploited for its ability to produce cellulase enzymes, whereas use of this fungus for over-expression of other proteins (homologous and heterologous) is still very limited. Identifying transformants expressing target protein is a tedious task due to low transformation efficiency, combined with highly variable expression levels between transformants. Routine methods for identification include PCR-based analysis, western blotting, or crude activity screening, all of which are time-consuming techniques.

Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins.

Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. Colorimetric assays for general glycoside hydrolase activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native cellulase preparations demonstrated low binding of endo- and exocellulases, high binding of xylanase, and moderate binding for β-D-glucosidases.

Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life.

Unlabelled: Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) are enzymes commonly employed in plant cell wall degradation across eukaryotic kingdoms of life, as they provide significant hydrolytic potential in cellulose turnover. To date, many fungal GH7 CBHs have been examined, yet many questions regarding structure-activity relationships in these important natural and commercial enzymes remain. Here, we present the crystal structures and a biochemical analysis of two GH7 CBHs from social amoeba: Dictyostelium discoideum Cel7A (DdiCel7A) and Dictyostelium purpureum Cel7A (DpuCel7A).

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover.

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall.

The catalytic mechanism and unique low pH optimum of Caldicellulosiruptor bescii family 3 pectate lyase.

The unique active site of the Caldicellulosiruptor bescii family 3 pectate lyase (PL3) enzyme has been thoroughly characterized using a series of point mutations, X-ray crystallography, pK(a) calculations and biochemical assays. The X-ray structures of seven PL3 active-site mutants, five of them in complex with intact trigalacturonic acid, were solved and characterized structurally, biochemically and computationally. The results confirmed that Lys108 is the catalytic base, but there is no clear candidate for the catalytic acid.

A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina.

Background: One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult.

High temperature pre-digestion of corn stover biomass for improved product yields.

Introduction: The efficient conversion of lignocellulosic feedstocks remains a key step in the commercialization of biofuels. One of the barriers to cost-effective conversion of lignocellulosic biomass to sugars remains the enzymatic saccharification process step. Here, we describe a novel hybrid processing approach comprising enzymatic pre-digestion with newly characterized hyperthermophilic enzyme cocktails followed by conventional saccharification with commercial enzyme preparations.

Heterologous protein expression in Hypocrea jecorina: a historical perspective and new developments.

Hypocrea jecorina, the sexual teleomorph of Trichoderma reesei, has long been favored as an industrial cellulase producer, first utilizing its native cellulase system and later augmented by the introduction of heterologous enzymatic activities or improved variants of native enzymes. Expression of heterologous proteins in H. jecorina was once considered difficult when the target was an improved variant of a native cellulase.

Identification and characterization of core cellulolytic enzymes from Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) critical for hydrolysis of lignocellulosic biomass.

Background: Enzymatic hydrolysis of pretreated lignocellulosic biomass is an essential process for the production of fermentable sugars for industrial use. A better understanding of fungal cellulase systems will provide clues for maximizing the hydrolysis of target biomass. Talaromyces cellulolyticus is a promising fungus for cellulase production and efficient biomass hydrolysis.

Engineering towards a complete heterologous cellulase secretome in Yarrowia lipolytica reveals its potential for consolidated bioprocessing.

Background: Yarrowia lipolytica is an oleaginous yeast capable of metabolizing glucose to lipids, which then accumulate intracellularly. However, it lacks the suite of cellulolytic enzymes required to break down biomass cellulose and cannot therefore utilize biomass directly as a carbon source. Toward the development of a direct microbial conversion platform for the production of hydrocarbon fuels from cellulosic biomass, the potential for Y.

Predicting enzyme adsorption to lignin films by calculating enzyme surface hydrophobicity.

The inhibitory action of lignin on cellulase cocktails is a major challenge to the biological saccharification of plant cell wall polysaccharides. Although the mechanism remains unclear, hydrophobic interactions between enzymes and lignin are hypothesized to drive adsorption. Here we evaluate the role of hydrophobic interactions in enzyme-lignin binding.

Cellulose-inducible xylanase Xyl10A from Acremonium cellulolyticus: Purification, cloning and homologous expression.

Cellulose-inducible endo-β-1,4-xylanase (Xyl10A) from the mesophilic fungus Acremonium cellulolyticus was purified, characterized, and expressed by a homologous expression system. A. cellulolyticus CF-2612 produces a high level of xylanase upon induction by Solka-Floc cellulose.

Genomic, proteomic, and biochemical analyses of oleaginous Mucor circinelloides: evaluating its capability in utilizing cellulolytic substrates for lipid production.

Lipid production by oleaginous microorganisms is a promising route to produce raw material for the production of biodiesel. However, most of these organisms must be grown on sugars and agro-industrial wastes because they cannot directly utilize lignocellulosic substrates. We report the first comprehensive investigation of Mucor circinelloides, one of a few oleaginous fungi for which genome sequences are available, for its potential to assimilate cellulose and produce lipids.

Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose.

Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers.

Construction of a starch-inducible homologous expression system to produce cellulolytic enzymes from Acremonium cellulolyticus.

A starch-inducible homologous expression system in Acremonium cellulolyticus was constructed to successfully produce recombinant cellulolytic enzymes. A. cellulolyticus Y-94 produced amylolytic enzymes and cellulolytic enzymes as major proteins in the culture supernatant when grown with soluble starch (SS) and Solka-Flock cellulose (SF), respectively.

Analysis of transgenic glycoside hydrolases expressed in plants: T. reesei CBH I and A. cellulolyticus EI.

Plant cell walls are composed of three basic structural biomolecules: cellulose, hemicellulose, and lignin with cellulose being the most abundant biopolymer on earth. Cellulose is composed of cellodextrins, which are linear polymers of glucose, and considered to be microcrystalline in structure. The conversion of cellulose to free glucose is one of the primary steps in the fermentative conversion of biomass to fuels and chemicals.

Natural paradigms of plant cell wall degradation.

Natural processes of recycling carbon from plant cell walls are slow but very efficient, generally involving microbial communities and their secreted enzymes. Efficient combinations of microbial communities and enzymes act in a sequential and synergistic manner to degrade plant cell walls. Recent understanding of plant cell wall ultra-structure, as well as the carbon metabolism, ATP production, and ecology of participating microbial communities, and the biochemical properties of their cellulolytic enzymes have led to new perspectives on saccharification of biomass.

Heterologous expression of glycosyl hydrolases in planta: a new departure for biofuels.

The concept of expressing non-plant glycosyl hydrolase genes in plant tissue is nearly two decades old, yet relatively little work in this field has been reported. However, resurgent interest in technologies aimed at enabling processes that convert biomass to sugars and fuels has turned attention toward this intuitive solution. There are several challenges facing researchers in this field, including the development of better and more specifically targeted delivery systems for hydrolytic genes, the successful folding and post-translational modification of heterologous proteins and the development of cost-effective process strategies utilizing these transformed plants.

Complete genome sequence of the complex carbohydrate-degrading marine bacterium, Saccharophagus degradans strain 2-40 T.

The marine bacterium Saccharophagus degradans strain 2-40 (Sde 2-40) is emerging as a vanguard of a recently discovered group of marine and estuarine bacteria that recycles complex polysaccharides. We report its complete genome sequence, analysis of which identifies an unusually large number of enzymes that degrade >10 complex polysaccharides. Not only is this an extraordinary range of catabolic capability, many of the enzymes exhibit unusual architecture including novel combinations of catalytic and substrate-binding modules.

Complete cellulase system in the marine bacterium Saccharophagus degradans strain 2-40T.

Saccharophagus degradans strain 2-40 is a representative of an emerging group of marine complex polysaccharide (CP)-degrading bacteria. It is unique in its metabolic versatility, being able to degrade at least 10 distinct CPs from diverse algal, plant and invertebrate sources. The S.

Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagus degradans 2-40.

Saccharophagus degradans 2-40 (formerly Microbulbifer degradans 2-40) is a marine gamma-subgroup proteobacterium capable of degrading many complex polysaccharides, such as agar. While several agarolytic systems have been characterized biochemically, the genetics of agarolytic systems have been only partially determined. By use of genomic, proteomic, and genetic approaches, the components of the S.

Saccharophagus degradans gen. nov., sp. nov., a versatile marine degrader of complex polysaccharides.

Gammaproteobacteria belonging and related to the genus Microbulbifer are an emerging group of complex carbohydrate-degrading marine bacteria. Previously, all of the representatives were placed within Microbulbifer or were unclassified. Recently, a new genus, Teredinibacter, represented by a single species, Teredinibacter turnerae, was formed to include an endosymbiotic branch of these organisms.