Improving the thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed evolution.

Secreted mixtures of Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which exhibited a 10.4 °C increase in Tm and a 44-fold greater half-life compared with the wild-type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 °C. The X-ray crystal structure of the catalytic domain was determined at 2.1 Å resolution and showed that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations revealed that the catalytic domain of wild-type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, whereas at elevated temperature (475 and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites.

Expression, crystal structure and cellulase activity of the thermostable cellobiohydrolase Cel7A from the fungus Humicola grisea var. thermoidea.

Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) play a key role in biomass recycling in nature. They are typically the most abundant enzymes expressed by potent cellulolytic fungi, and are also responsible for the majority of hydrolytic potential in enzyme cocktails for industrial processing of plant biomass. The thermostability of the enzyme is an important parameter for industrial utilization. In this study, Cel7 enzymes from different fungi were expressed in a fungal host and assayed for thermostability, including Hypocrea jecorina Cel7A as a reference. The most stable of the homologues, Humicola grisea var. thermoidea Cel7A, exhibits a 10°C higher melting temperature (T(m) of 72.5°C) and showed a 4-5 times higher initial hydrolysis rate than H. jecorina Cel7A on phosphoric acid-swollen cellulose and showed the best performance of the tested enzymes on pretreated corn stover at elevated temperature (65°C, 24 h). The enzyme shares 57% sequence identity with H. jecorina Cel7A and consists of a GH7 catalytic module connected by a linker to a C-terminal CBM1 carbohydrate-binding module. The crystal structure of the H. grisea var. thermoidea Cel7A catalytic module (1.8 Šresolution; R(work) and R(free) of 0.16 and 0.21, respectively) is similar to those of other GH7 CBHs. The deviations of several loops along the cellulose-binding path between the two molecules in the asymmetric unit indicate higher flexibility than in the less thermostable H. jecorina Cel7A.

The crystal structure of the core domain of a cellulose induced protein (Cip1) from Hypocrea jecorina, at 1.5 Å resolution.

In an effort to characterise the whole transcriptome of the fungus Hypocrea jecorina, cDNA clones of this fungus were identified that encode for previously unknown proteins that are likely to function in biomass degradation. One of these newly identified proteins, found to be co-regulated with the major H. jecorina cellulases, is a protein that was denoted Cellulose induced protein 1 (Cip1). This protein consists of a glycoside hydrolase family 1 carbohydrate binding module connected via a linker region to a domain with yet unknown function. After cloning and expression of Cip1 in H. jecorina, the protein was purified and biochemically characterised with the aim of determining a potential enzymatic activity for the novel protein. No hydrolytic activity against any of the tested plant cell wall components was found. The proteolytic core domain of Cip1 was then crystallised, and the three-dimensional structure of this was determined to 1.5 Å resolution utilising sulphur single-wavelength anomalous dispersion phasing (sulphor-SAD). A calcium ion binding site was identified in a sequence conserved region of Cip1 and is also seen in other proteins with the same general fold as Cip1, such as many carbohydrate binding modules. The presence of this ion was found to have a structural role. The Cip1 structure was analysed and a structural homology search was performed to identify structurally related proteins. The two published structures with highest overall structural similarity to Cip1 found were two poly-lyases: CsGL, a glucuronan lyase from H. jecorina and vAL-1, an alginate lyase from the Chlorella virus. This indicates that Cip1 may be a lyase. However, initial trials did not detect significant lyase activity for Cip1. Cip1 is the first structure to be solved of the 23 currently known Cip1 sequential homologs (with a sequence identity cut-off of 25%), including both bacterial and fungal members.

The structure of a bacterial cellobiohydrolase: the catalytic core of the Thermobifida fusca family GH6 cellobiohydrolase Cel6B.

Cellulases, glycoside hydrolases that catalyze the degradation of cellulose, are classified as either endoglucanases or cellobiohydrolases (CBHs) based on their architecture and mode of action on the cellulose. CBHs bind the cellulose chain in a more or less closed tunnel and cleave off cellobiose units processively from one end of the cellulosic polymer, while endoglucanases have their active sites in a more or less open cleft and show a higher tendency to cut bonds internally in the polymer. The CBH Cel6A (also called CBH2) from the ascomycete Hypocrea jecorina has a much shorter substrate-binding tunnel and seems less processive than the CBH Cel7A (CBH1), from the same fungus. Here, we present the X-ray crystal structure of the catalytic domain of the CBH Cel6B, also called E3, from the soil bacterium Thermobifida fusca, both in its apo form and co-crystallized with cellobiose. The enzyme structure reveals that the Cel6B enzyme has a much longer substrate-binding site than its fungal GH6 counterparts. The tunnel is comparable in length to that of GH7 CBHs. In the ligand structure with cellobiose, the tunnel exit is completely closed by a 13-residue loop not present in fungal GH6 enzymes. The loop needs to be displaced to allow cellobiose product release for a processive action by the enzyme. When ligand is absent, seven of these residues are not visible in the electron density and the tunnel exit is open.

Hypocrea jecorina CEL6A protein engineering.

The complex technology of converting lignocellulose to fuels such as ethanol has advanced rapidly over the past few years, and enzymes are a critical component of this technology. The production of effective enzyme systems at cost structures that facilitate commercial processes has been the focus of research for many years. Towards this end, the H. jecorina cellobiohydrolases, CEL7A and CEL6A, have been the subject of protein engineering at Genencor. Our first rounds of cellobiohydrolase engineering were directed towards improving the thermostability of both of these enzymes and produced variants of CEL7A and CEL6A with apparent melting temperatures above 70°C, placing their stability on par with that of H. jecorina CEL5A (EG2) and CEL3A (BGL1). We have now moved towards improving CEL6A- and CEL7A-specific performance in the context of a complete enzyme system under industrially relevant conditions. Achievement of these goals required development of new screening strategies and tools. We discuss these advances along with some results, focusing mainly on engineering of CEL6A.

Comparative sugar recovery and fermentation data following pretreatment of poplar wood by leading technologies.

Through a Biomass Refining Consortium for Applied Fundamentals and Innovation among Auburn University, Dartmouth College, Michigan State University, the National Renewable Energy Laboratory, Purdue University, Texas A&M University, the University of British Columbia, and the University of California at Riverside, leading pretreatment technologies based on ammonia fiber expansion, aqueous ammonia recycle, dilute sulfuric acid, lime, neutral pH, and sulfur dioxide were applied to a single source of poplar wood, and the remaining solids from each technology were hydrolyzed to sugars using the same enzymes. Identical analytical methods and a consistent material balance methodology were employed to develop comparative performance data for each combination of pretreatment and enzymes. Overall, compared to data with corn stover employed previously, the results showed that poplar was more recalcitrant to conversion to sugars and that sugar yields from the combined operations of pretreatment and enzymatic hydrolysis varied more among pretreatments. However, application of more severe pretreatment conditions gave good yields from sulfur dioxide and lime, and a recombinant yeast strain fermented the mixed stream of glucose and xylose sugars released by enzymatic hydrolysis of water washed solids from all pretreatments to ethanol with similarly high yields. An Agricultural and Industrial Advisory Board followed progress and helped steer the research to meet scientific and commercial needs.

The first structure of a glycoside hydrolase family 61 member, Cel61B from Hypocrea jecorina, at 1.6 A resolution.

The glycoside hydrolase (GH) family 61 is a long-recognized, but still recondite, class of proteins, with little known about the activity, mechanism or function of its more than 70 members. The best-studied GH family 61 member, Cel61A of the filamentous fungus Hypocrea jecorina, is known to be an endoglucanase, but it is not clear if this represents the main activity or function of this family in vivo. We present here the first structure for this family, that of Cel61B from H. jecorina. The best-quality crystals were formed in the presence of nickel, and the crystal structure was solved to 1.6 A resolution using a single-wavelength anomalous dispersion method with nickel as the source of anomalous scatter. Cel61B lacks a carbohydrate-binding module and is a single-domain protein that folds into a twisted beta-sandwich. A structure-aided sequence alignment of all GH family 61 proteins identified a highly conserved group of residues on the surface of Cel61B. Within this patch of mostly polar amino acids was a site occupied by the intramolecular nickel hexacoordinately bound in the solved structure. In the Cel61B structure, there is no easily identifiable carbohydrate-binding cleft or pocket or catalytic center of the types normally seen in GHs. A structural comparison search showed that the known structure most similar to Cel61B is that of CBP21 from the Gram-negative soil bacterium Serratia marcescens, a member of the carbohydrate-binding module family 33 proteins. A polar surface patch highly conserved in that structural family has been identified in CBP21 and shown to be involved in chitin binding and in the protein's enhancement of chitinase activities. The analysis of the Cel61B structure is discussed in light of our continuing research to better understand the activities and function of GH family 61.

An investigation of the substrate specificity of the xyloglucanase Cel74A from Hypocrea jecorina.

The substrate specificity of the xyloglucanase Cel74A from Hypocrea jecorina (Trichoderma reesei) was examined using several polysaccharides and oligosaccharides. Our results revealed that xyloglucan chains are hydrolyzed at substituted Glc residues, in contrast to the action of all known xyloglucan endoglucanases (EC 3.2.1.151). The building block of xyloglucan, XXXG (where X is a substituted Glc residue, and G is an unsubstituted Glc residue), was rapidly degraded to XX and XG (k(cat) = 7.2 s(-1) and Km = 120 microM at 37 degrees C and pH 5), which has only been observed before with the oligoxyloglucan-reducing-end-specific cellobiohydrolase from Geotrichum (EC 3.2.1.150). However, the cellobiohydrolase can only release XG from XXXGXXXG, whereas Cel74A hydrolyzed this substrate at both chain ends, resulting in XGXX. Differences in the length of a specific loop at subsite + 2 are discussed as being the basis for the divergent specificity of these xyloglucanases.

Structural and biochemical studies of GH family 12 cellulases: improved thermal stability, and ligand complexes.

In this review we will describe how we have gathered structural and biochemical information from several homologous cellulases from one class of glycoside hydrolases (GH family 12), and used this information within the framework of a protein-engineering program for the design of new variants of these enzymes. These variants have been characterized to identify some of the positions and the types of mutations in the enzymes that are responsible for some of the biochemical differences in thermal stability and activity between the homologous enzymes. In this process we have solved the three-dimensional structure of four of these homologous GH 12 cellulases: Three fungal enzymes, Humicola grisea Cel12A, Hypocrea jecorina Cel12A and Hypocrea schweinitzii Cel12A, and one bacterial, Streptomyces sp. 11AG8 Cel12A. We have also determined the three-dimensional structures of the two most stable H. jecorina Cel12A variants. In addition, four ligand-complex structures of the wild-type H. grisea Cel12A enzyme have been solved and have made it possible to characterize some of the interactions between substrate and enzyme. The structural and biochemical studies of these related GH 12 enzymes, and their variants, have provided insight on how specific residues contribute to protein thermal stability and enzyme activity. This knowledge can serve as a structural toolbox for the design of Cel12A enzymes with specific properties and features suited to existing or new applications.

Crystal complex structures reveal how substrate is bound in the -4 to the +2 binding sites of Humicola grisea Cel12A.

As part of an ongoing enzyme discovery program to investigate the properties and catalytic mechanism of glycoside hydrolase family 12 (GH 12) endoglucanases, a GH family that contains several cellulases that are of interest in industrial applications, we have solved four new crystal structures of wild-type Humicola grisea Cel12A in complexes formed by soaking with cellobiose, cellotetraose, cellopentaose, and a thio-linked cellotetraose derivative (G2SG2). These complex structures allow mapping of the non-covalent interactions between the enzyme and the glucosyl chain bound in subsites -4 to +2 of the enzyme, and shed light on the mechanism and function of GH 12 cellulases. The unhydrolysed cellopentaose and the G2SG2 cello-oligomers span the active site of the catalytically active H.grisea Cel12A enzyme, with the pyranoside bound in subsite -1 displaying a S31 skew boat conformation. After soaking in cellotetraose, the cello-oligomer that is found bound in site -4 to -1 contains a beta-1,3-linkage between the two cellobiose units in the oligomer, which is believed to have been formed by a transglycosylation reaction that has occurred during the ligand soak of the protein crystals. The close fit of this ligand and the binding sites occupied suggest a novel mixed beta-glucanase activity for this enzyme.

The Humicola grisea Cel12A enzyme structure at 1.2 A resolution and the impact of its free cysteine residues on thermal stability.

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have extended our previous work on the structural and biochemical diversity of GH 12 homologs to include the most stable fungal GH 12 found, Humicola grisea Cel12A. The H. grisea enzyme was much more stable to irreversible thermal denaturation than the Trichoderma reesei enzyme. It had an apparent denaturation midpoint (T(m)) of 68.7 degrees C, 14.3 degrees C higher than the T. reesei enzyme. There are an additional three cysteines found in the H. grisea Cel12A enzyme. To determine their importance for thermal stability, we constructed three H. grisea Cel12A single mutants in which these cysteines were exchanged with the corresponding residues in the T. reesei enzyme. We also introduced these cysteine residues into the T. reesei enzyme. The thermal stability of these variants was determined. Substitutions at any of the three positions affected stability, with the largest effect seen in H. grisea C206P, which has a T(m) 9.1 degrees C lower than that of the wild type. The T. reesei cysteine variant that gave the largest increase in stability, with a T(m) 3.9 degrees C higher than wild type, was the P201C mutation, the converse of the destabilizing C206P mutation in H. grisea. To help rationalize the results, we have determined the crystal structure of the H. grisea enzyme and of the most stable T. reesei cysteine variant, P201C. The three cysteines in H. grisea Cel12A play an important role in the thermal stability of this protein, although they are not involved in a disulfide bond.

Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei.

The filamentous fungus Trichoderma reesei produces and secretes profuse quantities of enzymes that act synergistically to degrade cellulase and related biomass components. We partially sequenced over 5100 random T. reesei cDNA clones. Among the sequences whose predicted gene products had significant similarity to known proteins, 12 were identified that encode previously unknown enzymes that likely function in biomass degradation. Microarrays were used to query the expression levels of each of the sequences under different conditions known to induce cellulolytic enzyme synthesis. Most of the genes encoding known and putative biomass-degrading enzymes were transcriptionally co-regulated. Moreover, despite the fact that several of these enzymes are not thought to degrade cellulase directly, they were coordinately overexpressed in a cellulase overproducing strain. A variety of additional sequences whose function could not be ascribed using the limited sequence available displayed analogous behavior and may also play a role in biomass degradation or in the synthesis of biomass-degrading enzymes. Sequences exhibiting additional regulatory patterns were observed that might reflect roles in regulation of cellulase biosynthesis. However, genes whose products are involved in protein processing and secretion were not highly regulated during cellulase induction.

Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability.

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have studied the biochemical diversity of several GH 12 homologs. The H. schweinitzii Cel12A enzyme differs from the T. reesei Cel12A enzyme by only 14 amino acids (93% sequence identity), but is much less thermally stable. The bacterial Cel12A enzyme from S. sp. 11AG8 shares only 28% sequence identity to the T. reesei enzyme, and is much more thermally stable. Each of the 14 sequence differences from H. schweinitzii Cel12A were introduced in T. reesei Cel12A to determine the effect of these amino acid substitutions on enzyme stability. Several of the T. reesei Cel12A variants were found to have increased stability, and the differences in apparent midpoint of thermal denaturation (T(m)) ranged from a 2.5 degrees C increase to a 4.0 degrees C decrease. The least stable recruitment from H. schweinitzii Cel12A was A35S. Consequently, the A35V substitution was recruited from the more stable S. sp. 11AG8 Cel12A and this T. reesei Cel12A variant was found to have a T(m) 7.7 degrees C higher than wild type. Thus, the buried residue at position 35 was shown to be of critical importance for thermal stability in this structural family. There was a ninefold range in the specific activities of the Cel12 homologs on o-NPC. The most and least stable T. reesei Cel12A variants, A35V and A35S, respectively, were fully active. Because of their thermal tolerance, S. sp. 11AG8 Cel12A and T. reesei Cel12A variant A35V showed a continual increase in activity over the temperature range of 25 degrees C to 60 degrees C, whereas the less stable enzymes T. reesei Cel12A wild type and the destabilized A35S variant, and H. schweinitzii Cel12A showed a decrease in activity at the highest temperatures. The crystal structures of the H. schweinitzii, S. sp. 11AG8, and T. reesei A35V Cel12A enzymes have been determined and compared with the wild-type T. reesei Cel12A enzyme. All of the structures have similar Calpha traces, but provide detailed insight into the nature of the stability differences. These results are an example of the power of homolog recruitment as a method for identifying residues important for stability.