A quantitative linear model accurately (R 2 ‫؍‬ 0.88) describes the thermostabilities of 54 characterized members of a family of fungal cellobiohydrolase class II (CBH II) cellulase chimeras made by SCHEMA recombination of three fungal enzymes, demonstrating that the contributions of SCHEMA sequence blocks to stability are predominantly additive. Thirty-one of 31 predicted thermostable CBH II chimeras have thermal inactivation temperatures higher than the most thermostable parent CBH II, from Humicola insolens, and the model predicts that hundreds more CBH II chimeras share this superior thermostability. Eight of eight thermostable chimeras assayed hydrolyze the solid cellulosic substrate Avicel at temperatures at least 5°C above the most stable parent, and seven of these showed superior activity in 16-h Avicel hydrolysis assays. The sequence-stability model identified a single block of sequence that adds 8.5°C to chimera thermostability. Mutating individual residues in this block identified the C313S substitution as responsible for the entire thermostabilizing effect. Introducing this mutation into the two recombination parent CBH IIs not featuring it (Hypocrea jecorina and H. insolens) decreased inactivation, increased maximum Avicel hydrolysis temperature, and improved long time hydrolysis performance. This mutation also stabilized and improved Avicel hydrolysis by Phanerochaete chrysosporium CBH II, which is only 55-56% identical to recombination parent CBH IIs. Furthermore, the C313S mutation increased total H. jecorina CBH II activity secreted by the Saccharomyces cerevisiae expression host more than 10-fold. Our results show that SCHEMA structure-guided recombination enables quantitative prediction of cellulase chimera thermostability and efficient identification of stabilizing mutations.SCHEMA is a computational approach to identifying blocks of sequence that minimize structural disruption when they are recombined in chimeric proteins (1). SCHEMA recombination of eight blocks from three fungal cellobiohydrolase class II (CBH II) 2 genes was used in our previous work to create a library of 3 8 ϭ 6,561 chimeric sequences, all having the native Hypocrea jecorina cellulose binding module and linker and observed to feature a degree of glycosylation similar to that found in native CBH IIs secreted by fungi (2). Synthesis and characterization of selected CBH II chimeras expressed in Saccharomyces cerevisiae revealed enzymes with thermostabilities and cellulose hydrolysis performance superior to those of the parent enzymes from Humicola insolens, H. jecorina, and Chaetomium thermophilum.Our prior analysis showed that a qualitative model based on sequence-stability data from 23 functional chimeras (categorizing blocks as destabilizing, stabilizing, or neutral) could identify highly stable chimeras in the SCHEMA library (2). When studying SCHEMA recombination of a bacterial cytochrome P450, we previously estimated that building a quantitative regression model would require stability measurements for at least 35 re...

BackgroundThe concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. Exoglucanase Cel48S and endoglucanase Cel8A, both key elements of the natural cellulosome from this bacterium, were engineered previously for increased thermostability, either by SCHEMA, a structure-guided, site-directed protein recombination method, or by consensus-guided mutagenesis combined with random mutagenesis using error-prone PCR, respectively. A thermostable β-glucosidase BglA mutant was also selected from a library generated by error-prone PCR that will assist the two cellulases in their methodic deconstruction of crystalline cellulose. The effects of a thermostable scaffoldin versus those of a largely mesophilic scaffoldin were also examined. By improving the stability of the enzyme subunits and the structural component, we aimed to improve cellulosome-mediated deconstruction of cellulosic substrates.ResultsThe results demonstrate that the combination of thermostable enzymes as free enzymes and a thermostable scaffoldin was more active on the cellulosic substrate than the wild-type enzymes. Significantly, “thermostable” designer cellulosomes exhibited a 1.7-fold enhancement in cellulose degradation compared to the action of conventional designer cellulosomes that contain the respective wild-type enzymes. For designer cellulosome formats, the use of the thermostabilized scaffoldin proved critical for enhanced enzymatic performance under conditions of high temperatures.ConclusionsSimple improvement in the activity of a given enzyme does not guarantee its suitability for use in an enzyme cocktail or as a designer cellulosome component. The true merit of improvement resides in its ultimate contribution to synergistic action, which can only be determined experimentally. The relevance of the mutated thermostable enzymes employed in this study as components in multienzyme systems has thus been confirmed using designer cellulosome technology. Enzyme integration via a thermostable scaffoldin is critical to the ultimate stability of the complex at higher temperatures. Engineering of thermostable cellulases and additional lignocellulosic enzymes may prove a determinant parameter for development of state-of-the-art designer cellulosomes for their employment in the conversion of cellulosic biomass to soluble sugars.Graphical abstractConversion of conventional designer cellulosomes into thermophilic designer cellulosomesElectronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0577-z) contains supplementary material, which is available to authorized users.

We introduce a method for identifying elements of a protein structure that can be shuffled to make chimeric proteins from two or more homologous parents. Formulating recombination as a graph-partitioning problem allows us to identify noncontiguous segments of the sequence that should be inherited together in the progeny proteins. We demonstrate this noncontiguous recombination approach by constructing a chimera of b-glucosidases from two different kingdoms of life. Although the protein's alpha-beta barrel fold has no obvious subdomains for recombination, noncontiguous SCHEMA recombination generated a functional chimera that takes approximately half its structure from each parent. The X-ray crystal structure shows that the structural blocks that make up the chimera maintain the backbone conformations found in their respective parental structures. Although the chimera has lower b-glucosidase activity than the parent enzymes, the activity was easily recovered by directed evolution. This simple method, which does not rely on detailed atomic models, can be used to design chimeras that take structural, and functional, elements from distantly-related proteins.

Sequence diversity within a family of functional enzymes provides a platform for elucidating structure-function relationships and for protein engineering to improve properties important for applications. Access to nature's vast sequence diversity is often limited by the fact that only a few enzymes have been characterized in a given family. Here, we recombined the catalytic domains of three glycoside hydrolase family 48 bacterial cellulases (Cel48; EC 3.2.1.176) -Clostridium cellulolyticum CelF, Clostridium stercorarium CelY, and Clostridium thermocellum CelS -to create a diverse library of Cel48 enzymes with an average of 106 mutations from the closest native enzyme. Within this set, we found large variations in properties such as the functional temperature range, stability, and specific activity on crystalline cellulose. We showed that functional status and stability were predictable from simple linear models of the sequence-property data: recombined protein fragments contributed additively to these properties in a given chimera. Using this, we correctly predicted sequences that were as stable as any of the native Cel48 enzymes described to date. The characterization of 60 active Cel48 chimeras expands the number of characterized Cel48 enzymes from 13 to 73. Our work illustrates the role that structure-guided recombination can play in helping to identify sequencefunction relationships within a family of enzymes by supplementing natural diversity with synthetic diversity.

Noncontiguous recombination (NCR) is a method to identify pieces of structure that can be swapped among homologous proteins to create new, chimeric proteins. These “blocks” are encoded by elements of sequence that are not necessarily contiguous along the polypeptide chain. We used NCR to design a library in which blocks of structure from Hypocrea jecorina cellobiohydrolase I (Cel7A) and its two thermostable homologues from Talaromyces emersonii and Chaetomium thermophilum are shuffled to create 531,438 possible chimeric enzymes. We constructed a maximally informative subset of 35 chimeras to analyze this library and found that the blocks contribute additively to the stability of a chimera. Within two highly stabilizing blocks, we uncovered six single amino acid substitutions that each improve the stability of H. jecorina cellobiohydrolase I by 1–3 °C. The small number of measurements required to find these mutations demonstrates that noncontiguous recombination is an efficient strategy for identifying stabilizing mutations.