SCHEMA Recombination of a Fungal Cellulase Uncovers a Single Mutation That Contributes Markedly to Stability

Abstract

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...

Computational Investigation of Glycosylation Effects on a Family 1 Carbohydrate-binding Module

“…Several routes exist for increasing cellulase performance, including rational and evolutionary methods to improve specific activity (8), screening for improved thermal tolerance (11,12), and the often overlooked strategy of increasing binding affinity to carbohydrates via CBM engineering (13)(14)(15)(16). In the lattermost case, several groups have shown that by increasing CBM binding affinity to cellulose via single point mutations or by swapping complete CBMs from other enzymes with higher binding affinity, cellulase enzyme activity can be improved (15,16).…”

Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins

“…This suggests that each sequence/structural block behaves differently in different contexts. For certain soluble protein properties (e.g., thermostability), it has been shown that block contributions are additive, that is, context independent, and that chimera stability can be predicted using linear regression (28,29,49,50). Our data suggest that ChR localization and photocurrent properties, however, require a more complex model to account for the nonlinear dependence of function on block sequence.…”

Recombinant Cellulase and Cellulosome Systems

“…Interestingly, the recombinant enzyme displayed resistance to glucose inhibition in addition to thermotolerance. Others have reported on the expression of thermotolerant cellulases in yeast using a mutagenesis and recombination strategies rather than a discovery approach to further improve stability and activity of the recombinant enzymes [23][24][25].…”