(509a) A Family of Thermostable Fungal Cellulases Created by Structure-Guided Recombination

We have used structure-guided recombination to create a family containing over 6,500 fungal cellobiohydrolase class II (CBH II) chimera genes. Approximately half the chimeras in a sample set of these genes were secreted in active form from a recombinant S. cerevisiae host, suggesting that this CBH II family contains over 3,000 catalytically active members. The majority of these novel CBH IIs differ from their closest natural counterparts by dozens of mutations. We constructed and validated a linear regression model for predicting chimera thermostability. This model predicted the sequences of chimeras with stabilities greater than that of the most stable parent CBH II with 100% accuracy. Coupled with our secretion results, the model predicts that the chimera family contains over 1,000 CBH IIs that are more stable than all of the parent enzymes. All of the thermostable chimeras tested were found to hydrolyze both crystalline and amorphous cellulose at higher temperatures than the parent CBH IIs. CBH II chimeras were also able to hydrolyze more cellulose than the parent enzymes in long time hydrolysis assays and were active across a broad range of pH values. We identified a single point mutation as the key determinant of chimera thermostability. Making this mutation in the backgrounds of the two recombination parent CBH IIs and selected chimeras that do not contain it markedly increased thermostability. This mutation also considerably increased the stability of a native CBH II gene not in the recombination parent set. Our findings show that structure-guided recombination enables the creation of diverse families of thermostable cellulases and represents the first step toward creating a large inventory of cellulases from which optimized application-specific mixtures can be formulated.