(119e) Efficient Screening of Natural Fungal Cellobiohydrolase Genes for Desirable Mutations by SCHEMA Structure-Guided Recombination

Heinzelman, P., California Institute of Technology
Komor, R., California Institute of Technology
Romero, P., California Institute of Technology
Mohler, S., California Institute of Technology
Kanaan, A., California Institute of Technology
Yu, X., California Institute of Technology
Arnold, F. H., California Institute of Technology
Snow, C. D., California Institute of Technology

A novel application of SCHEMA structure-guided recombination allows natural enzyme genes to be efficiently screened for contiguous blocks of amino acids that improve enzyme properties. This application offers a new means of improving enzymes that are difficult to engineer via high throughput screening methods, such as fungal cellobiohydrolase class I enzymes (CBH Is), the principal constituents of biocatalyst mixtures used in industrial biomass-to-fuel conversion processes. The enhancement of CBH I properties, particularly thermostability, that SCHEMA has enabled offers the prospect of reducing the amount of enzyme required for, and thus the cost of, converting cellulosic biomass into transportation fuel.

Constructing CBH I ?monomeras?, enzymes in which blocks of amino acid residues from wild type fungal CBH Is are substituted, one at a time, into a wild type fungal CBH I enzyme background that is well-secreted by a Saccharomyces cerevisiae recombinant expression host allows the thermostability contributions of 36 CBH I blocks to be determined by making just 28 stability measurements. This small set of data allows us to identify sequences of the CBH I chimeras, enzymes comprised of multiple blocks taken from two or more parents, predicted to be the most stable of the ~70,000 possible chimeras that can be constructed from the 36 characterized CBH I blocks.

The utility of SCHEMA for efficiently identifying desirable blocks is demonstrated by 16 of 16 predicted thermostable chimeras, comprised of blocks predicted to have positive or neutral stability contributions, being more stable than any of the parents. Five of 5 thermostable CBH I chimeras retained activity at higher temperatures than any of the parents in solid cellulose hydrolysis assays, showing that these stable chimeras could be useful in industrial biomass conversion processes. Furthermore, 6 of 6 selected CBH I chimeras have total yeast-secreted cellulase activity greater than or equal to that for any of the parent CBH Is, demonstrating the potential utility of these CBH I chimeras in consolidated biomass-to-biofuel process applications.

The observed improvements CBH I thermostability and total secreted activity show that SCHEMA is useful for engineering improved cellulases that could reduce the cost of biomass-to-fuel conversion processes. These results also motivate the future application of this monomera screening approach to the enhancement of other enzymes and demonstrate the feasibility of performing SCHEMA recombination with more than five parents.