CAZyme evolution and discovery: Ultrahigh throughput screening of carbohydrate-active enzymes in modular assays modular based on coupled reactions

Enzymes are at the core of the white biotechnologies which promise greener and more efficient processes. Transitioning from fossil to renewable carbon sources poses an environmental challenge that will necessitate a greater diversity of enzymes than currently available, making enzyme discovery and evolution crucial. Identifying new catalysts from ‘libraries’, whether created through directed evolution or sourced from environmental functional metagenomics, relies heavily on the scale of screening, as useful hits are rare. The selection criterion in such combinatorial campaigns also matters as the more similar the assay reaction is to the actual application, the more likely is the screening campaign to yield useful enzymes.

The project has developed a system to test over 10 million enzyme variants daily for the breakdown of natural substrates, utilizing coupled reaction systems to efficiently locate effective enzymes. The initial application focuses on evolving enzymes for degrading wood components like cellulose and discovering novel enzymes from metagenomic samples collected from hot springs, the Antarctic, and various domestic environments such as compost heaps, ponds, and soils. This approach aims to improve understanding of how cooperative interactions within enzymes affect evolution and to pinpoint areas of high activity within sequence landscapes.