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

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Enzymes are at the core of the white biotechnologies that will create greener, more efficient processes. The environmental challenge of switching our carbon source from fossil to renewable will require many more enzymes than are currently available, so enzyme discovery and evolution are necessary. Finding new catalysts in 'libraries' (person-made in directed evolution or environmental in functional metagenomics) is a numbers game: hits are rare, so technologies that can screen large numbers of library members will be more successful. The selection criterion in such combinatorial campaigns also matters: the more similar the assay reaction is to the actual application, the more likely is the screening campaign to yield useful enzymes. We have developed a system in which more than 10 million variants can be tested in a day for breakdown of natural substrates (and not for model substrates), by using coupled reaction systems, so we hope to find the right "needle in a haystack", more quickly. In a first application of coupled assays to carbohydrate-active enzymes, we will evolve enzymes used for the degradation of the main components of wood (carbohydrates, mainly in the form of cellulose) and also try to discover new enzymes in metagenomic samples collected in hot springs, the Antarctic and several domestic settings (local compost heaps, ponds, soils). The results of directed evolution will be interpreted with a sequencing technology that we have recently developed ('UMIC-Seq': Nat Commun 2020, 11 (1), 6023), that allows us to obtain full-length readouts of > 10,000 sequence per round of evolution (at a price of less than 1 penny per sequence). Equipped with this insight we hope to understand better how cooperative interactions in the enzymes enable evolution to proceed (or make it difficult in other cases), based on the reconstruction of sequence landscapes, in which areas of high activity are to be explored.