Mapping bacterial cutinase sequence space by high-throughput screening reveals that PET hydrolysis is a rare property
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Poly(ethylene terephthalate) (PET) is one of the most widely produced plastics, and enzymatic depolymerization offers a promising route to closed-loop recycling under mild conditions. However, most known bacterial PET hydrolases belong to a conserved canonical-fold cutinase family, leaving much of α/β-hydrolase diversity unexplored. Here, we mapped bacterial cutinase sequence space by combining bioinformatics-guided sequence selection with high-throughput secretion screening in Bacillus subtilis . A library of 1,120 genes encoding 954 unique bacterial cutinases, spanning canonical- and minimal-fold families, was screened for activity on Impranil DLN and semicrystalline PET. We identified 156 secreted cutinases with polyester activity, broadly distributed across sequence space, but only ten showed detectable PET hydrolysis, all from the canonical-fold family. These PET hydrolases were active at 40-50°C, preferred alkaline pH, and showed moderate thermostability. Our results demonstrate that PET activity is rare among bacterial cutinases and provide a scalable workflow for discovering diverse enzyme starting points.
Significance Statement
Poly(ethylene terephthalate) (PET) is a major plastic waste stream, and enzymatic depolymerization offers a route to closed-loop recycling under mild conditions. However, known PET-degrading enzymes occupy only a narrow region of bacterial cutinase sequence space, limiting discovery of new enzyme scaffolds. We combined bioinformatics-guided sequence selection with high-throughput screening in an industrial host to survey more than 1,100 bacterial cutinase genes. This approach identified 156 secreted polyester-active enzymes, including ten previously unreported PET hydrolases. This work provides an experimentally grounded map of polyester and PET hydrolysis across bacterial cutinase diversity, revealing that PET-degrading activity is relatively rare despite widespread polyester hydrolysis and expanding the foundation for future enzyme discovery and engineering.
