Engineering PHL7 for Improved Poly(ethylene Terephthalate) Depolymerization via Rational Design and Directed Evolution

Enzymatic depolymerization of poly(ethylene terephthalate) (PET) has emerged as a promising recycling approach for polyester recycling, and to date, many natural and engineered PET hydrolase enzymes have been reported. For industrial use, PET hydrolases must exhibit superior activity and thermostability in reactions with PET. Here, we engineered a natural, highly active PET hydrolase, Polyester Hydrolase Leipzig #7 (PHL7), through rational design and directed evolution using a previously reported high-throughput screening platform. Four new enzyme constructs were engineered with enhanced properties, including higher initial catalytic rates, higher total hydrolysis extents over time, better pH tolerance, and higher protein expression/solubility levels, compared to the parent enzyme, wildtype PHL7 (PHL7-WT), and other benchmark PET hydrolases, such as LCC-ICCG and PHL7-L93F/Q95Y, under tested conditions. In pH-controlled bioreactors, under different substrate loadings, the best engineered enzyme, PHL7-Jemez exhibited significantly improved ability to depolymerize amorphous PET film coupon substrate (~9.4% crystallinity) compared to PHL7-WT. In particular, comparing to PHL7-WT, PHL7-Jemez exhibited 86% higher hydrolysis at 24 h and 37% higher hydrolysis at 48 h, at 2.9% substrate loading. At 20% substrate loadings, PHL7-Jemez exhibited 383% higher hydrolysis at 24 h and 268% higher hydrolysis at 48 h comparing to PHL7-WT, respectively. This study develops several state-of-the-art PET hydrolases as candidates for evolution toward other engineering goals such as engineering new PET hydrolases at acidic pHs and low buffer concentration conditions and demonstrates a directed evolution platform to discover high-performance PET hydrolases, which can accelerate enzyme discovery, toward improved biocatalytic recycling.