We recently developed an HT screening platform for engineering PET hydrolases, which is capable of simultaneously screening large, random mutagenesis enzyme libraries for improved protein solubility, activity, and thermostability by coupling a split GFP assay and a BHET hydrolysis assay. Specifically, enzyme variants were expressed with C-terminal GFP11 tags using the vector indicated in Table S1, which allows their quantification in crude cell lysates, in solutions or on agar plates, via green fluorescent readout when complemented with GFP1-10, thus eliminating the need for protein purification during the screening steps, which ultimately increases the throughput and reduces the cost. Activity was evaluated stepwise, first on a model substrate using BHET, then on PET substrates. Enzyme activity was first screened on BHET agar plates based on clearing zones (halos) generated from BHET hydrolysis. Briefly, enzyme libraries were first grown on Durapore membranes on LB agar plates overnight, then protein expression was achieved by transferring the membranes to plates containing isopropyl β-ᴅ-1-thiogalactopyranoside (IPTG) for induction. Durapore membranes containing bacterial colonies were then moved to BHET agar screening plates, colonies were partially lysed by spraying with BugBuster, allowing the cell lysates to diffuse through the membranes onto the BHET agar plates, and membranes were finally returned to LB agar plates and stored at 4°C for later colony picking. The BHET agar plates were then incubated at the reaction temperature and colonies were selected based on higher enzyme activity (larger clearing zones) and/or greater expression levels (higher fluorescence intensities) after aligning BHET screening plates to the original colonies grown on the membranes. Coupling the fluorescence and colorimetric assays, we were able to quickly and precisely select enzyme variants with improved both activity and expression/thermostability concurrently. Selected enzymes were then expressed in small scale (2-25 mL) and validated on reactions with PET substrates, in solution, with soluble aromatic products measured in microwell plates using established, plate-based absorbance protocols. Enzyme variants displaying higher expression/solubility, activity, and/or thermostability compared with the parent enzymes from the previous round were sequenced and selected as parents for the next round of evolution. The workflow for directed evolution of PHL7 enzyme is illustrated in Figure 2.