Figure 2.

Workflow for engineering PHL7 in this study

In each round of directed evolution, PHL7 templates (from rational/semi-rational design or previous rounds of evolution) were chosen as parents for mutagenesis via DNA shuffling and screening. Enzyme libraries resulting from DNA library construction and transformation into E. coli were screened using the BHET/split GFP HT co-screening assay. Improved variants were then validated in assays with PET substrates, in solutions, via monitoring soluble aromatic products release over time. Confirmed “hits” were isolated and sequenced, then were used as parents for the next round of directed evolution. Once significantly improved enzymes were identified, they were chosen for large-scale production and thorough characterization, including monomer quantification with high-performance liquid chromatography (HPLC), evaluation in bioreactors, and differential scanning calorimetry (DSC).