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. 2025 Aug 21;5(8):101399. doi: 10.1016/j.checat.2025.101399

Figure 3.

Figure 3

Screening and evolutionary trajectory of evolved PHL7 variants

(A–D) Performance of evolved PHL7 variants across rounds of directed evolution. Activity is expressed in terms of equivalents of BHET, measuring aromatic products via UV absorbance. (A) Round 1. (B) Round 2. (C) Round 3. (D) Round 4. Reactions consisted of 0.05 or 0.1 μM enzyme in cell lysates with either 0.92% or 2.9% (w/v) PET amorphous coupons at 70°C in 1 M potassium phosphate buffer, pH 8. Activity was measured either after 6 or 24 h. Variants are shown across the bottom axes, with bars showing mean of n = 2 replicates and error bars representing ±1 S.D.

(E–H) Tables showing details of variants and screening conditions for each round of evolution. Note that screening conditions varied between each round. Names of variants are shown with corresponding mutations compared with PHL7-WT, as well as relative performance vs. PHL7-WT, expressed as fold change of activity vs. PHL7-WT, and relative expression vs. PHL7-WT, measured using split GFP complementation of cell lysates. Screening conditions include details on the colony plate-based coarse and fine screenings and validation screenings, including how many colonies were chosen from each round of evolution. Top selected, representative variants are shown that directly led to mutations in future rounds of evolution (namely, the mutations in final variants). In mutations listed, the asterisk denotes mutations originating from rational design (i.e., not random mutations). (E) Round 1. (F) Round 2. (G) Round 3. (H) Round 4.

(I) Evolutionary trajectory of final, engineered PHL7 variants. Mutations/scaffolds are shown that were introduced in each round of evolution. The dashed arrow denotes that scaffolds Q175E and R205K were introduced manually (not through DNA shuffling) between Round 2 and Round 3. Sequences of the final mutants are shown, as a combination of mutations incurred in Rounds 1–4.

(J) Table of mutations of each of the four engineered mutants from this paper, as well as the PHL7-L93F/Q95Y variant. For each variant, the mutation at a position compared to the PHL7-WT sequence is shown.