[P004] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P02-244] Functional Analysis of PETase Variants Identified from a Soil Metagenome

○Ilya Maisarah Binti Thariq¹, Eiko Yokota¹, Min Fey Chek¹, Yumiko Kawakami¹, Keita Katsuma², Masahito Hosokawa^3,4, Shosuke Yoshida¹ (1. Nara Institute of Science and Technology (Japan), 2. Bell Polyester Products, Inc. (Japan), 3. bitBiome, Inc. (Japan), 4. Waseda University (Japan))

Keywords:

PET,Biodegradation,PETase

The extensive use and improper disposal of polyethylene terephthalate (PET) plastic have resulted in widespread environmental pollution. PETase from Ideonella sakaiensis (IsPETase) hydrolyzes PET with high specificity under mild conditions; however, its limited thermostability hinders its industrial application. Accordingly, artificial protein engineering approaches have generated a variety of IsPETase mutants with dramatically improved activity and stability. In this study, we identified a gene encoding an IsPETase-like protein (>99% nucleotide sequence identity to Is-petase) through metagenomic sequencing of soil samples from a PET-contaminated site. Differential scanning fluorimetry revealed that the purified recombinant protein has a higher melting temperature (ΔT_m = 6.6 ºC) than IsPETase. Moreover, it exhibited a 1.8-fold increase in PET-degrading activity at 30 ºC, IsPETase’s optimal temperature. These findings prompted us to perform PCR-based Is-petase-targeted amplicon sequencing to explore additional naturally evolved IsPETase variants present in the soil samples. We selected the dominant sequence (SD1), containing six amino acid substitutions, for further investigation. SD1 exhibited a 19 ºC higher T_m compared with wild-type IsPETase. In PET film degradation assays, SD1 showed lower activity at 30 ºC but higher than the wild-type at 40 ºC (~3-fold) and 50 ºC (~9-fold), respectively. We generated two sets of single mutants: (i) six single-substitution variants introduced into the wild-type background and (ii) six corresponding single-reversion variants in the SD1 background. Activity profiling across 30-50 ºC indicated that four substitutions in SD1 were beneficial, one was neutral, and one resulted in loss of measurable activity. The beneficial mutations identified in this study overlap with those previously reported in engineered IsPETase variants, suggesting functional convergence between environmental and laboratory selection. The D186N mutant showed a notable increase in thermostability and PET degradation activity across 30–50 ºC compared with wild-type IsPETase, indicating that this mutation is a key contributor to SD1’s improved performance. In contrast, I218F was detrimental, showing no detectable activity across 30–50 ºC. However, reverting I218F in the SD1 background increased PET degradation by ~64-fold relative to IsPETase at 50 ºC, while SD1 retained substantial activity despite I218F, implying partial compensation by other SD1 mutation(s).

Together, these findings suggest that targeted screening of environmental samples could identify naturally occurring beneficial mutation combinations. This approach may offer an effective alternative strategy for guiding IsPETase optimization.