Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

Ancestral sequence reconstruction reveals highly thermostable ancestral PETase for efficient PET degradation (#104)

Yvonne Joho 1 2 , Matthew A Spence 2 , Vanessa Vongsouthi 2 , Alex T Caputo 1 , Tom S Peat 1 , Colin J Jackson 2 3 4 , Albert Ardevol 1 5
  1. Manufacturing, Commonwealth Scientific and Industrial Research Organisation, Clayton, Victoria, Australia
  2. Research School of Chemistry, Australian National University, Canberra , Australia
  3. ARC Centre of Excellence for Innovations in 3 Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
  4. ARC Centre of Excellence for Innovations in Synthetic Biology, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
  5. CSIRO Synthetic Biology Future Science Platform, ACT 2601, Canberra, Australia

Polyethylene terephthalate (PET) is increasingly used in our lives for various functions, and without proper waste management causes significant environmental pollution. Enzymatic PET recycling has inspired many protein engineers to explore the most efficient way to degrade this type of plastic. A breakthrough occurred in 2016 with the discovery of a PETase enzyme from Ideonella Sakaiensis with high PET degradation activity at moderate temperatures. Although significant efforts have been made to improve the enzyme mainly through rational protein engineering, the PETase enzyme is still unsuitable for degrading PET at an industrial scale. Ancestral sequence reconstruction is a useful method that often results in increased stability, solubility and activity while also giving a better evolutionary understanding of proteins. We are the first group to use ancestral sequence reconstruction to investigate the evolution and improve the activity of this newly discovered enzyme. We expressed and purified ten ancestral enzymes, measured their stability, and tested the activity against crystalline PET. Our ancestral reconstruction resulted in a 20˚C more stabilised enzyme with far higher PET degradation than the wildtype. The proposed strategy represents a significant achievement in understanding the evolution of PET enzymatic degradation. Furthermore, the stabilised ancestral enzyme has improved PET degradation efficiency, and together with future improvements, the enzyme could increase the suitability of PETase as an industrial enzyme.