Polyethylene terephthalate (PET) monomer metabolism and adaptation in environmentally isolated bacteria Alice M. Banks, José I. Jiménez Imperial College London, UK Isolation and analysis of candidate strains: Production of PET degrading enzymes: Pseudomonas_reinekei_SH_ben_homologue Pseudomonas_umsongensis_GO16_ben_cluster Isolate Species Rhodococcus_sp_DK17_ben_homologue Acinetobacter_schindleri_LK1_ben_homologue Rhodococcus_sp_DK17_tph_cluster LK1 Acinetobacter schindleri Pseudomonas_reinekei_SH_tph_homologue BHET Pseudomonas_vancouverensis_HM2_tph_homologue PETase Pseudomonas_umsongensis_GO16_tph_cluster Enzymatic PET LK2 Aeromonas media Acinetobacter_schindleri_LK1_hca_homologue Pseudomonas_umsongensis_GS_tph_homologue MHET Comamonas_sp_E6_tph_cluster degradation and consumption HM1 Rhodococcus sp. Rhodococcus_sp_HM1_tph_homologue Escherichia_coli_K12_MG1655_hca_cluster of PET monomers TA Rhodococcus_sp_HM1_ben_homologue HM2 Pseudomonas vancouverensis Pseudomonas_pudida_KT2440_ben_cluster Pseudomonas_vancouverensis_HM2_ben_homologue O EG Pseudomonas_umsongensis_GS_ben_homologue SH Pseudomonas reinekei OH + TA O 35 ºC 0.2 O GS Pseudomonas umsongensis OH + EG 30 ºC 32.5 ºC Genomes analysed to O bis(2-hydroxyethyl) terephthalate (BHET) Bacteria are engineered TS Ralstonia sp. identify candidate genes Engineered bacteria secrete enzymes to Bacteria isolated from soil to produce PET relating to plastic degrade PET into its constituent monomers and water under selective FS Ochrobactrum intermedium degradation PETase degrading enzymes pressures Isolates identified by genome sequencing O O O OH H O O OH HO O O PET monomers as microbial feedstock: polyethylene terephthalate (PET) mono-(2-hydroxyethyl) terephthalate (MHET) Adapted laboratory evolution: MHETase 5 mM TA 10 mM TA 15 mM TA 20 mM TA Bacteria are subjected LK1 LK2 HM1 HM2 TA 30 ºC 0.6 to artificial selection TA 32.5 ºC TA 35 ºC pressures to drive 0.4 600 O EG 30 ºC HO adaptation towards OD OH EG 32.5 ºC OH 0.2 HO ethylene glycol (EG) growth on target EG 35 ºC ALE 1.1 ALE 1.2 ALE 1.3 ALE 1.4 O substrates 0.0 terephthalic acid (TA) GS1 GS TS SH 0.6 Strains are characterised for 0.4 Unadapted wild-type Tolerance to PET monomers as a sole carbon source 0.4 growth using PET monomers as 600 0.3 Adapted sub-culture 1 OD a sole carbon source, across a improves across successive generations 0.2 Adapted sub-culture 7 range of increasing temperatures 600 0.2 0.0 OD Adapted strains achieve greater growth rates following 0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72 Time (hours) Time (hours) Time (hours) Time (hours) 0.1 prolonged exposure to PET monomers 0.0 0 1000 2000 3000 4000 5000 Time (mins) Impacts and future use: Acknowledgements: Bacteria capable of degrading waste PET, and utilising PET monomers, can be integrated into the circular economy to produce high-value products from PET waste.