Prospecting for Plastic Degrading Biocatalysts

1. Prospecting for Plastic Degrading Biocatalysts Eric S. Boyd, Montana State Univ. National Acad. Sciences May 9th, 2019

2. Polyethylene terephthalate (PET) • >45 million tons produced annually • <31% recycled in US • Much ends up in landfills where strength of ester bonds in polymer limit biodegradation

3. PET Biodegradation Thermobifidafusca Growth on PET at 55°C Ideonellasakaiensis Growth on PET at 30°C

4. Enzymatic Degradation of PET Likely the rate- limiting step Oxidative decarboxylation Ring-cleaving deoxygenation 5 steps to a linear tricarboxylic acid that can be integrated into cellular metabolism

5. Increased Accessibility: Heat, chemical pre-treatment Crystalline PET Amorphous PET 70°C heat • Operating above the glass transition temperature improves substrate access • The “Hot Polyester” problem: We also need an enzyme/organism to degrade at >70°C

6. Towards a Thermal PETase • Engineer PETase from I. sakaiensis(Topt = 30°C) orT. fusca (Topt = 55°C)to achieve thermotolerance • → Limitations: limited modification of protein chassis • 2). Allow evolution to do the work for you: Probe natural systems for a thermophilic PETase homolog or PET degrading organism • → Limitations: PET has only been around for ~70 years, has evolution had time to “capitalize” on PET? I. sakaiensis PDB: 5XJH

7. PETases are cutinases (α/β hydrolase) Cuticle – waxy protective outer layer of plants, invertebrates, shells, etc. Cutin – heterogeneous network of esters Wax : long chain ester Cutinases Where can we bio-prospect for thermophilic PETase?

8. Yellowstone National Park Bozeman, MT Magma Plume

9. YNP: A Diverse Natural Laboratory Siliceous, Carbonate Hydrothermal Systems Pyritic Hydrothermal Systems • >14,000 features • pH: 0.8 to 9.8 • Temp.: ~20 to 93°C • Variable minerals • S2-: ~0 to >165 µM • Fe2+ ~ 0 to > 80 µM • Ni: ~0 to >280 nM • CO: ~ 0 to > 750 nM • H2: ~ 0 to >1 µM • CH4: ~ 0 to 10 µM Sulfidic HydrothermalSystems Fe-BiomineralizingSystems

10. Geochemical Variation → Biological Variation Crystalline → Amorphous

11. Exploring Biodiversity: Identifying Target Enzymes Genes (genomes) encode all of the proteins and enzymes that a cell uses Genome

12. Identifying Target Enzymes Diverse Hot Spring Community (plant matter present - cutinase) DNA extraction (Meta)genomic sequencing Genes, proteins Informatics

13. Protein clustering:A tool for comparative genomics Stringency Esterases 1). Sequenced gene fragments [i.e., (meta)genome)] 2). Assemble into larger gene fragments (overlap in reads) 3). Organize genes based on relatedness (gene bins) 4). Gene bins corresponds to unique functions (e.g., esterases)

14. Sifting Through the Genes: Identifying Putative PETases Among Esterases PETase Esterase Cutinase Tf_Cutinase Unc_Cutinase Catalytic Triad PETase Esterase Cutinase Tf_Cutinase Unc_Cutinase PETase Esterase Cutinase Tf_Cutinase Unc_Cutinase PETase Esterase Cutinase Tf_Cutinase Unc_Cutinase Orange: major changes among PETase and esterases/cutinases Fecker et al., 2018

15. Overall Approach BLASTp: Bait with I. sakaiensisPETase sequence

16. Needles in a Haystake: Differentiating PETases from Esterases in YNP Metagenomes Identified 46 homologs Not evolutionarily conserved(do not form a tight cluster)

17. Diversity of 46 Sequences • Motifs Present • Clamp 1 • Clamp 2 • Clamp 3 • Oxyanion Hole Mesophilic PETases Sequences spanning diversity of this phylogeny AND possessing functionally important sequence motifs • Initial selection: 8 Seqs

18. Diversity of 46 Sequences • Motifs Present • Clamp 1 • Clamp 2 • Clamp 3 • Oxyanion Hole Mesophilic PETases Sequences spanning diversity of this phylogeny AND possessing functionally important sequence motifs Green: homology model Blue: PETase from I. sakaiensis Orange: homology catalytic triad Red: PETase catalytic triad Yellow: binding pocket • Initial selection: 8 Seqs

19. Diversity of 46 Sequences • Motifs Present • Clamp 1 • Clamp 2 • Clamp 3 • Oxyanion Hole Mesophilic PETases Sequences spanning diversity of this phylogeny AND possessing functionally important sequence motifs Green: homology model Blue: PETase from I. sakaiensis Orange: homology catalytic triad Red: PETase catalytic triad Yellow: binding pocket • Initial selection: 8 Seqs

20. Heterologous Expression Synthesize Gene with histidine tag Insert into expression plasmid and E. coli Activity screen Ni2+-column purification

21. Big Questions • Can we quickly screen diverse a/b-hydrolases, sorting them into lipases, cutinases, and PETases, using solution-state assays? • Can we correlate any easy-to-identify motif or functional feature(s) with PETase activity? • Can we use (1) and (2) to identify a PETase that is stable at high temperature?

22. Para-Nitrophenyl-Based Soluble Assays for Activity • Monitor pNPate formation at 415 nm over time

23. Para-Nitrophenyl-Based Soluble Assays for Activity (2) long-chain esterase function (cutinase-/*PETase-like) (1) lipase function (2) Interfacial activation (triton) disrupts lid of lipase (3) pH optimum (4) Thermal stability of protein (5) Thermal stability of activity Protein purification = rate limiting step

24. Results from Pilot Studies: Chain Length kcat(s-1) KM(mM) kcat /KM (M-1 s-1) kcat(s-1) KM(mM) kcat /KM IA? (M-1 s-1) Enzyme source 0.50 820 610 no - - 0.26 T. fusca I. sakaiensis 0.27 1100 250 no - - 0.00011 Spouter 2 0.0067 60 114 no 0.0012 10 120 0.20 1800 110 no - - 0.000031 Jinze 1 Jinze 2 0.0074 1100 7.0 no 0.0074 1100 0.0000029 Jinze 3 6,800 1100 6.2 no 0.00018 20 9.0 • All 6 are cutinase-like with no interfacial activation • Spouter2 and Jinze3 have preference for long chains (PETase-like?)

25. Pilot Studies: pH and T pHoptimum stability (6h) active lifetime at T (min) pH kcat (s-1) kcat /KM (M-1s-1) T(ºC) Enzyme source T. fusca 8.0 0.96 2700 80 < 2 I. sakaiensis 6.0 1.1 330 60 n.d. Spouter 2 8.0 0.00097 230 80 > 300 7.5 0.20 110 80 n.d. Jinze 1 Jinze 2 7.5 0.0074 1100 80 n.d. Jinze 3 5.0 0.0024 50 80 > 300 • Spouter2 and Jinze3 proteins and activities are stable for hours at 80 °C

26. Where we are heading • Additional metagenomics – our metagenomes were not selected with PET degradation in mind • Thermostable proteins identified with cutinase activity – solid state assays with PET (Beckham) • Can we use solution state assays (quick) to predict activities with solid state assays (time-consuming)? • Enzyme structure/function studies – improve PETase activity in a thermostable enzyme chassis? • Engineer thermophile (e.g., Thermus) with PETase? • MHETase, TPAdo– thermostable enzymes necessary? • Bioreactors, scale up

27. Thanks! Jennifer DuBois Bennett Streit Saroj Poudel Gregg Beckham