TY - JOUR
T1 - Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity
AU - Erickson, Erika
AU - Gado, Japheth E.
AU - Avilan, Luisana
AU - Bratti, Felicia
AU - Brizendine, Richard K.
AU - Cox, Paul
AU - Gill, Raj
AU - Graham, Rosie
AU - Kim, Dong-Jin
AU - Koenig, Gerhard
AU - Michener, William E.
AU - Poudel, Saroj
AU - Ramirez, Kelsey J.
AU - Shakespeare, Thomas Jack
AU - Zahn, Michael
AU - Boyd, Eric S.
AU - Payne, Christina
AU - DuBois, Jennifer L.
AU - Pickford, Andrew R.
AU - Beckham, Gregg T.
AU - McGeehan, John
PY - 2022/12/21
Y1 - 2022/12/21
N2 - Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5–9.0 and temperatures from 30–70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.
AB - Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5–9.0 and temperatures from 30–70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.
UR - http://www.scopus.com/inward/record.url?scp=85144545463&partnerID=8YFLogxK
U2 - 10.1038/s41467-022-35237-x
DO - 10.1038/s41467-022-35237-x
M3 - Article
C2 - 36543766
AN - SCOPUS:85144545463
SN - 2041-1723
VL - 13
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 7850
ER -