Discovery and Genetic Code Expansion of a Polyethylene Terephthalate (PET) Hydrolase from the Human Saliva Metagenome for the Degradation and Bio‐Functionalization of PET

Abstract

We report a bioinformatic workflow and subsequent discovery of a new polyethylene terephthalate (PET) hydrolase, which we named MG8, from the human saliva metagenome. MG8 has robust PET plastic degradation activities under different temperature and salinity conditions, outperforming several naturally occurring and engineered hydrolases in degrading PET. Moreover, we genetically encoded 2,3‐diaminopropionic acid (DAP) in place of the catalytic serine residue of MG8, thereby converting a PET hydrolase into a covalent binder for bio‐functionalization of PET. We show that MG8(DAP), in conjunction with a split green fluorescent protein system, can be used to attach protein cargos to PET as well as other polyester plastics. The discovery of a highly active PET hydrolase from the human metagenome—currently an underexplored resource for industrial enzyme discovery—as well as the repurposing of such an enzyme into a plastic functionalization tool, should facilitate ongoing efforts to degrade and maximize reusability of PET.

Efficient depolymerization of polyethylene terephthalate (PET) and polyethylene furanoate by engineered PET hydrolase Cut190

“…Most recently, Pfaff et al ( 2022 ) constructed L136F/Q138Y mutant of PES-H1 as the best performing variant. Although the importance of the Q138 position in PET hydrolases has no doubt, type I (TfCut2, Cut190 and LCC) and type II ( Is PETase, PES-H1) hydrolases (Eiamthong et al 2022 ) showed different replacement effects. Considering these results, we made several Cut190 variants, as listed in Table 1 and compared their PET hydrolyzing activities (Table 4 ).…”

“…Most recently, a novel PET hydrolase was cloned from a compost metagenome (Sonnendecker et al 2022 ) and its variant indicated the comparable activity to the LCC variant (Pfaff et al 2022 ). In addition, PET hydrolases are cloned even from human saliva metagenome (Eiamthong et al 2022 ). Thermostable mutants of PETase from mesophilic Ideonella sakaiensis (Yoshida et al 2016 ) have been documented so far.…”

Microbial Enzyme Biotechnology to Reach Plastic Waste Circularity: Current Status, Problems and Perspectives

“…Consequently, known PHEs seem unsuitable for efficient degradation of untreated highly crystalline PET at room temperature [ 128 , 129 ]. This prompted a search for thermostable PHEs produced by protein engineering [ 87 , 90 , 91 , 93 , 130 , 131 ] or enzyme discovery [ 92 , 132 , 133 , 134 ]. Among such attempts, the company Carbios, in a public–private partnership with the Toulouse Biotechnology Institute and other global partners, was the first to produce a thermostabilized engineered variant of the leaf–branch compost cutinase (LC-cutinase) suitable for an industrial-scale recycling process of PET in 2020 [ 135 ].…”

MGnify: the microbiome sequence data analysis resource in 2023

“…Examples include: (i) the protein database was cited as a crucial source of additional sequences for multiple sequence alignments (MSAs) used by AlphaFold2 ( 14 ), with sequences from metagenomic sources enriching poorly represented protein families in more classical protein databases; (ii) Eiamthong et al. ( 15 ) successfully mined the protein database in search of novel polyethylene terephthalate (PET) hydrolases using sequence homology to a known PETase; (iii) Inoue et al. ( 16 ) used the sequence set to determine the relationship between specific clades of metabolically important Ni-containing carbon monoxide dehydrogenases (Ni-CODHs) and their biome distribution and (iv) Kazlauskas et al.…”