The metagenome‐derived esterase PET40 is highly promiscuous and hydrolyses polyethylene terephthalate (PET)

Abstract

Polyethylene terephthalate (PET) is a widely used synthetic polymer and known to contaminate marine and terrestrial ecosystems. Only few PET‐active microorganisms and enzymes (PETases) are currently known and it is debated whether degradation activity for PET originates from promiscuous enzymes with broad substrate spectra that primarily act on natural polymers or other bulky substrates, or whether microorganisms evolved their genetic makeup to accepting PET as a carbon source. Here, we present a predicted diene lactone hydrolase designated PET40, which acts on a broad spectrum of substrates, including PET. It is the first esterase with activity on PET from a GC‐rich Gram‐positive Amycolatopsis species belonging to the Pseudonocardiaceae (Actinobacteria). It is highly conserved within the genera Amycolatopsis and Streptomyces. PET40 was identified by sequence‐based metagenome search using a PETase‐specific Hidden Markov Model (HMM). Besides acting on PET, PET40 has a versatile substrate spectrum, hydrolyzing δ‐lactones, β‐lactam antibiotics, the polyester‐polyurethane Impranil® DLN, and various para‐nitrophenyl (pNP) ester substrates. Molecular docking suggests that the PET degradative activity is likely a result of the promiscuity of PET40, as potential binding modes were found for substrates encompassing mono(2‐hydroxyethyl) terephthalate (MHET), bis(2‐hydroxyethyl) terephthalate (BHET), and a PET trimer (PET3). We also solved the crystal structure of the inactive PET40 variant S178A to 1.60 Å resolution. PET40 is active throughout a wide pH (pH 4‐10) and temperature range (4‐65 °C) and remarkably stable in the presence of 5% sodium dodecyl sulfate (SDS), making it a promising enzyme as a starting point for further investigations and optimization approaches.

Polyester degradation by soil bacteria: identification of conserved BHETase enzymes in Streptomyces

“…Three variants, SclipA (present in S. coelicolor ), S92 l ipA (present in MBT92), and S2 l ipA (present all other active strains) were further investigated. This situation is comparable to the presence of PET40 in several Amycolatopsis and other Streptomyces species 28 . However, in this study, the BHET-degrading activity in the original host strains was not investigated.…”

“…All enzyme variants were found to have an optimal pH of 7 and an optimal temperature of 25 °C. The enzymes were active in a range from pH 5-10, similar to the PET40 28 . However, in contrast with the PET40, the LipA variants appeared to lose most activity above 40 °C.…”

“…Several promiscuous actinobacterial enzymes exhibiting BHET or PET-degrading activity have been identified such as the leaf compost cutinase (LCC), Tf Cut2 from Thermobifida fusca and PET40, present in both Amycolatopsis and some Streptomyces species, earning them their place in the PAZY database 19 – 26 . For the entire genus of Streptomyces , only three PHEs have been described, namely SM14est, Sub1, and PET40 11 , 27 , 28 . Hence, limited data is available concerning the ability of Streptomyces to degrade BHET and PET and thus on the emergence, distribution, conservation, and activity of their corresponding BHET-degrading enzymes.…”

Proteomic examination of polyester‐polyurethane degradation by Streptomyces sp. PU10: Diverting polyurethane intermediates to secondary metabolite production

“…This enzyme was shown to degrade commercially available thermoplastic polyester‐PU at 60°C (Schmidt et al., 2017 ). Additionally, Est_PU10 is homologous to various PETases most closely related to metagenome‐derived PETases Enzyme 607 (Erickson et al., 2022 ) PET40 (Zhang et al., 2023 ) and PHL1 and PHL2 (Sonnendecker et al., 2022 ) which display increased thermotolerance, a highly coveted characteristic for PETases. But Est_PU10 is also related to the benchmark enzymes LCC (Sulaiman et al., 2012 ), IsPETase (Yoshida et al., 2016 ) and PHL7 (Sonnendecker et al., 2022 ) as well.…”

Seek and you shall find—news on the quest for novel PET‐degrading enzymes

“…To increase our understanding of PETases from a biodiverse range of microbial hosts, in this issue, Zhang et al [14] set out to search for PETase candidates from GC-rich Gram-positive bacteria in publicly available genomes and metagenomes (Fig. 1).…”