Evaluation of PET Degradation Using Artificial Microbial Consortia

Polyethylene terephthalate (PET) biodegradation is regarded as an environmentally friendly degradation method. In this study, an artificial microbial consortium composed of Rhodococcus jostii, Pseudomonas putida and two metabolically engineered Bacillus subtilis was constructed to degrade PET. First, a two-species microbial consortium was constructed with two engineered B. subtilis that could secrete PET hydrolase (PETase) and monohydroxyethyl terephthalate hydrolase (MHETase), respectively; it could degrade 13.6% (weight loss) of the PET film within 7 days. A three-species microbial consortium was further obtained by adding R. jostii to reduce the inhibition caused by terephthalic acid (TPA), a breakdown product of PET. The weight of PET film was reduced by 31.2% within 3 days, achieving about 17.6% improvement compared with the two-species microbial consortium. Finally, P. putida was introduced to reduce the inhibition caused by ethylene glycol (EG), another breakdown product of PET, obtaining a four-species microbial consortium. With the four-species consortium, the weight loss of PET film reached 23.2% under ambient temperature. This study constructed and evaluated the artificial microbial consortia in PET degradation, which demonstrated the great potential of artificial microbial consortia in the utilization of complex substrates, providing new insights for biodegradation of complex polymers.

Development of a targeted gene disruption system in the PET-degrading bacterium Ideonella sakaiensis and its applications to PETase and MHETase genes

Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however its non-biodegradability results in a large amount of waste accumulation that has a negative impact on the environment. Recently, a PET-degrading bacterium Ideonella sakaiensis 201-F6 strain was isolated and the enzymes involved in PET-digestion, PET hydrolase (PETase) and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase (MHETase), were identified. Despite the great potentials of I. sakaiensis in bioremediation and biorecycling, approaches to studying this bacterium remain limited. In this study, to enable the functional analysis of PETase and MHETase genes in vivo , we have developed a gene disruption system in I. sakaiensis . The pT18 mobsacB -based disruption vector harboring directly connected 5'- and 3'-flanking regions of the target gene for homologous recombination was introduced into I. sakaiensis cells via conjugation. First, we deleted the orotidine 5'-phosphate decarboxylase gene ( pyrF ) from the genome of the wild-type strain, producing the Δ pyrF strain with 5-fluoroorotic acid (5-FOA) resistance. Next, using the Δ pyrF strain as a parent strain, and pyrF as a counterselection marker, we disrupted the genes for PETase and MHETase. The growth of both Δ petase and Δ mhetase strains on terephthalic acid (TPA, one of the PET hydrolytic products) was comparable to that of the parent strain. However, these mutant strains dramatically decreased the growth level on PET to that on no carbon source. Moreover, the Δ petase strain completely abolished PET degradation capacity. These results demonstrate that PETase and MHETase are essential for I. sakaiensis metabolism of PET. IMPORTANCE The poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis possesses two unique enzymes able to serve in PET hydrolysis. PET hydrolase (PETase) hydrolyzes PET into mono(2-hydroxyethyl) terephthalic acid (MHET) and MHET hydrolase (MHETase) hydrolyzes MHET into terephthalic acid (TPA) and ethylene glycol (EG). These enzymes have attracted global attention as they have potential to be used for bioconversion of PET. Compared to many in vitro studies including the biochemical and crystal structure analyses, few in vivo studies have been reported. Here, we developed a targeted gene disruption system in I. sakaiensis , which was then applied for constructing Δ petase and Δ mhetase strains. Growth of these disruptants revealed that PETase is a sole enzyme responsible for PET degradation in I. sakaiensis , while PETase and MHETase play essential roles in its PET assimilation.

In silico binding affinity analysis of microplastic compounds on PET hydrolase enzyme target of Ideonella sakaiensis

Background The world today is faced with the humongous challenge of removing the numerous plastic wastes in our environment. Efforts in the removal or remediation of these materials from the ecosystem are presently at the budding stage. Some researchers have shown that certain bacterial enzymes have the ability to hydrolyze and further degrade these plastic compounds. In this study, the ability of PET hydrolase enzyme to hydrolyze polyvinylchloride, polyurethane, polymethyl methacrylate, polyamide, polyethylene terephthalate, and polycarbonate was investigated in silico. Results The binding affinity values of polycarbonate (− 5.7 kcal/mol) and polyethylene terephthalate (− 5.2 kcal/mol) on the enzyme targets were the highest and showed that they are likely to be efficiently hydrolyzed by this bacteria in the environment. The binding affinity of polyvinylchloride was the lowest (− 2.2 kcal/mol) and suggested that it would show resistance to hydrolysis by the PET hydrolase enzyme. Conclusion The findings from this study showed that PET hydrolase enzyme from Ideonella sakaiensis could be efficient in the hydrolysis of plastic wastes composed mainly of polycarbonate and polyethylene terephthalate.

Multiple structural states of Ca2+-regulated PET hydrolase, Cut190, and its correlation with activity and stability

An enzyme, Cut190, from a thermophilic isolate, Saccharomonospora viridis AHK190 could depolymerize polyethylene terephthalate (PET). The catalytic activity and stability of Cut190 and its S226P/R228S mutant, Cut190*, are regulated by Ca2+ binding. We previously determined the crystal structures of the inactive mutant of Cut190*, Cut190*S176A, in complex with metal ions, Ca2+ and Zn2+, and substrates, monoethyl succinate and monoethyl adipate. In this study, we determined the crystal structures of another mutant of Cut190*, Cut190**, in which the three C-terminal residues of Cut190* are deleted, and the inactive mutant, Cut190**S176A, in complex with metal ions. In addition to the previously observed closed, open and engaged forms, we determined the ejecting form, which would allow the product to irreversibly dissociate, followed by proceeding to the next cycle of reaction. These multiple forms would be stable or sub-stable states of Cut190, regulated by Ca2+ binding, and would be closely correlated with the enzyme function. Upon the deletion of the C-terminal residues, we found that the thermal stability increased while retaining the activity. The increased stability could be applied for the protein engineering of Cut190 for PET depolymerization as it requires the reaction above the glass transition temperature of PET.