A Bacterium That Degrades And Assimilates Poly (Ethylene Terephthalate)
Despite the immeasurable advantages and advancements that the implementation of plastics has afforded us, their cheap availability and disposable nature has led to an accumulation of waste that is cause for global concern. This inescapable and imperative issue is of central focus to this article and the authors’ research. Poly (ethylene terephthalate) (PET) is the most common thermoplastic resin used in the production of plastics and imparts a near indestructibility to the products its used in. Although, the benefits of this material are directly proportional to its most pressing drawback, that is, they exhibit negligible environmental degradation once disposed of. The authors stress the importance of their discovery and work by elucidating the limited microbes identified that are capable of producing esterases, lipases, and cutinases enzymes that would make remediation a viable solution.
The search for a solution to this challenge directed Kohei Oda of the Kyoto Institute of Technology and Kenji Miyamoto of Keio University to lead a team of researchers into the yard of a bottle-recycling factory in Osaka Japan. They endeavored to locate microorganisms capable of using PET films as a primary carbon source for growth. The team collected 250 PET-contaminated samples of sediment, soil, waste, water, and activated sludge to screen for microorganisms capable of such a task. They cultivated each of the samples in test tubes with various nutrients, of which, PET film served as the primary carbon source. One sample identified as “no. 46, ” contained a conglomeration of microorganisms that were observed to degrade the PET film surface. To isolate the microorganisms nutritionally dependent on PET, serial dilutions of the no. 46 consortium were cultured with PET film as the primary carbon source. In doing so, the researchers isolated a new species of bacteria of the Ideonella genre, possessing unique and novel enzymes capable of catabolizing and assimilating PET.
The researchers observed that the species appeared to be connected by appendages, with shorter appendages present between the cell and the PET film. The authors hypothesize that these shorter appendages assist in the transport of secreted enzymes into the film. Initial experiments, testing the efficiency of this organism’s enzymes to catabolize PET resulted in the film being almost completely degraded after 6 weeks of exposure, at 30°C. It should be noted that the authors do acknowledge that the PET enrichment of their sampling site and cultures may be the cause for the isolation of a bacterium whose ability to catabolize PET was acquired through lateral gene transfer. Although, they are quick to point out that there are only a limited and select mutations that are capable of modifying a hydrolase enzyme to produce the PETase enzyme observed in this research. The article notes that in the course of isolating the species termed Ideonella sakaiensis, several subcultures of no. 46 exhibited complete inability to degrade PET. While the methods by which the researchers determined that I. sakaiensis was not present in these subcultures is not detailed in this article, its implications would be strong evidence that I. sakaiensis is indeed the microbe functionally active in the degradation of PET film.
I’m not sure as to what level of summarization of the researcher’s methods this critique’s guidelines intend, but suffice it to say, a whole genome analysis of this bacterium was prepared to explore the genes involved in PET hydrolysis. One open reading frame (ISF6_4831) encoding a putative lipase exhibited a high degree of similarity in both amino acid sequence identity and catalytic residues with a hydrolase capable of PET-hydrolytic activity from Thermobifida fusca. When the I. sakaiensis proteins encoded by ISF6-4831 were incubated with PET film at 30°C for 18 hours, prominent pitting developed on the film. The major product reported from this protein’s reaction with the PET film, was mono(2-hydroxyethyl) terephthalic acid (MHET), with small quantities of TPA and bis(2-hydroxyethyl) TPA (BHET). The importance of this is that ISF6_4831 proteins seem to hydrolyze both PET and BHET, to yield MHET.
Furthermore, MHET was reported as such a minor component of the supernatant to conclude that it was rapidly metabolized by I. sakaiensis. The researchers constructed a phylogenetic tree of three evolutionary divergent PET-hydrolytic enzymes to compare against the activity of the ISF6_4831 protein. The evolutionary homologs compared, TfH from a thermophilic actinomycete, cutinase homolog from leaf-branch compost metagenome, and F. solani cutinase from a fungus, were commercially synthesized with codon optimization for expression in Escherichia coli cells. The enzymes harvested from each of the organisms were purified and then incubated with PET film. The team’s comparison of the activities of these three enzymes was quite thorough and included their reactions rate with p-nitrophenol-linked aliphatic esters, PET film, and BHET at 30°C and pH 7. 0. The only comparison where ISF6_4831 did not outperform the other three by a factor of at least five, was with its activities towards the p-nitrophenol-linked aliphatic esters. ISF6_4831 proteins showed such catalytic preference for PET, even when tested for its activities against highly crystallized commercial bottle-derived PET, that it has been designated a PET hydrolase (PETase). While PETase was more active at low temperatures than the three homologs it was compared against, it was shown to be somewhat heat-labile.
Additionally, comparisons of PETase activity ratios against the other three homologs showed that it was able to hydrolyze PET with less enzyme diffusion. To identify additional enzymes involved in PET degradation, the researchers’ RNA-sequenced transcriptomes of I. sakaiensis cultures growing on maltose, disodium terephthalate (TPA-Na), BHET, and PET film. This test showed dramatic up-regulation of the genes recognized as responsible for the catabolism of TPA and protocatechuic acid (PCA, a metabolite), in the cells cultured on TPA-Na, BHET, and PET film. Transcription levels of the PETase gene for cells cultured on PET film were reported to be 15, 31, 41 times higher than those cultured on maltose, TPA-Na, and BHET, respectively. From these results the authors suggest that the presence of PET films induced the bacterial expression of PETase. The potential bioremediation benefits of real-world application for these findings cannot be understated, as it may provide. They suggest that we may in the near future be able to deposit cultures of these microbes at PET waste collection sites.
One of the more fascinating topics in this article, albeit also the most disappointing, was the researchers’ efforts to elucidate the evolutionary origins for the metabolism of PET. With disappointing results, they queried the Integr8 fully sequenced genome database for other organisms with gene homologs capable of metabolizing similar compounds. Although, they were able to identify 92 microbes in the database possessing MHETase homologs, with 33 possessing homologs of both TPA and PCA dioxygenases. Although these results do not offer any clear origin to the evolutionary origin for the metabolism of PET, they do imply that analogs for the metabolism of MHET are ancestral to that of PET.
I found this article to be thorough and thoughtful towards all its goal and have no doubt the results of this research will have far reaching and significant implications. But, this publication’s lack of methods or evidence verifying their claims of that PET was assimilated by I. sakaiensis and serves as a carbon source for growth, is a clear oversight.
