by Janie Kim
Science and speculative fiction works may warn of imminent threats like hostile extraterrestrial beings or the artificial general intelligence of the technological singularity. But the more humdrum overabundance of plastic waste, an issue closer to home and our everyday lives, is just as deserving of fear. Plastic has been a simultaneous boon to and bane of humankind. The double-edged material that spurred innovation and creativity has an insidious nature that is becoming starker, as the size of ocean garbage patches becomes less of patches and more of countries. Plastic’s invention in the 1900s drew great praise and excitement – now, we seek ways to destroy what we created.
In 2016, Christoph wrote a post about the discovery of microorganisms that possess the rare ability to degrade polyethylene terephthalate (or simply PET) plastic: the bacterium Ideonella sakiensis and the fungus Pestalotiopsis microspora. Now, a team of researchers based at the universities of Hamburg and Leipzig added a handful of more plastic-munching microbes to the list. Danso et al. discovered previously unknown PET hydrolases, the enzymes that are directly responsible for degrading PET, that are produced by members of the phyla Actinobacteria, Proteobacteria, and Bacteroidetes.
The researchers took an interdisciplinary approach to identifying these new microbes, moving from in silico analyses to in vitro experiments. They first used computational tools to identify potential new PET hydrolases. First, they generated a hidden Markov model computer algorithm to filter through existing genome and metagenome databases to seek out signs of PET hydrolase sequences. The researchers took the catalytic site sequences of nine known PET hydrolases, popped the sequences into their algorithm, and identified eight conserved regions. They then ran the massive UniProtKB and GenBank databases through this genomic "filter" and performed BLAST searches for PET hydrolase candidates identified from this screen. The result? 504 potential PET hydrolase genes. The team then sifted through this pool of candidates and, using the alignments from the BLAST search, hand-picked thirteen that had the closest alignment to known PET hydrolases. All thirteen possess eight key motifs characteristic of known PET hydrolases – a very promising start.
It is worth noting that they selected mainly non-actinobacterial proteins for the final thirteen candidates. Because most PET hydrolases identified to date are from Actinobacteria, they reasoned that it would be particularly interesting to identify hydrolases from species previously unknown to harbor PET hydrolase genes.
Backed by their bioinformatic results, the researchers moved onto testing these findings at the lab bench. They cloned four of the thirteen PET hydrolase genes into plasmids to express them in bacteria. They overproduced and purified the enzymes using the T7 system. They added tiny bits of PCL (a plastic similar to PET, but less complex and easier to degrade) and PET to agar and poured that mix into plates. They then spotted solutions of the purified PET hydrolases on the agar surface. After incubating the plates for a day they were able to see that all four enzymes had plastic-degrading activity, that is they produced clearing zones around the enzyme spot. This was excellent news: the bioinformatics predictions had yielded tangible results in the lab.
Narrowing down the number of PET hydrolase candidates even further and trying to find the most effective ones, the researchers then selected the enzymes that showed the best activity. These were PET2 and PET6, from a marine bacterium and from a Vibrio, respectively. These two enzymes were not only able to degrade para-nitrophenyl esters, which have shorter carbon backbones, but also longer-chain substrates. The preferred reaction conditions for these two enzymes – 70°C for PET2 and 55°C for PET6 at a slightly alkaline pH – were similar to other PET hydrolases.
With their reliable prediction algorithm and successful identification of new PET hydrolases behind them, the researchers moved on to take a look at the bigger picture. They zoomed out of the molecular level to investigate the distribution of PET hydrolases at a global scale, identifying homologs from both marine and terrestrial metagenomes. Interrogating 31 marine and 11 terrestrial metagenomes resulted in 349 hits. Upon closer inspection of these hits, they found that in terrestrial environments PET hydrolases are most commonly produced by Actinobacteria and by Bacteroidetes in marine habitats
The final verdict? The new PET hydrolases identified in this paper hold potential for development of better tools to combat the plastic epidemic. But the real gem here appears to be the novel PET hydrolase search algorithm. This new algorithm and the workflow used to identify the enzymes – using computational tools to winnow out the proverbial grain from the chaff and then using wet-lab tools to pick out the real PET hydrolases – holds great promise for identifying more plastic-degrading enzymes.
Janie Kim is a sophomore at Princeton University who will be majoring in molecular biology. She is doing research on peptides secreted by algae-symbiont bacteria in the Donia Lab. She likes to bring her interest in microbes and sci-fi futurism into as many aspects of her life as possible, including her fiction writing and her sculpting (her Etsy truly fits with the idea of "niche marketplace").