Developing Dictyostelium Discoideum as a Bioremediation Tool for Polyethylene-Terephthalate (PET) Microplastic

The accumulation of plastic in the terrestrial environment has been occurring for around a century and we are now understanding the problems that plastic build-up can cause. Due to the long lifetime that many polymers exhibit these materials will continue to cause problems even if the plastic waste entering the environment is reduced. Polyethylene terephthalate (PET) is one of the most prevalent plastics found in the natural environment. In 2016, PET hydrolase (PETase) an enzyme produced by the bacterium Ideonella sakaiensis was found to exhibit PET degrading properties that could be utilized for bioremediation. Dictyostelium discoideum is a model organism among amoeba and is a prolific inhabitant within the terrestrial environment. This study investigated the development of D. discoideum as a vehicle for PET bioremediation, by exploring whether this organism could be a suitable PETase expression system for deployment in plastic-contaminated environments. Here we show D. discoideum growth is not significantly inhibited in environments containing PET microplastics, interestingly NC4 exhibited a significant increase in growth rate (mean difference=0.049, p=0.032) when incubated with 2.5% PET w/v compared with a control. The increased growth rate could be due an increase in surface area within the environment or a specific chemical property of the PET microplastic. Transformation of D. discoideum with plasmids containing the PETase expression cassette yielded positive results after antibiotic selection, although complications arose during further cultivation. To improve our ability to produce efficient recombinant genes for expression in D. discoideum, we produced tRNA profiles using HydroSeq a published deep sequencing approach. The tRNA profile generated had no significant correlation with tRNA gene count frequency (R2=0.027, p=0.297) or codon-usage frequency IX (R2=0.002, p=0.795). Because of the low correlation between these intrinsically linked biological factors it appears that the tRNA read profile generated throughout this investigation is inaccurate. Poor decipherability of post-transcriptional modifications was judged to affect the construction of an accurate tRNA profile for D. discoideum, this was supported by evidence that tRNAs known to contain post-translational modifications within the anti-codon region displayed extremely low read counts regardless of gene count. This study supports the suitability of D. discoideum to become a vehicle for bioremediation due to tolerance of PET microplastics, however, difficulties in particular with the transformation and continued cultivation of wild-type (non-axenic) strains will have to be overcome for successful development of this organism into a recombinant bioremediation tool. Further investigation of the HydroSeq data and technique could yet produce a viable tRNA profile for D. discoideum.