Advancing Microbial Polyethylene Terephthalate Degradation with Ideonella sakaiensis: Insights into its TPA Catabolism and Genome Modification

Plastic pollution is one of the greatest environmental challenges of the 21st Century due to the failure of contemporary recycling pipelines to valorize more than 9% of waste plastics and the recalcitrance of these materials to environmental degradation. As anthropogenic climate change threatens the life-sustaining planetary boundaries of Earth and necessitates a further reduction in the extraction of petroleum resources, recovering these vital chemical building blocks from discarded plastic waste will be essential. Using the tools of Synthetic Biology to harness microbes that are already capable of degrading and assimilating environmental plastics is one means of expanding a circular plastics economy. By converting environmental isolates into industrially robust chassis organisms, the production of essential chemical resources from waste materials is possible, alongside competition with the ecologically damaging petroleum industry. While many plastics are biologically inaccessible, the enzymatic degradation of polyethylene terephthalate (PET) is widespread, making it a promising substrate for a future plastics bioeconomy. However, only one known wildtype bacterium, Ideonella sakaiensis 201-F6, is capable of both PET degradation and assimilation of its constituent monomers, terephthalic acid (TPA) and ethylene glycol (EG), into microbial metabolism. Despite this unique metabolic ability, the Synthetic Biology community has largely neglected this terrestrial microbe due to its lack of genetic tools and in-depth physiological characterization. Yet, I. sakaiensis has untapped potential for turning waste PET into valuable chemical resources. In this thesis, the unique physiology of PET-catabolizing bacterium I. sakaiensis was examined through a combination of genomics, proteomics, and in vitro biochemistry. A closed genome structure for the microbe was deduced with hybrid short- and long-read sequencing. Proteomics provided experimental evidence for the predicted metabolic pathways for TPA and protocatechuic acid assimilation as well as transcription factors active during TPA consumption. A series of in vitro biochemistry techniques subsequently confirmed the DNA binding of the identified transcription factors to the promoters of key PET catabolism genes. One allosteric transcription factor with the ability to sense TPA pathway metabolites was characterized further. Finally, strides towards I. sakaiensis genetic tractability were made via the identification of a functional origin of replication using a high-throughput screen and an electroporation method. Taken together, this work advances our genomic and biochemical understanding of TPA catabolism in the non-model microbe I. sakaiensis, broadening the scope of Synthetic Biology strategies available for microbial plastic degradation and upcycling.