Project description:Poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis produces hydrolytic enzymes that convert PET, via mono(2-hydroxyethyl) terephthalate (MHET), into the monomeric compounds, terephthalic acid (TPA) and ethylene glycol (EG). Understanding PET metabolism is critical if this bacterium is to be engineered for bioremediation and biorecycling. TPA uptake and catabolism in I. sakaiensis have previously been studied, but EG metabolism remains largely unexplored despite its importance. First, we identified two alcohol dehydrogenases (IsPedE and IsPedH) and one aldehyde dehydrogenase (IsPedI) in I. sakaiensis as the homologs of EG metabolic enzymes in Pseudomonas putida KT2440. IsPedE and IsPedH exhibited EG dehydrogenase activities with Ca2+ and a rare earth element (REE) Pr3+, respectively. We further found an upregulated dehydrogenase gene when the bacterium was grown on EG, whose gene product (IsXoxF) displays a minor EG dehydrogenase activity with Pr3+. IsPedE displayed a similar level of activity toward various alcohols. In contrast, IsPedH was more active toward small alcohols, whereas IsXoxF was the opposite. Structural analysis with homology models revealed that IsXoxF had a larger catalytic pocket than IsPedE and IsPedH, which could accommodate relatively bulkier substrates. Pr3+ regulated the protein expression of IsPedE negatively; IsPedH and IsXoxF were positively regulated. Taken together, these results indicated that the combination of IsPedH and IsXoxF complements the function of IsPedE in the presence of REEs. IsPedI exhibited dehydrogenase activity toward various aldehydes with the highest activity toward glycolaldehyde (GAD). This study demonstrated a unique alcohol oxidation pathway of I. sakaiensis, which could be efficient in EG utilization.
Project description:The scale of plastic pollution boggles the mind. Nearly 400 megatons of virgin plastics are produced annually, with an environmental release rate of 80%, and plastic waste, including microplastics and nanoplastics, is associated with a plethora of problems. The naturally evolved abilities of plastic-degrading microbes offer a starting point for generating sustainable and eco-centric solutions to plastic pollution-a field of endeavor we term eco-microbiology. Here, we developed an iterative discovery procedure coupling faster polyethylene terephthalate (PET)-dependent bioactivity screens with longer-term PET biodegradation assays to find biochemical boosters of PET consumption by the bacterium Piscinibacter sakaiensis. We discovered multiple hits supporting the enhancement of PET biodegradation, with a 0.39% dilution of growth medium #802, a rich medium similar to Luria-Bertani broth, on average more than doubling the rate of PET biodegradation both alone and in combination with 0.125% ethylene glycol. In addition, we identified other chemical species (sodium phosphate, L-serine, GABA) worth further exploring, especially in combination with growth medium #802, for enhanced PET biodegradation by P. sakaiensis. This work represents an important step toward the creation of a low-cost PET fermentation process needed to help solve PET plastic pollution.ImportancePlastic pollution is an urgent issue. Adding to the well-known problems of bulk plastic litter, shed microplastics and nanoplastics are globally distributed, found in diverse organisms including human foodstuffs and tissues, and increasingly associated with chronic disease. Solutions are needed and the microbial world offers abundant help via naturally evolved consumers of plastic waste. We are working to accelerate polyethylene terephthalate (PET) plastic biodegradation by Piscinibacter sakaiensis, a recently described bacterium that evolved to slowly but completely consume PET, one of the most common types of plastic pollution. We used a combination of PET-dependent bioactivity screens and biodegradation tests to find stimulators of PET biodegradation. Out of hundreds, we found a small number of biochemical conditions that more than double the PET biodegradation rate. Our work provides a foundation for further studies to realize a fermentation process needed to help solve PET plastic pollution.