PF-5

Global accumulation of difficult-to-recycle plastics, including polyurethane (PU), poses a significant environmental threat, as current recycling strategies are insufficient to meet the growing demand. Pseudomonas protegens is capable of degrading PU and may offer a promising nature-inspired solution. However, the genetic and regulatory mechanisms underlying microbial PU biodegradation remain poorly understood. To address this, we employed transposon insertion sequencing (TIS), a high-throughput functional genomics approach, to identify genes essential for PU degradation under plastic exposure conditions. A transposon mutant library of P. protegens Pf-5 was enriched on two types of PU: a polyester-polyurethane dispersion (Impranil DLN) and a thermoplastic polyurethane (Avalon AE). Through this approach, we identified a key global regulator encoding a sensor kinase, gacS, that plays an inhibitory role in PU degradation, so that P. protegens Pf-5 lacking gacS displayed enhanced capacity to degrade PU. Transcriptomic and phenotypic analyses revealed that disruption of gacS resulted in increased activity of siderophore-mediated iron acquisition, while negatively impacting sulfur homeostasis and oxidative stress responses. We propose that this imbalance in iron homeostasis and oxidative stress in the gacS mutant triggers a Fenton reaction, leading to the accumulation of reactive oxygen species, which in turn enhances PU degradation via oxidative processes. These findings establish that siderophores involved in iron acquisition, such as pyoverdine, could act as catalysts for plastic degradation. Our results not only provide a deeper understanding of the genetic and regulatory basis of PU biodegradation but also open new avenues for leveraging bacterial pathways and siderophores for innovative plastic recycling strategies.

The polyurethane (PU) market is projected to reach $94 billion USD by 2028 and spans many industries, including automotive, construction, medical, furniture, and fashion. However, less than 30% of PU plastics are recycled, with the remainder going to landfill. The inherent recalcitrant nature of PU makes it challenging to study naturally occurring routes of PU degradation, like microbial biodegradation and its underlying genetic mechanisms. To address this, we developed a high-throughput screening method using transposon-directed insertion site sequencing (TraDIS) on the known PU-degrading strain Pseudomonas protegens Pf-5. This approach identified a key global regulatory gene (GacS), the mutant of which acts as a “hyperdegrader” with a faster PU degradation rate. Subsequent transcriptomic and phenotypic analyses revealed an unsuspected PU-degradation mechanism involving a siderophore-mediated Fenton reaction. These findings highlight the importance of using high-throughput functional genomics to uncover novel genes and pathways involved in plastic biodegradation. Our findings not only advance the understanding of PU degradation but also open new avenues for developing innovative solutions for plastic waste management.