An Ultra-Sensitive <i>Comamonas thiooxidans</i> Biosensor for the Rapid Detection of Enzymatic Polyethylene Terephthalate (PET) Degradation

Polyethylene terephthalate (PET) is a prevalent synthetic polymer that is known to contaminate marine and terrestrial environments. Currently, only a limited number of PET-active microorganisms and enzymes (PETases) are known. This is in part linked to the lack of highly sensitive function-based screening assays for PET-active enzymes. Here, we report on the construction of a fluorescent biosensor based on <i>Comamonas thiooxidans</i> strain S23. <i>C. thiooxidans</i> S23 transports and metabolizes TPA, one of the main breakdown products of PET, using a specific tripartite tricarboxylate transporter (TTT) and various mono- and dioxygenases encoded in its genome in a conserved operon ranging from <i>tphC-tphA1.</i> TphR, an IclR-type transcriptional regulator is found upstream of the <i>tphC-tphA1</i> cluster where TPA induces transcription of <i>tphC-tphA1</i> up to 88-fold in exponentially growing cells. In the present study, we show that the <i>C. thiooxidans</i> S23 wild-type strain, carrying the sfGFP gene fused to the <i>tphC</i> promoter, senses TPA at concentrations as low as 10 μM. Moreover, a deletion mutant lacking the catabolic genes involved in TPA degradation <i>thphA2-A1 (</i>Δ<i>tphA2A3BA1</i>) is up to 10,000-fold more sensitive and detects TPA concentrations in the nanomolar range. This is, to our knowledge, the most sensitive reporter strain for TPA and we demonstrate that it can be used for the detection of enzymatic PET breakdown products. <b>IMPORTANCE</b> Plastics and microplastics accumulate in all ecological niches. The construction of more sensitive biosensors allows to monitor and screen potential PET degradation in natural environments and industrial samples. These strains will also be a valuable tool for functional screenings of novel PETase candidates and variants or monitoring of PET recycling processes using biocatalysts. Thereby they help us to enrich the known biodiversity and efficiency of PET degrading organisms and enzymes and understand their contribution to environmental plastic degradation.