Fig. 6: Construction of the sensor P. putida SENS·P strain for detecting PET-degradation products.

A Enabling ethylene glycol (EG) and monohydroxyethyl terephthalate (MHET) assimilation to construct the sensor P. putida SENS·P strain. The growth of strain SENS·T was improved through adaptive laboratory evolution, yielding SENS·TE; subsequent genomic integration of a MHETase module enables constitutive production and secretion of this hydrolytic enzyme (P. putida SENS·TM). Deletion of the gclR regulator gene lifted the repression of the catabolic pathway that transforms glyoxylate into pyruvate, allowing for complete assimilation of PET-breakdown products; these modifications gave rise to strain SENS·P. B Combinatorial titration of MHET and EG for the sensor strains SENS·TE, which can only consume TPA, and SENS·P, engineered for MEHT and EG consumption. The msfGFP fluorescence profile of the sensor strains was evaluated in DBM medium with 10 mM TPA and increasing MHET and EG concentrations (concn.). C A suspension of 20 g L^–1 PET was treated with the leaf and branch compost cutinase (LCC) at different enzyme concentrations (concn.), incubation times, and temperatures. After enzymatic treatment, the supernatant was inoculated with the sensor strain P. putida SENS·TE (left) or SENS·P (right). The msfGFP fluorescence signal was recorded after 24 h. D Relationship between the concentration of PET degradation products (TPA and MHET, independently quantified by HPLC) and the msfGFP fluorescence signal by sensor strain SENS·TE (left) and SENS·P (right). In all cases, results represent average values ± standard deviation of three independent experiments. Abbreviations: AU, arbitrary units.