The enzymatic degradation of polyethylene terephthalate (PET) is a promising new approach for the environmentally friendly recycling of PET waste, but so far, low degradation rates paired with releatively high costs have limited the economic feasibility of this method. Here we present the construction of a new bacterial whole‐cell biocatalyst utilizing a new inverse autotranspoter based surface display of PETase in E. coli. The resulting catalyst allows an extremely easy production of large amounts of enzyme and a five times more effective degradation of PET at a lower temperature of 25 °C compared to free PETase at 30 °C. Additionally, we demonstrate how rhamnolipids, which are environmentally benign and can be cost‐effectively produced using strains of E. coli, can be used to amplify the hydrolysis rates of PET even further, presumably by acting as mediators between PETase and PET. Cells displaying PETase in combination with externally supplied rhamnolipids outperformed free PETase by a factor of 16, allowing the degradation of highly crystalline post‐consumer PET waste to an extent of 8 % within 3 days at room temperature.

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Summary Inducible promoters such as Plac are of limited usability for industrial protein production with Pseudomonas putida. We therefore utilized cell density‐dependent auto‐inducible promoters for recombinant gene expression in P. putida KT2440 based on the RoxS/RoxR Quorum Sensing (QS) system of the bacterium. To this end, genetic regions upstream of the RoxS/RoxR‐regulated genes ddcA (PRox132) and PP_3332 (PRox306) were inserted into plasmids that mediated the expression of superfolder green fluorescent protein (sfGFP) and surface displayed mCherry, confirming their promoter functionalities. Mutation of the Pribnow box of PRox306 to the σ70 consensus sequence (PRox3061) resulted in a more than threefold increase of sfGFP production. All three promoters caused cell density‐dependent expression, starting transcription at optical densities (OD578) of approximately 1.0 (PRox132, PRox306) or 0.7 (PRox3061) as determined by RT‐qPCR. The QS dependency of PRox306 was further shown by cultivating P. putida in media that had already been used for cultivation and thus contained bacterial signal molecules. The longer P. putida had grown in these media before, the earlier protein expression in freshly inoculated P. putida appeared with PRox306. This confirmed previous findings that a bacterial compound accumulates within the culture and induces protein expression.

Summary The application of enzymes as biocatalysts in industrial processes has great potential due to their outstanding stereo‐, regio‐ and chemoselectivity. Using autodisplay, enzymes can be immobilized on the cell surface of Gram‐negative bacteria such as Escherichia coli. In the present study, the surface display of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO) on E. coli was investigated. Displaying these enzymes on the surface of E. coli resulted in whole‐cell biocatalysts accessible for substrates without further purification. An apparent maximal reaction velocity VMAX(app) for the oxidation of cyclohexanol with the ADH whole‐cell biocatalysts was determined as 59.9 mU ml−1. For the oxidation of cyclohexanone with the CHMO whole‐cell biocatalysts a VMAX(app) of 491 mU ml−1 was obtained. A direct conversion of cyclohexanol to ε‐caprolactone, which is a known building block for the valuable biodegradable polymer polycaprolactone, was possible by combining the two whole‐cell biocatalysts. Gas chromatography was applied to quantify the yield of ε‐caprolactone. 1.12 mM ε‐caprolactone was produced using ADH and CHMO displaying whole‐cell biocatalysts in a ratio of 1:5 after 4 h in a cell suspension of OD578nm 10. Furthermore, the reaction cascade as applied provided a self‐sufficient regeneration of NADPH for CHMO by the ADH whole‐cell biocatalyst.