A cell-free platform for the high-efficiency synthesis and screening of PET hydrolase

In this study, cell-free system (CFS) was employed to establish a platform for the high-efficiency synthesis and screening of PET hydrolase (PETase). This platform integrates the in vitro PETase expression using linear DNA templates, the in-situ protein quantification by fluorometry, and the evaluation of catalytic activity by colorimetry. Upon implementing on three naturally occurring PETases (IsPETase, LCC and TfCut2) as well as 16 variants, we demonstrated the ratio of absorbance to fluorescence is positively correlated with the specific activity of the enzyme, which thus verified the feasibility of this platform in the preliminary screening of effective PETase variants. Compared with conventional in vivo expression and HPLC-based activity assay, this study provided a more convenient and cost-effective screening platform for accelerating the design and evolution of PETase.