Fig. 4

Engineering PET hydrolase secretion. A Overview of plasmid design to screen a 30-mer SP library for LCC, HiC, and IsP secretory expression in P. putida TA7-EG. We used the RK2 low copy number plasmid, the P_rhaB induction system, BCD2 translational coupler, and a SP library cloned at the 5’ end of the LCC, HiC, and IsP ORFs. B Sequence view of the 3’ end of the BCD2 translational coupler, where we introduced a silent point mutation (red) to create a unique BbsI restriction site. Together with a unique PstI site preceding the PET hydrolase ORFs, this allowed facile in-frame cloning of the SP library. C SP library screening results, showing culture OD600 (measured 20 h post-induction) plotted against culture supernatant esterase activity (measured 3 h post-induction). Each dot represents a single clone (one well of a 96-well culture plate); grey dots are e.v. controls, red dots are sequenced clones, with the identified SPs indicated. D Shake flask validation of P. putida TA7-EG expressing the best identified SP-PET hydrolase constructs. Data depict OD600 (top) and supernatant or cell-associated esterase activity measured for 5 h following induction (bottom). The * in (D) indicates that esterase activity was not measured at the t = 5 h timepoint due to obvious cell lysis for most samples. Data shown are derived from one representative experiment with duplicate cultures