Increased Recombinant Protein Expression of Petase Enzyme in E. coli

In 2015, more than 380 million metric tons of plastic were produced, with a large quantity being polyethylene terephthalate, or PET, that is commonly used for disposable water bottles. PET is engineered for durability, leading to its slow natural breakdown in the environment. A recently discovered enzyme, PETase, speeds up depolymerization of PET; however, it’s turnover rate is still too slow for practical recycling application. Many groups have sought to engineer PETase for faster catalysis and elevated stability. Considerably less effort has been focused toward expressing large quantities of the protein, which is necessary for large-scale application. Our previous work proved that making PETase in a disulfide bond promoting strain of E. coli, SHuffle T7 Express, results in higher active expression of the enzyme compared to standard E. coli production strains, as PETase is known to require disulfide bonds to be functional. The goal of this work is to further enhance and scale-up production of PETase by studying different medias, carbon sources, induction times, and bioreactor feeding strategies. PETase activity was measured in clarified lysates by quantifying catalysis of p-Nitrophenyl acetate. Using the common laboratory media, 2xLB, PETase was produced in a shake flask with an activity of 10 units/L (unit = umol pNitrophenol produced/s). In a controlled bioreactor, using the same media, expression was increase to 15U/L. Switching to a complex media, with glycerol as its main carbon source, and early induction time, we observed a further 3-fold increase in expression, up to 45U/L, for a batch time of 48 hours. We hope these advances in PETase production will improve the economics to apply this industrially relevant enzyme.