Plastic degradation using genetically engineered microorganisms

Abstract

The usage of PET plastics in daily life have excessively increased in the last decade. The increased usage of PET is accompanied with the massive amount of PET waste accumulating rapidly. Environmental pollution caused by this waste has reached a critical point with pollutants being found even in the most remote parts of the world. Causing massive damage to ecosystems and even human health, PET plastic waste needs to be handled urgently. Although there are ongoing PET recycling and treatment efforts, the current methods in use are insufficient. The techniques currently used are either costly, leave a significant carbon footprint or are lacking in their ability to recycle microplastics. However, with the discovery of microorganisms which have the ability of degrading PET, biodegradation of PET products has emerged as a promising green alternative. In this thesis we designed bacterial tools to utilize the PET hydrolyzing enzyme, PETase. For this purpose, living bacterial platforms were engineered. The first system employed E. coli as the host to display PETase on the cellular surface. With PETase molecules anchored on its surface, aiding in the stability and the activity of the enzyme, the system will be a useful tool for PET degradation. For the surface display system, the Ag43 autotransporter protein is used. The system was cloned, and expression was analyzed using immunocytochemistry labeling. The activity of the system was analyzed with chromatography and mass spectrometry. The second system proposed uses E. coli once again as a workhorse for PETase secretion, creating a simple yet effective tool for the bioremediation of PET. For secretion of the enzyme, the disruption of Braun’s lipoprotein to create a leaky outer membrane is exploited. The system was cloned, and the cloning was verified. Also, the activity of native PETase was analyzed with HPLC and mass spectrometry. With this analysis, the PET degrading activity of PETase was confirmed.