PET is one of the most widely used plastics, so WUST-China developed a Rhodococcus that can degrade and recycle PET by using PET as a degradation target.PETase and MHETase have been found in recent years to completely degrade PET plastic to terephthalic acid TPA and ethylene glycol EG.
In addition, the high concentration of terephthalic acid is toxic to microorganisms. Rhodococcus Opacus is more tolerant to ketones and aromatic compounds than engineered bacteria such as Escherichia coli and Bacillus subtilis. Rhodococcus Opacus PD630 is a natural oil producer and can accumulate a large number of microbial oils and fats from a single carbon source (e.g. gluconate, glucose or dextrose), so we have engineered Rhodococcus Opacus to degrade PET plastic and convert the degraded PET into microbial oils and fats.
Various types of promoters, signal peptides, and enhancers were screened to efficiently express PETase and MHETase and promote the degradative enzymes' expression and secretion. In addition, PE Tase-MHE Tase fusion proteins were constructed using a linker peptide to improve the efficiency of PET degradation. The thermal stability of PETase is insufficient to enable its use in the degradation of plastics. Homology modeling is proposed to improve the thermal stability of PETase and to identify sites that potentially affect its thermal stability. Based on this premise, targeted mutagenesis was used to modifty PE Tase genetically, and the mutant enzyme's thermal stability was subsequently verified to corroborate the modeling results.
To convert PET waste into usable substrates for Rhodococcus Opacus, PET degradation products must be converted into simpler forms that the bacteria can more easily consume. However, Rhodococcus Opacus cannot rapidly consume TPA and EG to produce microbial lipids. To enable Rhodococcus Opacus to use TPA to produce microbial lipids rapidly, we introduced the Tph operon from Comamonas Testosterone YZW-D. This operon converts TPA to protocatechuic acid (PCA)，resulting in faster accumulation of degradation products through the tricarboxylic acid cycle and the production of microbial oils and fats. The practical resource conversion of PET waste solves environmental problems and addresses the energy crisis through bioremediation.