Background

Poly(ethylene terephthalate) (PET), an extensively used and manufactured plastic, has become a major global concern due to its accumulation and resistance to degradation in the natural environment. Current strategies for handling waste PET, such as mechanical and chemical recycling, have been applied to alleviate PET pollution. However, some recycling processes can potentially cause secondary pollution by consuming massive amounts of energy. Moreover, the amount of recycled PET is quite limited and certainly much less than the amount of PET that ends up in the landfill. Consequently, the majority of PET waste is further broken down into small pieces during the landfilling process and then dispersed in the environment. These plastic pieces with sizes smaller than 5 mm are classified as microplastics. The leakage of microplastics into the ocean can severely threaten marine lives and ecosystems, and further jeopardize human health through the food web. Our project focuses on developing a remedy for PET microplastics by applying metabolic engineering to construct a whole-cell biocatalyst for degrading and assimilating waste PET in situ. Vibrio natriegens, a fast-growing, nonpathogenic, salt-tolerant marine bacteria, is the ideal chassis to achieve this bioremediation purpose.

Methods and Results

Firstly, we engineered V. natriegens to express PET-degrading enzymes. Previous studies revealed several microorganisms and enzymes that hydrolyze PET. Among them, a two-enzyme system composed of PETase and MHETase was discovered in Ideonella sakaiensis and was demonstrated to degrade PET efficiently at ambient temperature. We transferred the I. sakaiensis PETase and MHETase into V. natriegens after codon optimization, and fused PETase to a surface anchor, successfully constructing a V. natriegens that displays PETase on its outer membrane. We verified the surface display by using immunocytochemistry and demonstrated the ability of the displayed PETase to degrade the PET monomer bis(2-hydroxyethyl) terephthalate (BHET) using liquid chromatography.

After hydrolysis by PETase and MHETase, PET is broken down into terephthalic acid (TPA) and ethylene glycol (EG). Therefore, we constructed a metabolic pathway for assimilating TPA in V. natriegens. TPA is commonly metabolized via enzymes that convert TPA into protocatechuic acid (PCA), followed by further catabolism through the benzoate pathway. V. natriegens proved to be an ideal chassis for this project as it demonstrated cell growth in minimal media with PCA as the sole carbon source. We will present our efforts to enable TPA catabolism in V. natriegens by first prototyping the necessary transporters and metabolic enzymes in E. coli, demonstrating growth of E. coli using TPA as a sole carbon source for the first time.

Finally, we will describe our efforts to apply directed evolution for improving the surface display, PETase, and TPA degradation efficiency. For this purpose, we will describe a combination of error-prone PCR and in vivo mutagenesis (using temperate phage), followed by fluorescence-activated cell sorting and growth-based selections. The ability of these evolved strains to grow on PET as a sole carbon source in seawater media will also be presented.

Implications

Our project proposes a sustainable in situ bioremediation strategy for PET microplastic pollution. The engineered V. natriegens with the two-enzyme system may be able to perform as a whole-cell catalyst that can be easily separated or recycled by centrifugation and proliferated by cultivation so that it can overcome the major limitation of enzymatic PET recycling - product inhibition. Our research also contributes to the advancement of synthetic biology by expanding the genetic toolkit of V. natriegens, an under-characterized bacterium with superior growth rates compared with E. coli and yeast. Specifically, we have enabled surface display of heterologous proteins by V. natriegens for the first time. Taken together, this work expands the knowledge about engineering V. natriegens, and establishes a promising strategy for mitigating PET accumulation in seawater.