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Synthetic polymers are widely used in basic day to day activities given the wide range of uses associated with their advantageous material properties. Polyethylene terephthalate (PET) is a widely used synthetic polymer with annual production exceeding 73.39 million tons. Out of all the PET material generated, only 30% PET is recycled because current mechanical and chemical recycling methods are not techno-economically viable. This leads to the accumulation of a large amounts of PET waste in the environment causing significant damage to terrestrial and aquatic ecosystems. An alternative to recycling is PET upcycling approaches strategize of converting PET waste into high-value products. This development enables a circular material economy for PET. There are several reports of PET upcycling strategies that describe hybrid-chemo biological approaches. However, efficient whole-cell microbial catalysts capable of selectively degrading PET into its original monomers of ethylene glycol (EG) and terephthalic acid (TPA), and simultaneously upcycling these monomers into high-value compounds is yet to be developed. The selection of an appropriate host strain for plastic upcycling is vital in developing industrially applicable whole-cell biocatalysts. Use of non-model organisms in industrial applications has gained attention over the recent years. The work presented here illustrates comprehensive genomic and phenomic investigations suggesting that the metabolic pathways of the newly identified, Erwinia aphidicola LJJL01, is a promising candidate for upcycling PET-degraded substrates. First, we performed a comprehensive phenomic characterization of E. aphidicola LJJL01 including SEM imaging, pH, optimal temperature, toxicity tolerance, antibiotic tolerance, and fatty acid profile. The metabolic capability of the strain was shown using a substrates utilization assay that includes 29 substrates which comprise C-6 sugars, C-5 sugars, sugar alcohols, acids, alcohols. Secondly, we developed an efficient system for plasmid-based expression and secretion of heterologous proteins. We established synthetic microbiology tools, including CRISPR/Cas9-based genomic editing, to engineer the E. aphidicola LJJL01 strain. Thirdly, we demonstrated successful heterologous expression of PET hydrolyzing enzymes such as PETase and MHETase from Ideonella sakaiensis together with their secretion signal peptides in E. aphidicola LJJL01. We assessed the strain's PET hydrolyzing activity using Bis(2-Hydroxyethyl) terephthalate (BHET), an intermediate molecule of PET as the model substrate. The strain yields 0.88 ±0.10 mol of TPA/mol of BHET in minimal salt medium within 48 hours and outperforms the commonly used platform organisms such as Pseudomonas putida KT2440. We also successfully expressed the thermostable leaf branch compost cutinase (LCC) in E. aphidicola LJJL01. For the first time we were able to demonstrate the synergistic activity of LCC and MHETase enzymes at 30 °C. Since the developed strains didn't show considerable PET degradation at ambient conditions, we developed a novel process to hydrolyze amorphous and commercial grade PET using cell-free supernatant of secreted LCC enzyme at 72°C (the glass transition temperature of PET). Finally, we further engineered the aromatic catabolism of the strain to demonstrate the potential of upcycling PET-degraded TPA into high-value platform chemicals such as cis, cis-muconate. Taken together, we demonstrated E. aphidicola LJJL01, a promising microbial chassis to develop whole-cell biocatalysts to upcycle PET and enable circular material economy. |
