The escalating global plastic pollution crisis demands innovative solutions beyond traditional mechanical, thermal, and chemical recycling methods. Microbial-based bioconversion offers a promising alternative, capable of simplifying processing and integrating waste degradation with product generation. Polyethylene terephthalate (PET), a ubiquitous plastic, has been a focus of research, with several microbial isolates and engineered enzymes showing potential for its breakdown and assimilation. However, current biotransformation approaches using monocultures face challenges: (1) complete PET depolymerization yields a mixture of TPA and EG, leading to catabolic cross-talk and inefficient substrate utilization; (2) depolymerization products can inhibit microbial metabolism; and (3) upcycling plastics into valuable products requires complex, multi-step pathways needing sophisticated system-level fine-tuning, which is difficult to achieve in single strains. This study proposes the use of designer microbial consortia to overcome these limitations, leveraging the principle of division of labor (DOL) to enhance efficiency and flexibility in PET upcycling.

Numerous studies have explored microbial degradation of PET. Several bacterial isolates, including *Ideonella sakaiensis*, *Thermobifida fusca*, and *Saccharomonospora viridis*, have demonstrated the ability to break down PET. Simultaneously, significant progress has been made in enzyme engineering, using techniques like random mutagenesis and machine learning to enhance the efficiency of PET depolymerization. Several bacterial and fungal strains have also been engineered to convert PET waste into valuable chemicals and products. However, these primarily monoculture-based approaches face challenges due to the complex nature of PET depolymerization products and the need for robust, multi-step metabolic pathways.

The researchers engineered a synthetic microbial consortium composed of two *Pseudomonas putida* strains: Pp-T, a TPA specialist, and Pp-E, an EG specialist. Pp-T was created by deleting the *ped* gene cluster (responsible for EG utilization) from EM42 and introducing the *tpa* cluster from *Rhodococcus jostii* RHA1 for TPA degradation. Pp-E was constructed from M31 by deleting the *gclR* gene to enhance EG assimilation and improving the expression of the *glcDEF* operon. A single-strain counterpart, Pp-TE, capable of both TPA and EG utilization, was also engineered for comparison. Batch fermentations were performed using various substrate combinations (TPA, EG, and mixtures) to assess the efficiency of the consortium and the single strain. An integrated workflow combining chemical hydrolysis with microbial consumption was developed for complete PET degradation. Alkaline hydrolysis (using NaOH) was used to depolymerize PET, and the resulting hydrolysate was then used as a substrate for fermentation. To demonstrate upcycling, the researchers further engineered the consortium and single strain to produce mcl-PHA. They overexpressed genes (*phaG*, *alkK*, *phaC1*, *phaC2*) involved in mcl-PHA biosynthesis and deleted genes (*fadBA*, *fadBAxE*, *phaZ*) to limit competing pathways. Finally, to illustrate system-level optimization through DOL, the researchers engineered a TC-EM consortium for MA production. The MA pathway was split between Pp-TC (TPA to catechol) and Pp-EM (catechol to MA), allowing for independent optimization via population modulation and inoculation timing.

The study revealed several key findings: (1) The T-E consortium significantly outperformed the single-strain Pp-TE in co-utilizing TPA and EG, especially at high substrate concentrations and with crude hydrolysate. (2) The T-E consortium exhibited simultaneous substrate consumption, unlike Pp-TE which showed sequential consumption due to catabolic cross-talk. (3) The integrated chemical and microbial process effectively degraded PET, with the consortium showing faster degradation than the single strain, even with crude hydrolysate. (4) Engineering the consortium for mcl-PHA production resulted in significantly higher titers and yields compared to the single strain, especially in fed-batch fermentations. (5) The TC-EM consortium, engineered for MA production, demonstrated flexible system optimization through manipulation of inoculation ratios and time delays, leading to substantially higher MA titers compared to the single strain Pp-TEM. The optimal conditions minimized catechol accumulation, a toxic intermediate, leading to efficient MA production. The use of varying initial inoculation ratios and time delays for inoculation of different strains showed a significant effect on the overall efficiency of the processes.

The results demonstrate the significant advantages of engineered microbial consortia over monocultures for PET upcycling. The division of labor strategy effectively reduced catabolic cross-talk and improved substrate utilization efficiency, particularly with complex substrates like crude hydrolysate. The superior performance in mcl-PHA and MA production further highlights the potential of DOL for optimizing bioconversion pathways. The flexible optimization through population modulation and inoculation timing underscores the power of DOL for system-level engineering. The findings align with observations of multispecies communities in natural plastic degradation environments, suggesting that consortia may represent a more realistic and effective approach for industrial applications.

This study provides compelling evidence for the efficacy of engineered microbial consortia in upcycling PET. The division of labor strategy resulted in superior performance in substrate utilization and product synthesis compared to monoculture approaches. The flexible optimization strategies highlight the potential for scaling up these processes for industrial applications. Future research should focus on further minimizing metabolic crosstalk, exploring the use of different chassis organisms and pathway designs, and expanding this approach to other types of plastic waste.

The study primarily focused on laboratory-scale experiments. Further research is needed to evaluate the scalability and economic feasibility of these consortia for industrial-scale PET upcycling. The optimization strategies were explored for specific products and may need further investigation for other valuable products. The toxicity of PET hydrolysate at high concentrations warrants further study to explore ways to reduce its toxicity or improve microbial tolerance.