Mitigating cell lysis in PET hydrolase production: Nuclease supplementation and growth-decoupled co-cultivation enable scalable enzymatic PET recycling

Enzymatic recycling of polyethylene terephthalate (PET) provides a sustainable alternative to conventional PET waste treatment, but the scalable production of PET-degrading enzymes remains a challenge. A key bottleneck is intense cell lysis during production, caused by the PET hydrolases attacking the host cell membrane. This lysis leads to elevated extracellular DNA content, increased viscosity, excessive foaming, reduced oxygen transfer during fermentation, and difficulties in downstream processing such as filtration and centrifugation. In this study, we explored two strategies to address these issues: (1) the addition of nucleases during fermentation, and (2) co-cultivation of PETase- and nuclease-expressing E. coli strains in varying inoculum ratios. To stabilize population distribution during the production phase of co-cultivation, we employed a growth-decoupled expression system. Both strategies reduced extracellular DNA, lowered broth viscosity, and improved cell settling and foam control, while maintaining or even enhancing PET hydrolase activity and titer. While adding nuclease to produce PETase may increase production costs, co-cultivation provides a promising alternative to simplify processing. Our findings offer a scalable and cost-efficient strategy for producing lysis-prone enzymes, enabling robust fermentation processes for industrial enzymatic PET recycling. • NucA from co-cultivation or dosing reduces extracellular DNA and broth viscosity • Lower DNA levels reduce antifoam use and improve oxygen transfer in fermentation • Improved settling enhances separation of cells and debris from supernatant in primary recovery • Co-cultivation maintains PET hydrolase activity and titer despite NucA expression • Co-cultivation offers a cost-efficient, scalable strategy for enzyme production