Schwanemann, T. | Search bioRxiv

bioRxiv2026

Engineering Pseudomonas putida KT2440 for open-loop upcycling of mixed plastics

Current mechanical and chemical recycling strategies address less than 10% of global plastic waste, necessitating alternative valorization routes. Biological upcycling via enzymatic depolymerization combined with microbial conversion of the resulting monomers offers a promising pathway to transform mixed plastic waste into valuable alternatives. Here, we employed a single engineered Pseudomonas putida KT2440 for simultaneous co-utilization of five plastic monomers including ethylene glycol, terephthalic acid, adipic acid, 1,4-butanediol, and L-lactic acid, which can be derived from enzymatic hydrolysis of polyethylene terephthalate (PET), polybutylene adipate-co-terephthalate (PBAT), polyester-polyurethanes (PUs), and polylactic acid (PLA). Continuous fermentation over 21 days with alternating mixed-monomer feeds achieved steady state growth and complete substrate depletion, yielding adaptive mutations that informed iterative strain improvement. Further engineering enabled the biosynthesis of (R)-3-hydroxybutyrate (R-3HB), and 0.70 g L-1 R-3HB was produced directly from enzymatic hydrolysates of blended PET, PBAT, and TPU. These results establish a viable bio-based approach for upcycling realistic mixed plastics into value-added bioproducts.

microbiology↗

bioRxiv2025

Engineering of Pseudomonas putida KT2440 for Broad-Chain-Length 3-Hydroxy Fatty Acid Biosynthesis

3-Hydroxy fatty acids (3-HFAs) are versatile intermediates for bio-based polymers, fuels, and surfactants, and are advancing circular economy manufacturing. We built an acyl- CoA ligase-deficient chassis of Pseudomonas putida KT2440, thereby blocking 3-HFA activation and preventing its use as carbon source. This strategy decoupled synthesis from catabolism and enabled 3-HFA accumulation. We then compared two different routes for modifying the free 3-HFA composition. 1. In a direct route, overexpressing native PhaG produced C8, C10, C12, and C14 3-HFAs, achieving a total titer of 0.73 g/L in shake flasks. Functional analyses under our tested conditions support a re-assignment of PhaG: rather than acting primarily as a 3-hydroxyacyl-ACP:CoA transacylase, it functions mainly as a thioesterase, liberating free 3-HFAs from hydroxyacyl-ACP. 2. In an indirect route, overexpressing RhlA variants generated hydroxyalkanoyl-alkanoates (HAAs) that were converted to free 3-HFAs by endogenous esterase(s): RhlA from Pseudomonas aeruginosa PAO1 favored C8-C12 and yielded 0.47 g/L 3-HFAs, whereas RhlA from Burkholderia plantarii PG1 favored C10-C14 with 0.14 g/L, of which 80% was C14. Finally, we demonstrated process feasibility by up-scaling the PhaG pathway in a stirred-tank reactor. These results establish modular, stable, chassis-compatible routes for tailoring 3-HFA chain-length distributions, thereby providing a foundation for scalable, bio-based monomer supply in a circular economy.

synthetic biology↗