Development of an ethanol-based microbial platform for 3-hydroxypropionic acid production using engineered Pseudomonas putida KT2440

Ethanol is an attractive C2 feedstock for microbial biomanufacturing because it is directly oxidized to acetyl-CoA with favorable redox balance. 3-Hydroxypropionic acid (3-HP), a versatile platform chemical, can be synthesized via the malonyl-CoA and β-alanine pathways. An inducer-free ethanol-to-3-HP process in Pseudomonas putida KT2440 was developed by enabling constitutive expression of both pathways and deleting native 3-HP catabolism and polyhydroxyalkanoate (PHA) synthesis to redirect flux toward the target product. Each route and their co-activation were evaluated, and a genome-scale model constrained with transcriptomic data was applied to identify metabolic nodes governing pathway choice and performance. Shake-flask experiments with 1% (v/v) ethanol showed that the malonyl-CoA pathway yielded higher 3-HP titers than the β-alanine route. Co-activation of both pathways in a PHA-deficient strain improved production to 15.9 mM (1.42 g/L; 179 mg/g ethanol) while reducing acetate overflow. In a fed-batch with continuous ethanol feeding, the engineered strain reached 43.7 mM (3.92 g/L; 154 mg/g ethanol) 3-HP. Deletion of endogenous 3-HP catabolism and PHA synthesis redirected carbon flux toward the product, and additional acetyl-CoA supply was achieved by introducing a heterologous acetaldehyde dehydrogenase. Genome-scale modeling constrained with transcriptomic data revealed dominant flux routing through the glyoxylate shunt and limited oxaloacetate regeneration, explaining the advantage of the malonyl-CoA pathway and the acetate accumulation associated with β-alanine operation. An inducer-free ethanol-to-3-HP platform was established in P. putida KT2440 by co-activating the malonyl-CoA and β-alanine pathways while eliminating competing sinks such as 3-HP catabolism and PHA synthesis. This approach enhanced yield, reduced acetate overflow, and systems-level analysis identified the glyoxylate shunt and oxaloacetate limitation as key control points. Overall, the study demonstrates an efficient and scalable route for 3-HP production from ethanol.