De novo biosynthesis of putrescine from glucose using engineered Escherichia coli in a two-step process.

Putrescine, is a key monomer for the synthesis of high-performance polymer materials. However, its sustainable microbial production remains challenging in yield and efficiency. In this study, we developed a modular two-step biosynthesis process for the conversion of glucose to putrescine. In the first stage, an l-arginine-overproducing Escherichia coli ARGFM strain was constructed through metabolic engineering. Then, fermentation optimization in a 5 L fermenter achieved a titer of 97.6 g/L with a yield of 0.52 g/g, representing a 49% improvement over the initial process. Notably, by implementing a dynamic dissolved oxygen control strategy (20-30%), the l-arginine productivity was enhanced to 2.03 g/(L·h). In the second stage, the arginine was efficiently converted to putrescine via whole-cell catalysis by another engineered E. coli, reaching a final titer of 48.6 g/L, with a yield of 0.26 g/gGlucose which was the highest level reported for de novo biosynthesis of putrescine. Specifically, this catalytic step employed in situ CO2 generated from the decarboxylation reaction for pH self-regulation, minimizing viability loss caused by exogenous acid/base addition. Furthermore, the protocol can be scaled up to a 50 L reactor with similar yield. Finally, a downstream separation process was established, recovering high-purity putrescine from the fermentation broth with a final purity of 99%. Techno-economic analysis and life cycle assessment predict that putrescine, derived from a two-step process, can reduce carbon dioxide emissions by 30%. Overall, this study established an integrated fermentation-catalysis-separation process for putrescine, demonstrating a viable and efficient strategy for the bio-based production of this valuable diamine.