Microbial production of propionic acid through a novel β-alanine route

Propionic acid is a key three carbon platform chemical with broad applications in food preservation, pharmaceuticals, and polymer production. Traditional microbial production of propionic acid employing Propionibacterium species is constrained by slow growth, and limited genetic engineering tools, thereby restricting its industrial use. Here, we report the development of a novel biosynthetic pathway for propionic acid production via the β-alanine route. This pathway was engineered into two modules: an upstream β-alanine-forming module and a downstream propionic acid-forming module. The downstream pathway was first constructed and validated in Escherichia coli W3110. Subsequently, co-expression of the upstream module enabled de novo propionic acid production from glucose. Through enzyme screening, precursor flux enhancement, and optimization of phosphoenolpyruvate carboxylase (PPC) flux, the final engineered E. coli strain achieved 14.8 g/L of propionic acid in fed-batch fermentation. Furthermore, we explored Corynebacterium glutamicum ATCC 13032 as an alternative host due to its superior tolerance to propionic acid. The same downstream pathway was introduced into a previously developed β-alanine-overproducing C. glutamicum strain to enable propionic acid production from glucose. Additional engineering strategies, such as enzyme screening, disruption of competing pathways (ack-pta), and elimination of propionic acid catabolic pathways (prpD2B2C2), led to the production of 47.4 g/L of propionic acid in fed-batch fermentation, representing the highest reported titer of heterologous propionic acid production. This work establishes a novel and vitamin B₁₂-independent strategy for bio-based propionic acid production, offering a sustainable alternative to conventional processes.