Metabolic engineering of Propionibacterium freudenreichii subsp. shermanii for enhanced propionic acid fermentation: Effects of overexpressing three biotin-dependent carboxylases

• PYC, MMD, and MMC in the dicarboxylic acid pathway were overexpressed in P. shermanii.
• The mutant overexpressing PYC grew slower and produced more succinate but less propionate.
• Overexpressing MMC and MMD increased propionate yield and productivity in the fermentation.
• This is the first report on enhanced propionic acid production by MMC and MMD in propionibacteria.

Bio-based propionic acid production by propionibacteria is increasingly attractive because of its potential to replace petroleum-based chemical synthesis processes. Metabolic engineering of Propionibacterium freudenreichii subsp. shermanii DSM 4902 for enhanced propionic acid fermentation is made possible by the recently available whole genome sequence and genetic engineering tools. Pyruvate carboxylase (PYC), methylmalonyl-CoA decarboxylase (MMD), and methylmalonyl-CoA carboxyltransferase (MMC) are three biotin-dependent carboxylases in the dicarboxylic acid pathway controlling the carbon flux in the Wood–Werkman cycle. These carboxylases were overexpressed in P. shermanii to study their effects on propionic acid fermentation in serum bottles with glucose and glycerol as substrates. Compared to the wild-type, the mutants overexpressing MMC and MMD showed a significant increase in the metabolic fluxes toward the biosynthesis of propionic acid, against acetic and succinic acids, with significantly increased yield (up to 14% increase) and productivity (up to 17% increase), especially in the co-fermentation of glycerol and glucose. On the other hand, the mutant overexpressing PYC grew slower, produced more succinate, and had a lower production of propionate (up to 12% decrease in productivity). This is the first study demonstrating enhanced propionic acid production by overexpressing MMC and MMD in propionibacteria.