Engineering Escherichia coli for the Production of Propionic Acid through the Wood-Werkman Cycle

Biological production of propionic acid represents a viable alternative for the production of C3 chemicals. Propionic acid is natively produced through the Propionibacteria’s Wood-Werkman cycle. By coupling the electron transport chain during the reduction of fumarate to succinate in the dicarboxylic acid branch of the TCA cycle, the cycle offers an energetic advantage for cells. The unique functionality of one of the enzymes, the methylmalonyl-CoA carboxyltransferase, allows the direct exchange of a carboxyl group and a thiol group without ATP consumption, giving an even higher energy gain. These features make Propionibacteria an ideal candidate for the production of propionic acid. Unfortunately, engineering Propionibacteria has proven extremely challenging. Equipping Escherichia coli with the Wood-Werkman cycle is thus a viable alternative for heterologous propionic acid production.  In silico analysis predicts that E. coli can use the cycle and take advantage of its energy benefits. The genome-scale model predicts that cells harbouring the cycle can sustain growth at a 43% higher growth rate and a yield of 1.135 mol_propionate/mol_glucose. Thus, seven genes from P. acidipropionici were transformed into E. coli K12, W and MG1655. Fermentations were performed in bioreactors, and cells were grown on minimal media. Initial results showed that E. coli was able to produce propionic acid through the Wood-Werkman cycle despite a stress related effect resulting in lactate overproduction. Intracellular metabolomics and proteomics identified limitations in the expression system as two key enzymes, namely methylmalonyl-CoA mutase and methylmalonyl-CoA carboxylase, were under-expressed.

The initial design, comprising a single synthetic operon under a nar inducible T7 promoter was too strong causing cell death after induction. This problem was addressed by testing different promoters and modifying the structure of the synthetic operon. Our results show for the first time that E. coli can express a functional Wood-Werkman cycle leading to propionic acid production. The next step is to evolve cells to adapt to the new metabolism. Cells are currently under adaptive evolution, and alternative by-products pathways are being knocked-out. We anticipate that the knowledge generated here in a heterologous host can help overcoming the current limitations in the biological production of C3 chemicals.