Metabolic Engineering of Yeast for Commercial Production of Succinic Acid

DSM and ROQUETTE have started a joint venture by the name of “Reverdia” (www.reverdia.com) for the fermentative production and commercialization of succinic acid from renewable resources, to be marketed under the name Biosuccinium^TM. Succinic acid has been identified as a potential key building block for deriving both commodity and specialty chemicals from biomass. While existing markets for chemically produced succinic acid include pharmaceuticals, food, coatings and pigments, bio-based succinic acid is envisioned to drive the emergence of new applications such as polyester polyols for polyurethanes, polybutylene succinate (PBS), plasticizers, 1,4-butanediol and resins. The fermentative production of succinic acid at low pH results in a substantially lower environmental footprint compared to both the current petrochemical process, the near-neutral pH used in bacterial fermentation processes, as well as the process for petrochemical adipic acid, which is the conventional chemical used for production of for example polyester polyols.

We present the metabolic engineering strategy of the yeast used in this process. Heterologous genes, optimized for expression in the host, were introduced for achieving high level production of succinic acid from sugar. Expression of these genes was verified at the protein level with LC-MS. Systems-level analysis (transcripts, proteins and metabolic fluxes) provided insight into the physiology and metabolism of the strain during fermentation, and generated various new leads for strain and process optimization. The strain was additionally improved by classical means with mutagenesis and crossing. Re-sequencing of a selection of strains provided additional information and targets for further strain improvement.

Initial shake flask protocols for strain testing were successfully down-scaled to micro titer plates, and the process for production of succinic acid has been scaled up in our demonstration plant. In this process the strain produces succinic acid at a high titer at a low pH. Furthermore, Reverdia is the first in the world to have a large scale facility for the commercial production of bio-based succinic acid. The new facility, based in Cassano Italy, commenced operation in December 2012.