Metabolic engineering of Saccharomyces cerevisiae microbial cell factories for succinic acid production

Saccharomyces cerevisiae is a proven, robust, industrial production platform used for expression of a wide range of therapeutic agents, high added-value chemicals, and commodities. Central carbon metabolism in S. cerevisiae has been extensively investigated using a wide variety of substrates for determination of how glycolytic flux is distributed across C1 (CO2,g), C2 (ethanol, acetate), and C3 (glycerol, pyruvate) products.

For the S. cerevisiae CEN.PK113-7D strain cultivated under carbon-limited, aerobic, well-controlled batch fermentations, the distribution of carbon across biomass, C1, C2, and C3 products is 18, 14, 54, and 9 C-mol/C-mol-glucose, respectively, with <5 C-mol/C-mol glucose unaccounted for. A class of high added-value chemicals being targeted for biotechnology production is C4 organic acids, encompassing fumaric, malic, and succinic acid.

Succinic acid is a key building block molecule for further conversion to precursor molecules such as tetrahydrofuran, 1,4-butanediol, and butyrolactone. Succinic acid has the potential to become a commodity chemical, with world-wide annual demand exceeding $2 billion USD and over 160 million kg currently produced from petrochemical conversion of maleic anhydride.

There are several biomass platforms, all prokaryotic, for succinic acid production; however, overproduction of succinic acid in S. cerevisiae offers distinct process advantages. For example, S. cerevisiae has been awarded GRAS status for use in human consumables, grows well at low pH significantly minimizing purification and acidification costs associated with organic acid production, and can utilize diverse carbon substrates in chemically defined medium.

S. cerevisiae offers the unique advantage of being the most well characterized eukaryotic expression system. Here we describe the use of systems biology tools to drive C6 carbon flux to succinic acid by enhancement of the two native pathways for succinic acid production: the TCA and glyoxylate cycles.

S. cerevisiae does not naturally accumulate succinic acid; however, through the use of in silico metabolic predictions guiding targeted gene deletions and over-expression, mutants that overproduce succinic acid have been engineered and thoroughly characterised. Metabolic engineering approaches developed promise to have broad applicability to industrial biotechnology platforms, as well as enhancing fundamental understanding of central carbon metabolism in S. cerevisiae.