Proof-of-concept: Successful metabolic engineering strategy guided by genome-scale metabolic modeling.

Panel a shows the central carbon metabolism of S. cerevisiae, and the model-guided metabolic engineering strategy for succinate over-production. Succinate production is directly coupled to biomass formation based on three gene deletions: sdh3 (cytochrombe b subunit of succinate dehydrogenase complex), and ser3/ser33 (3-phosphoglycerate dehydrogenase isoenzymes). The remodeling of central carbon flux towards succinate requires minimizing the conversion of succinate to fumarate, and forcing the biomass-required amino acids L-glycine and L-serine to be produced from glyoxylate pools. Production of glyoxylate results from isocitrate conversion by Icl1p, producing equimolar succinate. As the biomass yield increases, the demand for L-glycine and L-serine increase proportionally, driving biomass-coupled succinate production. Legend: native reactions (blue solid line), lumped native reactions (blue dashed line), interrupted reactions (red solid line), up-regulated reactions (green solid line). Panel b demonstrates the proof of concept. The reference strain and genetically engineered mutant strain, 8D, supplemented with 500 mg L−1 glycine were physiologically characterized in 2L well-controlled stirred-tank fermentations. There was a 13.3X improvement in succinate titer.