18-04: Engineering Actinobacillus succinogenes for Succinate Production from Lignocellulosic Hydrolysates and Glycerol

If succinate was fermentatively produced at a price competitive with that of oil-based maleic anhydride, it could replace maleic anhydride as the precursor of many bulk chemicals, which a total $15 billion/year market. Actinobacillus succinogenes is among the best natural succinate producers, making it a natural choice for industrial succinate production. A. succinogenes grows on lignocellulose’s most abundant sugars, and it can ferment multiple carbon sources simultaneously, suggesting that it could be used in a lignocellulose-based succinate production process. A. succinogenes also grows on glycerol by aerobic and anaerobic respirations, suggesting that it can be used in a glycerol-based succinate production process as well. We have evolved A. succinogenes for fast growth on individual lignocellulosic sugars and on corn stover hydrolysates. The evolution process did not significantly affect the succinate yields of evolved strains. The mutations accumulated during evolution have been identified. A markerless gene knockout method was developed and a pyruvate formate-lyase deletion (ΔpflB) mutant was engineered. When grown anaerobically on glucose, strain ΔpflB produced 69% more succinate (in mol/mol glucose), less acetate, and less ethanol than the wild-type strain. During growth on glycerol, succinate yields were highest in microaerobic cultures, followed by dimethylsulfoxide-respiring, nitrate-respiring, and fully aerobic cultures. Succinate yields on glycerol reached up to 85% (wild-type strain) and 89% (ΔpflB strain) in chemostats gased with 5% O₂ + 95% CO₂. We have demonstrated that A. succinogenes can be engineered for increased succinate yields, and that it can produce succinate from agriculture and industrial waste products.