Improving succinate production from glycerol by Actinobacillus succinogenes

The EPA has proposed to maintain the production of biomass-based diesel at 1.28 billion gallons for 2015, maintaining a steady influx of crude glycerol (a by-product) in the market. Crude glycerol is used as a substrate for producing many bio-based chemicals such as 1,3-propanediol, citric acid, succinate. Our focus is on producing bio-based succinate from crude glycerol by Actinobacillus succinogenes both fermentatively and via respiration. A. succinogenes naturally produces high amounts of succinate, which, along with genetic engineering makes it one of the best catalysts for glycerol bioconversion to succinate. We have previously demonstrated that A. succinogenes can grow microaerobically on glycerol, producing succinate with a yield of 67% in batch and 75% under continuous culture conditions. The knockout strain ΔpflB can produce yields as high as 85% under batch conditions. Different oxygen concentrations are being investigated under continuous culture conditions to increase the yield further. We demonstrated earlier that A. succinogenes can ferment glycerol when it expresses a heterologous 1,3 propanediol (1,3-PD) pathway to maintain redox balance. However, our pilot strain expressed the 1,3-PD pathway from a plasmid under control of a strong promoter and accumulated 1,3-PD at the expense of succinate. We are currently identifying promoters of varying strengths using lacZ as the reporter gene in a ΔlacZ background. The 1,3-PD pathway will then be expressed under the control of a weaker promoter to test for increase in succinate production. We will present the continued progress in these efforts for improving succinate production under microaerobic and fermentative conditions.