P110: Biocatalysts to convert biofuel waste products, CO2 and glycerol, into value-added succinate through the use of metabolic engineering

Biodiesel and bioethanol have the growing potential to represent sustainable, secure, renewable, and environmentally safe alternatives to fossil fuels. However, these biofuels generate two main waste byproducts: glycerol and carbon dioxide (CO₂).  To add value to these low-priced glycerol streams and further reduce greenhouse gas emissions, we metabolic engineered the E. coli metabolism to produce novel biocatalysts capable of converting glycerol and CO₂ into succinic acid (succinate), a commodity feedstock used in the production of industrially important chemicals. Succinate production was greatly elevated by 1) blocking pathways for the synthesis of competing by-products lactate, ethanol, and acetate, and 2) expressing Lactococcus lactis pyruvate carboxylase to drive the generation of succinate from the pyruvate node (as opposed to that of phosphoenolpyruvate). Strategies utilizing a heterologous (Citrobacter freundii) ATP-dependent dihydroxyacetone kinase instead of E. coli’s native PEP-dependent ortholog were also employed.  Overall, these metabolic engineering strategies coupled cell growth to succinate production because the synthesis of succinate remained as the primary route of NAD+ regeneration. This feature enabled the operation of the succinate pathway in the absence of selective pressure (e.g. antibiotics). These biocatalysts yield some of the best performances to date in the production of succinate from glycerol, i.e., upwards of 30 g/L for succinate titers and molar yields of 70%. Results obtained from this work have the potential to help reduce CO₂ emissions, improve sustainability of the U.S. biofuels industry, and strongly contribute to the development of the concept of “biorefinery” by integrating the production of biofuels and higher-value chemicals.