Succinic acid tops the U.S. Department of Energy's list of value-added products from biomass, because it has the potential, if produced economically, to become the feedstock for a bulk chemical industry currently based on maleic anhydride, a petrochemical. In addition to the large market potential for succinate and its immediate derivatives, bio-based succinate production has the added environmental benefit of using CO2, a greenhouse gas, as a substrate. Actinobacillus succinogenes 130Z naturally produces among the highest levels of succinate from a variety of inexpensive carbon substrates. Previous reports of A. succinogenes's metabolic capabilities mainly used glucose as a feedstock and provided insight into several key factors controlling succinate production. Conversely, little is known about how A. succinogenes metabolizes glycerol, a waste product of biodiesel manufacture and an inexpensive feedstock with potential application in bio-based succinate production. As suggested by our manual annotation of its genome, A. succinogenes cannot ferment glycerol in defined minimal medium but it can metabolize glycerol by aerobic or anaerobic respiration. We investigated A. succinogenes's glycerol metabolism in a variety of respiratory conditions by comparing growth, metabolite production, and in vitro activity of terminal oxidoreductases. Under conditions of nitrate-respiration and fully aerobic respiration, acetate was the primary acid produced from glycerol. However, succinate was the primary product of dimethyl sulfoxide-respiring cultures and cultures grown in microaerobic conditions. The highest succinate yield observed was 0.69 mol succinate/mol glycerol (69% of the maximum theoretical yield) under microaerobic conditions. We also show that A. succinogenes can grow and produce succinate on partially refined glycerols obtained directly from biodiesel manufacture. We used recently developed genetic tools to create knockout mutants of A. succinogenes. The gene knockout strategy uses natural transformation to introduce linearized DNA into the cells. The isocitrate dehydrogenase gene (icd) from Escherichia coli was used as a selection marker, enabling positive selection of recombination events based on the glutamate auxotrophy of A. succinogenes. After successful deletion of the target gene, we employed the Saccharomyces cerevisiae flippase recombinase to remove the icd marker, enabling its re-use. With the aim of increasing succinate yields, the A. succinogenes pflB gene (encoding pyruvate formate-lyase, PFL) was targeted for deletion. Strain ÄpflB produced higher succinate yields than strain 130Z (0.85 mol/mol glycerol) under microaerobic conditions.In summary, in optimized respiratory conditions, A. succinogenes can conserve most of the reducing power available in glycerol for succinate production. The increased understanding of A. succinogenes's glycerol metabolism, combined with new genetic tools, sets the stage for future strain and process development towards a highly productive and economic glycerol-to-succinate conversion process.
Read
