(188bg) Development of Actinobacillus succinogenes 130Z As a Biotechnology Host for Succinic Acid Production

The co-production of high-value products, in addition to biofuel, could increase the profitability of biorefineries. Currently, utility of the high fiber fraction from corn is limited to low value feed products for cattle and other ruminant animals; however, the polysaccharides in fiber can be broken down into sugars that can then be converted by various microorganisms into valuable products. One such product is succinic acid, which is a versatile chemical that can be used in a variety of materials that are currently produced from petroleum, including plastics, clothing fibers, and biodegradable solvents. One bacterium of interest for the production of succinic acid from corn fiber is Actinobacillus succinogenes 130Z. This non-model bacterium is a high efficiency producer of succinic acid, but in its native form has limited tractability due to its inability to regulate internal pH, its production of biofilms, and the lack of knowledge pertaining to its metabolism and the impact of growth in monocultures. Here we will share our work developing A. succinogenes as a biotechnology host, including antibiotic selection, pH control strategies, and synthetic biology tools for predictable control of gene expression. A. succinogenes has demonstrated unexpected resistance to common antibiotic selectors which is further complicated by the organism’s common formation of biofilms. When using ampicillin resistance as a selection marker, biofilm formation masked the difference between colonies with and colonies without the marker. Additionally, our findings show pH levels can be moderately stabilized using phosphate buffer, although, cultures containing phosphate in concentrations that did not inhibit growth still dropped below tolerable pH levels for A. succinogenes after 24 hours. Furthermore, preliminary attempts to manipulate gene expression have included the development and characterization of heterologous promoters; both constitutive and inducible. The use of heterologous promoters has not been previously reported for this organism. Our results reveal necessary information for cultivation of A. succinogenes and demonstrate capabilities for genetic manipulation using exogenous promoters. Our findings lay a foundation for further development of A. succinogenes as a stable, efficient producer of succinic acid from corn fiber; a direction of research that will maximize the value obtained from corn and increase the profitability of corn ethanol facilities.