(86d) Development of a Non-Leaky Inducible System for Tunable Gene Expression in Actinobacillus Succinogenes 130 Z and Application for Increased Succinic Acid Production

Recent research endeavors have turned to generating useful chemicals from biological platforms as an environmentally responsible alternative to non-sustainable sources. One such chemical is succinic acid (SA), which is a versatile product 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 production of SA from corn fiber is Actinobacillus succinogenes 130Z. This non-model bacterium is a relatively high efficiency producer of SA but in its native form is not a competitive alternative to petroleum-based sources. Metabolic engineering would be required to increase SA production to sufficient levels. As part of the on-going work, potential in silico metabolic engineering strategies have been predicted, however, as A. succinogenes is a non-model bacterium that has not been well characterized, there is a limited set of protocols and practically no synthetic biology tools available to attempt these strategies. Here we share our development of a non-leaky inducible system for tunable gene control in A. succinogenes as well as ongoing work to explore double gene knockouts combined with synthetic gene tuning for increased SA production. It is commonly recognized that development of promoters, specifically inducible promoters that can be turned on and off, is one of the easiest and most effective ways to control gene expression. Therefore, we sought to develop an inducible system within A. succinogenes. The lac system from E. coli was modified by replacing the original promoter region, P_lac, with a promoter previously shown in our lab to have stronger expression in A. succinogenes. Characterization using a fluorescent reporter protein demonstrated maximum expression levels ~9X greater than the original lac system and flaunted a 165-fold change from the deactivated to activated state. The system was surprisingly non-leaky, exhibiting no significant difference between expression in the absence of the inducer molecule and a wild type control. This is not typical as even E. coli shows some leakiness in the absence of the inducer molecule. Additionally, expression in the induced state was equivalent to expression of the same promoter in a constitutive (always on) system previously characterized within our lab, thus demonstrating complete induction. Growth in the presence of varying concentrations of the inducer molecule exhibited a graded response, therefore providing a tunable and useful tool for future metabolic engineering of A. succinogenes. Current and future work within our lab is focusing on incorporating this regulatory element into explorations of engineering A. succinogenes' metabolism for increased SA production. Using a double homologous recombination method, we are in the process of creating two double knockout strains, predicted, from a genome scale metabolic model created within our lab, to show increased SA production. We plan to further explore the metabolic flux to SA by tuning expression of other key metabolic genes using our inducible system, thus adding to the understanding of A. succinogenes metabolism and optimizing SA production of our knockout strains. Our findings lay a foundation for further development of A. succinogenes as an efficient producer of succinic acid from corn fiber; a direction of research that will provide a sustainable, environmentally responsible, and economically beneficial method for succinic acid production.