Production of succinic acid with engineered genetic switches in methylobacterium extorquens

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Succinic acid is one of the 12 most important molecules in the modern chemical industry. It is used in many applications, including the production of polymers such as PBS (polybutylsuccinate). Currently, almost all of the succinic acid production originates from fossil-carbon. In order to find other sources and rely less on petroleum, so that the succinic industry can embrace a "green approach", many companies have started projects to produce succinic acid by fermentation from biological raw material, mainly sugars derived from crops. However, this greener product is controversial because it diverts agricultural land and ressources that could potentially be used for crops intended for food towards the chemical industry instead. For the past few years, this debate has been gaining in importance and thus industry is starting to envision second generation processes that would rely on cellulose or waste. In this project, the source of raw material used for fermentation does not compete with human or animal food, but rather uses bio-methanol derived from waste by gazeification (thermo-chimio-conversion). In an initial phase of the project, the feasibility of using Methylobacterium extorquens to produce succinic acid for industrial processes has been evaluated. The methylotrophic bacteria M. extorquens is capable of consuming methanol and has the citric acid cycle; it is thus capable of producing succinic acid. Therefore, a knockout of the sdh gene, which converts succinate to fumarate, has already been performed. However, because this gene is essential, the knockout strains survive only with the addition of malate to rescue the citric acid cycle. To circumvent this problem and achieve succinic acid production in a range close to 10-20g/L, we have three main objectives. 1) Engineer a sdh knockout strain complemented with an inducible sdh. This requires the design and implementation of new genetic switches with promoters compatible with M. extorquens. 2) Similarly, engineer inducible small RNAs that repress sdh to tune expression according to succinic acid production needs. 3) Convert the biomass accumulated in the form of poly-ß-hydroxybutyrate (PHB) to succinic acid by genetically rewiring the PHB degradation pathways.