This Small Business Innovation Research Phase I project aims to engineer novel biocatalysts for cost-effective production of malic acid from CO2. Malic acid is a key intermediate in energy metabolism of all living species. Additionally, presence of two carboxyl and one hydroxyl groups makes it an important building block for production of diverse chemicals and chemical intermediates. Attempts to engineer heterotrophic biocatalysts for production of malic acid have had limited success due to the inherent limitations of accessing the substrate required for the key biosynthetic enzyme. We have developed an innovative strategy to co-localize this key enzyme within carboxysomes naturally found in photosynthetic microorganisms. Carboxysomes are leveraged by ribulose-1,5-bisphosphate carboxylase/oxygenase to overcome their kinetic deficiency for CO2, a cosubstrate. The engineered metabolic pathway will allow conversion of 3- phosphoglycerate naturally produced inside carboxysome to malic acid. The anticipated result of this SBIR Phase 1 research project is establishment of a groundbreaking and cost-effective technology that will convert CO2 into malic acid using energy derive from sunlight. The Broader/Commercial outcome of the proposed research will be to offer multiple benefits and applications: 1) Development of an efficient biocatalyst that could significantly reduce the production cost of malic acid. Lower production cost and presence of multiple functional groups will open new market opportunities. 2) Production of enantiomerically pure L-malic acid from renewable feedstock, a preferred additive for food and pharmaceutical applications over the racemic mixtures produced by the chemical method. 3) An attractive and immediate opportunity to reduce carbon footprint of the existing biofuel industries. 4) Opportunity to target other CO2 utilizing enzymes to this locale within cells and to use CO2 more efficiently in making other important biomolecules thus opening new commercial possibilities. 5) Finally, it will open up the opportunity to link light harvesting more effectively to carbon fixation since enzymes that make malic acid have substantially higher turnover capability than RuBisCO. Success of the proposed process technology will contribute to the economic and energy security of the U.S. by reducing the dependence on fossil fuels and emission of greenhouse gas emission, and use of CO2 in the chemical industry instead of being an emitter
