The global demand for energy is rising, and one of the sectors projected to see the most growth is the transportation industry. Some of the increased demand can be replaced by electric vehicles running on renewable electricity. However, the aviation, shipping and long-haul trucking industries will still require liquid fuels. To address the increased demand for liquid fuels from sustainable resources, photosynthetic bacteria will be genetically engineered to convert carbon dioxide directly to limonene, a hydrocarbon which can be further processed into jet fuel. Limonene is one molecule in an important class of biologically-produced hydrocarbon molecules called terpenoids, which have desirable properties as fuels, pharmaceuticals, and as feedstocks for a variety of commercial chemicals. The goal of this project is to optimize the metabolic pathways within photosynthetic bacteria to enhance the flux of carbon dioxide into limonene production. The educational activities associated with the project will mentor a team of college and high school students to participate in the International Genetically Engineered Machines (iGEM) competition, a widely known and effective program for promoting active learning in the context of the scientific and societal aspects of the field of molecular biology.
The proposed research will engineer a photosynthetic bacterium, Synechococcus sp. PCC 7002, to act as a photocatalytic factory for the production of the monoterpene limonene directly from carbon dioxide. In bacteria, all terpenoids are produced through the methylerythritol 4-phosphate (MEP) pathway. The proposed research has three objectives designed to enhance this pathway. Objective 1 will seek to improve the production of limonene by engineering the increased availability of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAP). This will be accomplished by replacing the known rate-limiting enzymes with genetic material sourced from high terpenoid-producing organisms, including Populus trichocarpa (terrestrial plant) and Botryococcus braunii (alga), to circumvent endogenous regulation of these rate-limiting enzymes. Objective 2 will further improve limonene production by knocking out a suite of genes to divert flux toward terpenoid metabolism, as predicted by stoichiometric metabolic modeling. Top-performing strains will be selected based on superior limonene productivity, and isotope assisted metabolic flux analysis (13C-MFA) will be used to identify unknown bottlenecks and side product formation. Intracellular metabolic fluxes will be measured by using isotope-assisted metabolic flux analysis (isotope MFA) of the wild type and top-performing limonene-production strains. This will allow for the rational design of metabolic engineering strategies to further increase MEP pathway flux. Objective 3 will investigate the effect of nutrient limitation and altered carbon storage ability on limonene production. For example, a mutant that lacks the ability to synthesize glycogen, the main carbohydrate storage product in Synechococcus, may not perceive nitrogen-deprivation stress. As a result, the mutant maintains photosynthetic activity in the absence of cell growth and division, and spills energy by excreting organic acids, including terpenoid precursors. Isotopic 13C-MFA will be used to identify metabolic bottlenecks in the glycogen-deficient mutant that prevent the over-accumulating organic acids from entering terpenoid metabolism. These findings will suggest design engineering strategies to complete the redirection of flux from glycogen towards limonene. Overall, the proposed work will create a strain with enhanced carbon flux through the MEP pathway that can be used as a photosynthetic platform strain for the production of any terpenoid, thus introducing a new model organism for producing this wide and diverse class of chemicals. The platform strain will be shared freely amongst the academic community to enable faster development of production strains and increased knowledge of Synechococcus.
