Synthetic Crop for Direct Biofuel Production through Re-routing the Photorespiration Intermediates and Engineering Terpenoid Pathways#

During the initial 18-month funding period for the DOE-ARPA-E TAMU grant, the Chappell laboratory will be responsible for 5 research objectives: 1. The Chappell laboratory will first identify all the known, functionally characterized limonene and caryophyllene synthase genes to date and compile a list of their known biochemical features. These will include kinetic constants and reaction product specificity. From this initial list of gene candidates, the 3 best limonene and caryophyllene synthases as defined by their catalytic efficiency (kcat/Km) and reaction product specificity will be prioritized for further confirmation studies. 2. The Chappell laboratory will verify these biochemical characteristics experimentally for 3 limonene synthase and caryophyllene synthases. The genes encoding for these enzymes will be obtained by either direct cloning from their native hosts, or synthetic forms of the genes will be purchased from a private vendor. The genes will be incorporated into suitable bacterial expression vectors and the heterologous produced enzymes purified based on affinity tags appended to the amino or carboxy terminal regions of the genes. The purified proteins will then be used in standard terpene synthase enzyme assays, the activity of the enzymes determined, their kinetic constants calculated, and the profile of their reaction products determined by GC-MS. 3. In parallel to the biochemical assessments of these terpene synthase genes, the selected terpene synthase genes will be inserted into Ti-plasmid vectors for engineering these constructs into transgenic tobacco plants. The Ti-plasmid vectors will be similar to those described by Wu et al. (2006 Nature Biotech. 24:1441) but modified to effect constitutive and trichome specific gene expression with the encoded proteins targeted to either the cytosolic or chloroplast compartments. The various Ti-plasmid vectors will be inserted into Agrobacterium tumefaciens and these bacteria used to introduce the various gene constructs into transgenic tobacco lines as described by Wu et al. (2006 Nature Biotech. 24:1441). Twenty independent transgenic tobacco lines for each construct will be generated in this manner and the T0 generation propagated in the greenhouse and screened for their limonene and caryophyllene levels by GC-MS analysis. 4. The Chappell laboratory will also participate with other investigators in this project to perform genetic crosses between the various tobacco lines developed during the course of this project, generating hybrid lines. We will reposit these seed lines and distribute these seed lines to the various participating research groups as requested. 5. The Chappell laboratory will also perform field tests of select transgenic lines at the UK Spindle Top research farm. Typically experimental tests consisting of plants grown in triplicate settings and evaluating agronomic (biomass accumulation, high, leaf area) and physiological (photosynthetic measurements) performance, along with determining terpene chemical composition by GC-MS of tissue sample extracts are anticipated.