(602t) Transgenic Expression of Thermomyces Lanuginosus Lipase in Plants for the Enzymatic Production of Biodiesel

Diminishing petroleum reserves and increasing environmental concerns have led to an urgent need to develop environmentally friendly fuels, such as biodiesel. A relatively new and promising development in the production of biodiesel is via enzymatic transesterification with lipase as the catalyst. The motivation for this research arises from the need to develop inexpensive biocatalysts to replace environmentally harmful chemical catalysts. We are genetically engineering plants to constitutively express a lipase for biodiesel production from spent oils and non-edible plant oils. We have cloned the gene of a lipase with known transesterification activity from Thermomyces lanuginosus, a thermophylic fungus. Cloning involved isolation of total RNA, reverse transcription of the mRNA into cDNA and PCR amplification of the lipase gene using specific primers. The gene was first inserted into a cloning vector (pCR8/GW/TOPO) and sequenced to confirm its identity. The gene has been inserted into a plant destination vector (pGWB408 and pMDC83) via LR clonase reaction. Pieces of Nicotiana tabacum (tobacco) leaf was transformed with the lipase gene using Agrobacterium tumefaciens (strain GV3101) and transferred onto selection media plates.  We are currently growing Arabidopsis thaliana from seeds that have been transformed using the floral dip transformation method. The recombinant enzyme will be collected from the genetically engineered plants, purified, and tested for its activity. This approach should provide a relatively cheap and environmentally safe source of lipase for use as a biocatalyst.