| Limited fossil fuel resources and the increasing demand for energy are motivating the development of economically feasible alternatives for energy and renewable means of producing chemical feedstocks that are currently supplied by petroleum manufacturing. A recent development in the production of renewable fuels is the use of cyanobacteria as production hosts. Cyanobacteria are photosynthetic organisms that are able to utilize carbon dioxide as their sole carbon source and light as their energy source. Although cyanobacteria naturally produce hydrocarbons, the native yields from wild type strains are insufficient to be cost competitive with petroleum-derived chemicals. Therefore, a deeper understanding of the pathways involved in hydrocarbon biosynthesis in cyanobacteria is needed in order to rationally engineer and enhance this trait in modified organisms.;In this thesis we analyzed the hydrocarbon profile of the cyanobacterium Synechococcus sp. PCC 7002 (PCC 7002) and demonstrated the involvement of a gene (ols gene for olefin synthase) with modular organization, similar to a polyketide synthase, in the synthesis of medium chain olefins (1-nonadecene and 1, 14-nonadecadiene). Feeding studies suggested that the putative enzyme used an elongation decarboxylation-mechanism to convert fatty acyl-acyl carrier proteins (fatty acyl-ACPs) to the &agr;-olefins. We also studied PCC 7002's growth under various temperatures and demonstrated the involvement of a desaturase gene (desE) in the formation of the internal double bond in 1, 14-nonadecadiene in response to changes in temperature; suggesting that the olefins might play a role in responding to cold stress and maintaining membrane fluidity. After improving CO 2 delivery, increasing expression of the ols gene and expressing a thioesterase from Geobacillus sp. Y412MC10, a significant increase in olefin production was observed. The discovery and characterization of pathways involved in hydrocarbon biosynthesis, like the Ols pathway describe in this thesis, will be an important contribution to the development of sustainable routes for hydrocarbon production and rationally engineer their production in modified organisms.;Additionally, we developed a method for dissecting failed heterologous expression experiments in E. coli by designing a DNA cassette that couples translation of a target gene to a response gene that generates an easily monitored phenotype in vivo.. |
