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Jay D. Keasling
Department of
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"Metabolic
Engineering of Microbes for Production
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Abstract
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Plants and fungi produce a plethora of isoprenoids that function as aromas, flavors, and pest rejection. Many of these molecules have found use as antibacterial, anti-fungal, and anti-cancer agents, even though evolution has optimized them for function in plants. Unfortunately, many of these compounds are produced at extremely low levels. To increase their diversity and the level of production in E. coli , we have recruited genes from several organisms for complete reconstruction of the terpene biosynthetic pathways and have evolved sesquiterpene genes to increase the diversity of terpene olefins produced by this organism. The genes encoding the mevalonate-dependent isopentenyl pyrophosphate (IPP) biosynthetic pathway from Saccharomyces cerevisiae were cloned into operons and integrated into the chromosome, and their expression was optimized using laboratory evolution and promoter control to maximize the flux through the pathway. The genes encoding sesquiterpene cyclases from various plants were cloned and optimized for expression in E. coli . Although it was possible to detect sesquiterpenes from the culture extracts of the recombinant strains, the levels produced were very low (µg/L). Increases in the flux to the precursor (FPP) did not yield higher terpene production, indicating that poor terpene cyclase expression and/or activity was limiting.
To test this hypothesis, two cyclase genes optimized for expression in E. coli were synthesized in the laboratory. These enzymes (amorphadiene and epi -cedrol cyclases) did not show significantly higher yields of terpenes from endogenous levels of FPP but produced significantly higher yields (mg/L) when expressed in a host over-producing FPP from the mevalonate-dependent IPP pathway. To sample the potential terpene diversity produced from terpene cyclases, we constructed point mutation and chimeric libraries of cyclase genes and screened these libraries for the presence of sesquiterpenes. Results on the sesquiterpene diversity generated by these libraries will be presented. In addition, we have examined the physiology of the engineered E. coli strains using transcriptional and metabolic profiling and will present the results of these studies.
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Room: 1064 Duncan Hall . Time: 2:30 PM For more information contact: |
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