Sunday, May 3, 2009 - 4:00 PM
2-06
Metabolic Engineering of Saccharomyces Cerevisiae for the Production of n-Butanol
Eric J. Steen1, Rossana Chan Chan2, Nilu Prasad Prasad2, Samuel Myers1, Chris Petzold3, Alyssa Redding1, Mario Ouellet2, and Jay D. Keasling4. (1) Bioengineering, UC Berkeley, UC San Francisco, 2034 ashby ave, apt 12, Berkeley, CA 94703, (2) Physical Biosciences Division, Lawrence Berkeley National Lab, 717 Potter street, Berkeley, CA 94720, (3) Technology Division, Joint BioEnergy Institute, 5885 Hollis Street, Fourth Floor, Emeryville, CA 94608, (4) Departments of Chemical Engineering and Bioengineering, UC-Berkeley; Lawrence Berkeley National Laboratory, EmeryStationEast, 5885 Hollis St, 4th floor, Emeryville, CA 94608
Background
Increasing energy costs and environmental concerns have motivated engineering microbes for the production of “second generation” biofuels that have better properties than ethanol.
Results & Conclusions
Saccharomyces cerevisiae was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms (S. cerevisiae, Escherichia coli, Clostridium beijerinckii, and Ralstonia eutropha) were substituted for the Clostridial enzymes and their effect on n-butanol production was compared. By choosing the appropriate isozymes, we were able to improve production of n-butanol ten-fold to 2.5 mg/L. The most productive strains harbored the C. beijerinckii 3-hydroxybutyryl-CoA dehydrogenase, which uses NADH as a co-factor, rather than the R. eutropha isozyme, which uses NADPH, and the acetoacetyl-CoA transferase from S. cerevisiae or E. coli rather than that from R. eutropha. Surprisingly, expression of the genes encoding the butyryl-CoA dehydrogenase from C. beijerinckii (bcd and etfAB) did not improve butanol production significantly as previously reported in E. coli. Using metabolite analysis, we were able to determine which steps in the n-butanol biosynthetic pathway were the most problematic and ripe for future improvement.
Increasing energy costs and environmental concerns have motivated engineering microbes for the production of “second generation” biofuels that have better properties than ethanol.
Results & Conclusions
Saccharomyces cerevisiae was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms (S. cerevisiae, Escherichia coli, Clostridium beijerinckii, and Ralstonia eutropha) were substituted for the Clostridial enzymes and their effect on n-butanol production was compared. By choosing the appropriate isozymes, we were able to improve production of n-butanol ten-fold to 2.5 mg/L. The most productive strains harbored the C. beijerinckii 3-hydroxybutyryl-CoA dehydrogenase, which uses NADH as a co-factor, rather than the R. eutropha isozyme, which uses NADPH, and the acetoacetyl-CoA transferase from S. cerevisiae or E. coli rather than that from R. eutropha. Surprisingly, expression of the genes encoding the butyryl-CoA dehydrogenase from C. beijerinckii (bcd and etfAB) did not improve butanol production significantly as previously reported in E. coli. Using metabolite analysis, we were able to determine which steps in the n-butanol biosynthetic pathway were the most problematic and ripe for future improvement.
Web Page: www.jbei.org