| Summary: | <p>Abstract</p> <p>Background</p> <p>Increasing energy costs and environmental concerns have motivated engineering microbes for the production of "second generation" biofuels that have better properties than ethanol.</p> <p>Results and conclusion</p> <p><it>Saccharomyces cerevisiae </it>was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms (<it>S. cerevisiae</it>, <it>Escherichia coli</it>, <it>Clostridium beijerinckii</it>, and <it>Ralstonia eutropha</it>) 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 <it>C. beijerinckii </it>3-hydroxybutyryl-CoA dehydrogenase, which uses NADH as a co-factor, rather than the <it>R. eutropha </it>isozyme, which uses NADPH, and the acetoacetyl-CoA transferase from <it>S. cerevisiae </it>or <it>E. coli </it>rather than that from <it>R. eutropha</it>. Surprisingly, expression of the genes encoding the butyryl-CoA dehydrogenase from <it>C. beijerinckii </it>(<it>bcd </it>and <it>etfAB</it>) did not improve butanol production significantly as previously reported in <it>E. coli</it>. 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.</p>
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