Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol

Abstract

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 and conclusion: 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.

Figure 1

The n-butanol biosynthetic pathway. The enzymes in green are from Clostridium beijerinckii. Enzymes in black are from other organisms: AtoB, Escherichia coli; Erg10, S. cerevisiae; PhaA, Ralstonia eutropha; PhaB, Ralstonia eutropha; Ccr, Streptomyces collinus. Each enzyme candidate was evaluated in the pathway for n-butanol production (except thl, which is native to Clostridia).

Figure 2

Representative plasmids used in this study. Plasmids were constructed by the SLIC method, previously described. They contain the 2μ origin of replication, LEU2D or HIS3 genes for selection, the GAL1 or GAL10 promoters, and the CYC1, ADH1, or PGK1 transcription terminators. The first three genes of the n-butanol pathway were placed on the pESC-LEU2D plasmid and the last two or four (in the case of the etfAB, bcd bearing strain) genes were placed on the pESC-HIS3 plasmid.

Figure 3

n-Butanol production from engineered S. cerevisiae. Symbols and strains: black squares, ESY7; empty squares, ESY11; black circles, ESY2; the rest of the samples all produced approximately the same amount of n-butanol and are indicated on the graph. Symbols and error bars represent the mean and standard deviation of triplicate cultures.

Figure 4

n-Butanol pathway intermediates at 24 h. Bars and strains: black bars, ESY4; gray bars, ESY7; white bars, ESY11. (A) All pathway intermediates in strains ESY4, 7 and 11. (B) HbCoA, CrCoA and BtCoA intermediates in strains ESY4 and ESY7. Levels of AcCoA were similar except for strains ESY11 (A). Levels of 3-hydroxybutyryl-CoA (HbCoA) and butyryl-CoA (BtCoA) were notably higher in ESY7 compared to ESY4, while crotonyl-CoA (CrCoA) was relatively similar in the two strains. Values and error bars represent the mean and standard deviation of triplicate cultures.