Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli
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Fischer_2010-Assessment of heterologous.pdf
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Author(s)
Fischer, Curt R.
Tseng, Hsien-Chung
Tai, Mitchell
Stephanopoulos, Gregory
Prather, Kristala L. Jones
Date Issued
July 2010
Journal
Applied Microbiology and Biotechnology
Publisher
Springer-Verlag
Citation
Fischer, Curt R., Hsien-Chung Tseng, Mitchell Tai, Kristala L. J. Prather, and Gregory Stephanopoulos. “Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli.” Applied Microbiology and Biotechnology 88, no. 1 (September 13, 2010): 265-275.
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Final published version
Abstract
In clostridia, n-butanol production from carbohydrates at yields of up to 76% of the theoretical maximum and at titers of up to 13 g/L has been reported. However, in Escherichia coli, several groups have reported butyric acid or butanol production from recombinant expression of clostridial genes, at much lower titers and yields. To pinpoint deficient steps in the recombinant pathway, we developed an analytical procedure for the determination of intracellular pools of key pathway intermediates and applied the technique to the analysis of three sets of E. coli strains expressing various combinations of butyrate biosynthesis genes. Low expression levels of the hbd-encoded S-3-hydroxybutyryl-CoA dehydrogenase were insufficient to convert acetyl-CoA to 3-hydroxybutyryl-CoA, indicating that hbd was a rate-limiting step in the production of butyryl-CoA. Increasing hbd expression alleviated this bottleneck, but in resulting strains, our pool size measurements and thermodynamic analysis showed that the reaction step catalyzed by the bcd-encoded butyryl-CoA dehydrogenase was rate-limiting. E. coli strains expressing both hbd and ptb-buk produced crotonic acid as a byproduct, but this byproduct was not observed with expression of related genes from non-clostridial organisms. Our thermodynamic interpretation of pool size measurements is applicable to the analysis of other metabolic pathways.
MIT Department
Massachusetts Institute of Technology. Department of Chemical Engineering
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DOI of Published Version
https://doi.org/10.1007/s00253-010-2749-2
