Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli.

Applied Microbiology and Biotechnology
Curt R FischerGregory Stephanopoulos

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 ...Continue Reading

References

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Nov 3, 2009·Metabolic Engineering·Keith E J TyoGregory Stephanopoulos

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Citations

May 20, 2011·Biochemistry·Amy M Weeks, Michelle C Y Chang
Oct 17, 2012·Proceedings of the National Academy of Sciences of the United States of America·Hsien-Chung Tseng, Kristala L J Prather
Apr 27, 2012·The Journal of Biological Chemistry·Se Hyeuk Kim, Pyung Cheon Lee
Mar 15, 2011·Applied and Environmental Microbiology·Claire R ShenJames C Liao
Apr 28, 2012·PloS One·Paul OldhamGeoff Burton
Apr 19, 2011·Current Opinion in Biotechnology·Vasiliy A PortnoyKarsten Zengler
Apr 12, 2014·Microbiology·Alexandra R VolkerThomas A Bobik
Jul 29, 2015·Microbial Cell Factories·Eleni TheodosiouAndreas Schmid
Jul 13, 2005·PLoS Biology·Kate S CarrollCarolyn R Bertozzi
Apr 30, 2014·Journal of Agricultural and Food Chemistry·Mukesh SainiYun-Peng Chao
May 9, 2018·Bioprocess and Biosystems Engineering·Jong-Min JeonYung-Hun Yang
Feb 10, 2020·Biochemical and Biophysical Research Communications·Peng LiaoHua Lu

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Methods Mentioned

BETA
PCR
PBAD
dissection

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