Engineering of Ralstonia eutropha H16 for autotrophic and heterotrophic production of methyl ketones

Jana Müller, Daniel MacEachran, Helcio Burd, Noppadon Sathitsuksanoh, Changhao Bi, Yi Chun Yeh, Taek Soon Lee, Nathan J. Hillson, Swapnil R. Chhabra, Steven W. Singer, Harry R. Beller

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68 Citations (Scopus)

Abstract

Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H2 and CO2 under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces copious amounts of poly[(R)-3-hydroxybutyrate] (PHB). Its ability to utilize CO2 as a sole carbon source renders it an interesting new candidate host for the production of renewable liquid transportation fuels. We engineered R. eutropha for the production of fatty acid-derived, diesel-range methyl ketones. Modifications engineered in R. eutropha included overexpression of a cytoplasmic version of the TesA thioesterase, which led to a substantial (>150-fold) increase in fatty acid titer under certain conditions. In addition, deletion of two putative β-oxidation operons and heterologous expression of three genes (the acyl coenzyme A oxidase gene from Micrococcus luteus and fadB and fadM from Escherichia coli) led to the production of 50 to 65 mg/liter of diesel-range methyl ketones under heterotrophic growth conditions and 50 to 180 mg/liter under chemolithoautotrophic growth conditions (with CO2 and H2 as the sole carbon source and electron donor, respectively). Induction of the methyl ketone pathway diverted substantial carbon flux away from PHB biosynthesis and appeared to enhance carbon flux through the pathway for biosynthesis of fatty acids, which are the precursors of methyl ketones.

Original languageEnglish
Pages (from-to)4433-4439
Number of pages7
JournalApplied and Environmental Microbiology
Volume79
Issue number14
DOIs
Publication statusPublished - 2013 Jul 1

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

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    Müller, J., MacEachran, D., Burd, H., Sathitsuksanoh, N., Bi, C., Yeh, Y. C., Lee, T. S., Hillson, N. J., Chhabra, S. R., Singer, S. W., & Beller, H. R. (2013). Engineering of Ralstonia eutropha H16 for autotrophic and heterotrophic production of methyl ketones. Applied and Environmental Microbiology, 79(14), 4433-4439. https://doi.org/10.1128/AEM.00973-13