title: Engineering of Ralstonia eutropha H16 for autotrophic and heterotrophic production of methyl ketones.
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.
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Jana
authors.initials: J
authors.lastname: Müller
authors.forename: Daniel
authors.initials: D
authors.lastname: MacEachran
authors.forename: Helcio
authors.initials: H
authors.lastname: Burd
authors.forename: Noppadon
authors.initials: N
authors.lastname: Sathitsuksanoh
authors.forename: Changhao
authors.initials: C
authors.lastname: Bi
authors.forename: Yi-Chun
authors.initials: YC
authors.lastname: Yeh
authors.forename: Taek Soon
authors.initials: TS
authors.lastname: Lee
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Hillson
authors.forename: Swapnil R
authors.initials: SR
authors.lastname: Chhabra
authors.forename: Steven W
authors.initials: SW
authors.lastname: Singer
authors.forename: Harry R
authors.initials: HR
authors.lastname: Beller
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D052837:Chemoautotrophic Growth; D020118:Cupriavidus necator / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering; D052836:Heterotrophic Processes; D006885:Hydroxybutyrates / Q000378:metabolism*; D007659:Ketones / Q000378:metabolism*; D016982:Micrococcus luteus / Q000235:genetics; D010084:Oxidation-Reduction; D011091:Polyesters / Q000378:metabolism*; D000094122:Polyhydroxybutyrates
chemical_list: D001426:Bacterial Proteins; D002245:Carbon Dioxide; D005227:Fatty Acids; D006885:Hydroxybutyrates; D007659:Ketones; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates