Non-growing Rhodopseudomonas palustris Increases the Hydrogen Gas Yield from Acetate by Shifting from the Glyoxylate Shunt to the Tricarboxylic Acid Cycle

McKinlay, James B.; Oda, Yuji; Rühl, Martin; Posto, Amanda L.; Sauer, Uwe; Harwood, Caroline S.

doi:10.1074/jbc.m113.527515

Cited by 97 publications

(111 citation statements)

References 43 publications

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“…Recent metabolic flux analysis studies indicate growing R. palustris cells use the glyoxylate shunt to incorporate acetate when it is the only available carbon source (15,45). Here, we provided R. palustris with complex carbon Table S4 in the supplemental material).…”

Section: Discussionmentioning

confidence: 99%

Essential Genome of the Metabolically Versatile Alphaproteobacterium Rhodopseudomonas palustris

et al. 2016

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Rhodopseudomonas palustris is an alphaproteobacterium that has served as a model organism for studies of photophosphorylation, regulation of nitrogen fixation, production of hydrogen as a biofuel, and anaerobic degradation of aromatic compounds. This bacterium is able to transition between anaerobic photoautotrophic growth, anaerobic photoheterotrophic growth, and aerobic heterotrophic growth. As a starting point to explore the genetic basis for the metabolic versatility of R. palustris, we used transposon mutagenesis and Tn-seq to identify 552 genes as essential for viability in cells growing aerobically on semirich medium. Of these, 323 have essential gene homologs in the alphaproteobacterium Caulobacter crescentus, and 187 have essential gene homologs in Escherichia coli. There were 24 R. palustris genes that were essential for viability under aerobic growth conditions that have low sequence identity but are likely to be functionally homologous to essential E. coli genes. As expected, certain functional categories of essential genes were highly conserved among the three organisms, including translation, ribosome structure and biogenesis, secretion, and lipid metabolism. R. palustris cells divide by budding in which a sessile cell gives rise to a motile swarmer cell. Conserved cell cycle genes required for this developmental process were essential in both C. crescentus and R. palustris. Our results suggest that despite vast differences in lifestyles, members of the alphaproteobacteria have a common set of essential genes that is specific to this group and distinct from that of gammaproteobacteria like E. coli. IMPORTANCEEssential genes in bacteria and other organisms are those absolutely required for viability. Rhodopseudomonas palustris has served as a model organism for studies of anaerobic aromatic compound degradation, hydrogen gas production, nitrogen fixation, and photosynthesis. We used the technique of Tn-seq to determine the essential genes of R. palustris grown under heterotrophic aerobic conditions. The transposon library generated in this study will be useful for future studies to identify R. palustris genes essential for viability under specialized growth conditions and also for survival under conditions of stress.

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Section: Discussionmentioning

confidence: 99%

Essential Genome of the Metabolically Versatile Alphaproteobacterium Rhodopseudomonas palustris

et al. 2016

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“…Such cells are spared the imperative of carrying out biosynthetic reactions required for growth, which frees up electrons derived from the oxidation of external electron donors for use in reduction reactions (12). To test if diversion of electrons from biosynthesis leads to more CH 4 production by the remodeled nitrogenase, the cells of the R. palustris nifA* nifD V75AH201Q strain grown to midlogarithmic phase were harvested, resuspended in nitrogen-free medium, and tested for their ability to produce CH 4 .…”

Section: Nifdmentioning

confidence: 99%

“…2 Reduction to CH 4 by R. palustris. GC-MS analysis was conducted to confirm the production of CH 4 from CO 2 reduction using a Shimadzu GC-2010 Gas Chromatograph equipped with a programmed temperature vaporizing injector and a Shimadzu GCMS-QP2010S Mass Spectrometer by using 12 …”

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Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium

Fixen

Zheng

Harris

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Nitrogenase is an ATP-requiring enzyme capable of carrying out multielectron reductions of inert molecules. A purified remodeled nitrogenase containing two amino acid substitutions near the site of its FeMo cofactor was recently described as having the capacity to reduce carbon dioxide (CO 2 ) to methane (CH 4 ). Here, we developed the anoxygenic phototroph, Rhodopseudomonas palustris, as a biocatalyst capable of light-driven CO 2 reduction to CH 4 in vivo using this remodeled nitrogenase. Conversion of CO 2 to CH 4 by R. palustris required constitutive expression of nitrogenase, which was achieved by using a variant of the transcription factor NifA that is able to activate expression of nitrogenase under all growth conditions. Also, light was required for generation of ATP by cyclic photophosphorylation. CH 4 production by R. palustris could be controlled by manipulating the distribution of electrons and energy available to nitrogenase. This work shows the feasibility of using microbes to generate hydrocarbons from CO 2 in one enzymatic step using light energy.A n essential process for life and an important step in the biogeochemical nitrogen cycle is nitrogen fixation by nitrogenase, in which nitrogen gas (N 2 ) is converted to ammonia (NH 3 ) (1). The difficult reduction of N 2 to two NH 3 occurs at an FeMoS cluster called FeMo cofactor in Mo-dependent nitrogenase in a reaction that requires ATP hydrolysis and dihydrogen production as shown in 1Nitrogenase deprived of access to N 2 but provided with a source of electrons produces H 2 exclusively. Also, the ability of nitrogenase to carry out the multielectron reduction of an inert molecule is not limited to reduction of N 2 . This enzyme can also reduce other molecules with double and triple bonds, including carboncontaining compounds (reviewed in ref.2). Recently, we found that a remodeled nitrogenase with substitutions in two key amino acids near the FeMo cofactor is capable of reducing carbon dioxide (CO 2 ) to methane (CH 4 ) in vitro. This enzyme did not retain its ability to reduce N 2 but was active in H 2 production (3). It was unclear if the remodeled nitrogenase gene could confer to bacteria the ability to reduce CO 2 to CH 4 . Here, we describe a biocatalyst capable of generating the energyrich hydrocarbon CH 4 by reduction of CO 2 using a remodeled nitrogenase. Development of this biocatalyst required selection of an appropriate microbial host, because large amounts of cellular reductant and ATP are used by nitrogenase, and as a consequence, this energetically expensive enzyme is repressed by both transcriptional and posttranslational regulatory mechanisms when an alternative nitrogen source, like ammonium, is available (4). Use of nitrogenase to generate a product not used by the organism would require overcoming these regulatory constraints to achieve expression of active enzyme, while at the same time providing cells with ammonium for growth. We reasoned that the anoxygenic photosynthetic bacterium Rhodopseudomonas palustris would be a good ch...

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“…Mass isotopomer distributions were corrected for natural isotopic abundances using IsoCor software (31). Flux estimates were made using 13CFLUX2 (32) software with the nag_opt_nlp optimizer found in the Numerical Algorithms Group C library (www.nag.com/numeric/CL/CLdescription.asp) as previously described (33). Data from parallel labeling experiments were fit to a single metabolic model as previously described (34).…”

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N₂gas is an effective fertilizer for bioethanol production byZymomonas mobilis

Kremer

LaSarre

Posto

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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A nascent cellulosic ethanol industry is struggling to become costcompetitive against corn ethanol and gasoline. Millions of dollars are spent on nitrogen supplements to make up for the low nitrogen content of the cellulosic feedstock. Here we show for the first time to our knowledge that the ethanol-producing bacterium, Zymomonas mobilis, can use N 2 gas in lieu of traditional nitrogen supplements. Despite being an electron-intensive process, N 2 fixation by Z. mobilis did not divert electrons away from ethanol production, as the ethanol yield was greater than 97% of the theoretical maximum. In a defined medium, Z. mobilis produced ethanol 50% faster per cell and generated half the unwanted biomass when supplied N 2 instead of ammonium. In a cellulosic feedstock-derived medium, Z. mobilis achieved a similar cell density and a slightly higher ethanol yield when supplied N 2 instead of the industrial nitrogen supplement, corn steep liquor. We estimate that N 2 -utilizing Z. mobilis could save a cellulosic ethanol production facility more than $1 million/y.Zymomonas mobilis | nitrogenase | ethanol | cellulosic | biofuel

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Non-growing Rhodopseudomonas palustris Increases the Hydrogen Gas Yield from Acetate by Shifting from the Glyoxylate Shunt to the Tricarboxylic Acid Cycle

Cited by 97 publications

References 43 publications

Essential Genome of the Metabolically Versatile Alphaproteobacterium Rhodopseudomonas palustris

Essential Genome of the Metabolically Versatile Alphaproteobacterium Rhodopseudomonas palustris

Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium

N₂gas is an effective fertilizer for bioethanol production byZymomonas mobilis

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Non-growing Rhodopseudomonas palustris Increases the Hydrogen Gas Yield from Acetate by Shifting from the Glyoxylate Shunt to the Tricarboxylic Acid Cycle

Cited by 97 publications

References 43 publications

Essential Genome of the Metabolically Versatile Alphaproteobacterium Rhodopseudomonas palustris

Essential Genome of the Metabolically Versatile Alphaproteobacterium Rhodopseudomonas palustris

Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium

N2gas is an effective fertilizer for bioethanol production byZymomonas mobilis

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N₂gas is an effective fertilizer for bioethanol production byZymomonas mobilis