Conversion of 4-Hydroxybutyrate to Acetyl Coenzyme A and Its Anapleurosis in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Carbon Fixation Pathway

ABSTRACTThe extremely thermoacidophilic archaeonMetallosphaera sedula(optimum growth temperature, 73°C, pH 2.0) grows chemolithoautotrophically on metal sulfides or molecular hydrogen by employing the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation cycle. This cycle adds two CO2molecules to acetyl coenzyme A (acetyl-CoA) to generate 4HB, which is then rearranged and cleaved to form two acetyl-CoA molecules. Previous metabolic flux analysis showed that two-thirds of central carbon precursor molecules are derived from succinyl-CoA, which is oxidized to malate and oxaloacetate. The remaining one-third is apparently derived from acetyl-CoA. As such, the steps beyond succinyl-CoA are essential for completing the carbon fixation cycle and for anapleurosis of acetyl-CoA. Here, the final four enzymes of the 3HP/4HB cycle, 4-hydroxybutyrate-CoA ligase (AMP forming) (Msed_0406), 4-hydroxybutyryl-CoA dehydratase (Msed_1321), crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (Msed_0399), and acetoacetyl-CoA β-ketothiolase (Msed_0656), were produced recombinantly inEscherichia coli, combinedin vitro, and shown to convert 4HB to acetyl-CoA. Metabolic pathways connecting CO2fixation and central metabolism were examined using a gas-intensive bioreactor system in whichM. sedulawas grown under autotrophic (CO2-limited) and heterotrophic conditions. Transcriptomic analysis revealed the importance of the 3HP/4HB pathway in supplying acetyl-CoA to anabolic pathways generating intermediates inM. sedulametabolism. The results indicated that flux between the succinate and acetyl-CoA branches in the 3HP/4HB pathway is governed by 4-hydroxybutyrate-CoA ligase, possibly regulated posttranslationally by the protein acetyltransferase (Pat)/Sir2-dependent system. Taken together, this work confirms the final four steps of the 3HP/4HB pathway, thereby providing the framework for examining connections between CO2fixation and central metabolism inM. sedula.

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Genome Sequence of Serratia marcescens MSU97, a Plant-Associated Bacterium That Makes Multiple Antibiotics

Genome Announcements ◽

10.1128/genomea.01752-16 ◽

2017 ◽

Vol 5 (9) ◽

Cited By ~ 5

Author(s):

Miguel A. Matilla ◽

Zulema Udaondo ◽

Tino Krell ◽

George P. C. Salmond

Keyword(s):

Serratia Marcescens ◽

Genome Sequence ◽

Coenzyme A ◽

Acetyl Coa Carboxylase ◽

Acetyl Coa ◽

Acetyl Coenzyme A ◽

Content Type ◽

Acetyl Coenzyme ◽

Multiple Antibiotics

ABSTRACT Serratia marcescens MSU97 was isolated from the Guayana region of Venezuela due to its ability to suppress plant-pathogenic oomycetes. Here, we report the genome sequence of MSU97, which produces various antibiotics, including the bacterial acetyl-coenzyme A (acetyl-CoA) carboxylase inhibitor andrimid, the chlorinated macrolide oocydin A, and the red linear tripyrrole antibiotic prodigiosin.

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A Pathway for Degradation of Uracil to Acetyl Coenzyme A in Bacillus megaterium

Applied and Environmental Microbiology ◽

10.1128/aem.02837-19 ◽

2020 ◽

Vol 86 (7) ◽

Cited By ~ 2

Author(s):

Di Zhu ◽

Yifeng Wei ◽

Jinyu Yin ◽

Dazhi Liu ◽

Ee Lui Ang ◽

...

Keyword(s):

Energy Source ◽

Bacillus Megaterium ◽

Coenzyme A ◽

Catabolic Pathway ◽

Acetyl Coa ◽

Acetyl Coenzyme A ◽

Content Type ◽

Biochemical Pathways ◽

Acetyl Coenzyme

ABSTRACT Bacteria utilize diverse biochemical pathways for the degradation of the pyrimidine ring. The function of the pathways studied to date has been the release of nitrogen for assimilation. The most widespread of these pathways is the reductive pyrimidine catabolic pathway, which converts uracil into ammonia, carbon dioxide, and β-alanine. Here, we report the characterization of a β-alanine:pyruvate aminotransferase (PydD2) and an NAD+-dependent malonic semialdehyde dehydrogenase (MSDH) from a reductive pyrimidine catabolism gene cluster in Bacillus megaterium. Together, these enzymes convert β-alanine into acetyl coenzyme A (acetyl-CoA), a key intermediate in carbon and energy metabolism. We demonstrate the growth of B. megaterium in defined medium with uracil as its sole carbon and energy source. Homologs of PydD2 and MSDH are found in association with reductive pyrimidine pathway genes in many Gram-positive bacteria in the order Bacillales. Our study provides a basis for further investigations of the utilization of pyrimidines as a carbon and energy source by bacteria. IMPORTANCE Pyrimidine has wide occurrence in natural environments, where bacteria use it as a nitrogen and carbon source for growth. Detailed biochemical pathways have been investigated with focus mainly on nitrogen assimilation in the past decades. Here, we report the discovery and characterization of two important enzymes, PydD2 and MSDH, which constitute an extension for the reductive pyrimidine catabolic pathway. These two enzymes, prevalent in Bacillales based on our bioinformatics studies, allow stepwise conversion of β-alanine, a previous “end product” of the reductive pyrimidine degradation pathway, to acetyl-CoA as carbon and energy source.

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Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic Crenarchaeota and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation

Journal of Bacteriology ◽

10.1128/jb.181.4.1088-1098.1999 ◽

1999 ◽

Vol 181 (4) ◽

pp. 1088-1098 ◽

Cited By ~ 103

Author(s):

Castor Menendez ◽

Zsuzsa Bauer ◽

Harald Huber ◽

Nasser Gad’on ◽

Karl-Otto Stetter ◽

...

Keyword(s):

Phosphoenolpyruvate Carboxylase ◽

Carbon Fixation ◽

Coenzyme A ◽

Autotrophic Growth ◽

Acetyl Coa Carboxylase ◽

Acetyl Coa ◽

Carboxylase Activity ◽

Acetyl Coenzyme A ◽

Acetyl Coenzyme

ABSTRACT The pathway of autotrophic CO2 fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO2-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either 14CO2 or [14C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed forC. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus andAcidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula,S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO2 fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus.

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3-Hydroxypropionyl-Coenzyme A Dehydratase and Acryloyl-Coenzyme A Reductase, Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in the Sulfolobales

Journal of Bacteriology ◽

10.1128/jb.00068-09 ◽

2009 ◽

Vol 191 (14) ◽

pp. 4572-4581 ◽

Cited By ~ 51

Author(s):

Robin Teufel ◽

Johannes W. Kung ◽

Daniel Kockelkorn ◽

Birgit E. Alber ◽

Georg Fuchs

Keyword(s):

Alcohol Dehydrogenase ◽

Carbon Fixation ◽

Coenzyme A ◽

Chloroflexus Aurantiacus ◽

Reaction Sequence ◽

Acetyl Coenzyme A ◽

Sulfolobus Tokodaii ◽

Metallosphaera Sedula ◽

Acetyl Coenzyme ◽

Coa Reductase

ABSTRACT A 3-hydroxypropionate/4-hydroxybutyrate cycle operates in autotrophic CO2 fixation in various Crenarchaea, as studied in some detail in Metallosphaera sedula. This cycle and the autotrophic 3-hydroxypropionate cycle in Chloroflexus aurantiacus have in common the conversion of acetyl-coenzyme A (CoA) and two bicarbonates via 3-hydroxypropionate to succinyl-CoA. Both cycles require the reductive conversion of 3-hydroxypropionate to propionyl-CoA. In M. sedula the reaction sequence is catalyzed by three enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the CoA- and MgATP-dependent formation of 3-hydroxypropionyl-CoA. The next two enzymes were purified from M. sedula or Sulfolobus tokodaii and studied. 3-Hydroxypropionyl-CoA dehydratase, a member of the enoyl-CoA hydratase family, eliminates water from 3-hydroxypropionyl-CoA to form acryloyl-CoA. Acryloyl-CoA reductase, a member of the zinc-containing alcohol dehydrogenase family, reduces acryloyl-CoA with NADPH to propionyl-CoA. Genes highly similar to the Metallosphaera CoA synthetase, dehydratase, and reductase genes were found in autotrophic members of the Sulfolobales. The encoded enzymes are only distantly related to the respective three enzyme domains of propionyl-CoA synthase from C. aurantiacus, where this trifunctional enzyme catalyzes all three reactions. This indicates that the autotrophic carbon fixation cycles in Chloroflexus and in the Sulfolobales evolved independently and that different genes/enzymes have been recruited in the two lineages that catalyze the same kinds of reactions.

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Functional Analyses of Two Acetyl Coenzyme A Synthetases in the Ascomycete Gibberella zeae

Eukaryotic Cell ◽

10.1128/ec.05071-11 ◽

2011 ◽

Vol 10 (8) ◽

pp. 1043-1052 ◽

Cited By ~ 44

Author(s):

Seunghoon Lee ◽

Hokyoung Son ◽

Jungkwan Lee ◽

Kyunghun Min ◽

Gyung Ja Choi ◽

...

Keyword(s):

Sexual Development ◽

Coenzyme A ◽

Lipid Synthesis ◽

Gibberella Zeae ◽

Atp Citrate Lyase ◽

Acetyl Coa ◽

Acetyl Coenzyme A ◽

Content Type ◽

Temporal Precision ◽

Acetyl Coenzyme

ABSTRACTAcetyl coenzyme A (acetyl-CoA) is a crucial metabolite for energy metabolism and biosynthetic pathways and is produced in various cellular compartments with spatial and temporal precision. Our previous study on ATP citrate lyase (ACL) inGibberella zeaerevealed that ACL-dependent acetyl-CoA production is important for histone acetylation, especially in sexual development, but is not involved in lipid synthesis. In this study, we deleted additional acetyl-CoA synthetic genes, the acetyl-CoA synthetases (ACSgenesACS1andACS2), to identify alternative acetyl-CoA production mechanisms for ACL. TheACS1deletion resulted in a defect in sexual development that was mainly due to a reduction in 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol production, which is required for perithecium development and maturation. Another ACS coding gene,ACS2, has accessorial functions forACS1and has compensatory functions forACLas a nuclear acetyl-CoA producer. This study showed that acetate is readily generated during the entire life cycle ofG. zeaeand has a pivotal role in fungal metabolism. Because ACSs are components of the pyruvate-acetaldehyde-acetate pathway, this fermentation process might have crucial roles in various physiological processes for filamentous fungi.

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Investigation of Carbon Metabolism in “Dehalococcoides ethenogenes” Strain 195 by Use of Isotopomer and Transcriptomic Analyses

Journal of Bacteriology ◽

10.1128/jb.00085-09 ◽

2009 ◽

Vol 191 (16) ◽

pp. 5224-5231 ◽

Cited By ~ 62

Author(s):

Yinjie J. Tang ◽

Shan Yi ◽

Wei-Qin Zhuang ◽

Stephen H. Zinder ◽

Jay D. Keasling ◽

...

Keyword(s):

Genome Annotation ◽

Carbon Fixation ◽

Coenzyme A ◽

Citrate Synthase ◽

Tricarboxylic Acid Cycle ◽

Acetyl Coa ◽

Experimental Conditions ◽

Acetyl Coenzyme A ◽

Acetyl Coenzyme ◽

Dehalococcoides Ethenogenes

ABSTRACT Members of the genus “Dehalococcoides” are the only known microorganisms that can completely dechlorinate tetrachloroethene and trichloroethene to the innocuous end product, ethene. This study examines the central metabolism in “Dehalococcoides ethenogenes” strain 195 via 13C-labeled tracer experiments. Supported by the genome annotation and the transcript profile, isotopomer analysis of key metabolites clarifies ambiguities in the genome annotation and identifies an unusual biosynthetic pathway in strain 195. First, the 13C-labeling studies revealed that strain 195 contains complete amino acid biosynthesis pathways, even though current genome annotation suggests that several of these pathways are incomplete. Second, the tricarboxylic acid cycle of strain 195 is confirmed to be branched, and the Wood-Ljungdahl carbon fixation pathway is shown to not be functionally active under our experimental conditions; rather, CO2 is assimilated via two reactions, conversion of acetyl-coenzyme A (acetyl coenzyme A [acetyl-CoA]) to pyruvate catalyzed by pyruvate synthase (DET0724-0727) and pyruvate conversion to oxaloacetate via pyruvate carboxylase (DET0119-0120). Third, the 13C-labeling studies also suggested that isoleucine is synthesized from acetyl-CoA and pyruvate via citramalate synthase (CimA, EC 2.3.1.182), rather than from the common pathway via threonine ammonia-lyase (EC 4.3.1.19). Finally, evidence is presented that strain 195 may contain an undocumented citrate synthase (>95% Re-type stereospecific), i.e., a novel Re-citrate synthase that is apparently different from the one recently reported in Clostridium kluyveri.

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Synthetic Biology for Engineering Acetyl Coenzyme A Metabolism in Yeast

mBio ◽

10.1128/mbio.02153-14 ◽

2014 ◽

Vol 5 (6) ◽

Cited By ~ 47

Author(s):

Jens Nielsen

Keyword(s):

Coenzyme A ◽

Pyruvate Dehydrogenase Complex ◽

High Yield ◽

Cell Factory ◽

Efficient Production ◽

Acetyl Coa ◽

Acetyl Coenzyme A ◽

Content Type ◽

Acetyl Coenzyme ◽

High Yields

ABSTRACT The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels, chemicals, and pharmaceuticals. The use of this cell factory for cost-efficient production of novel fuels and chemicals requires high yields and low by-product production. Many industrially interesting chemicals are biosynthesized from acetyl coenzyme A (acetyl-CoA), which serves as a central precursor metabolite in yeast. To ensure high yields in production of these chemicals, it is necessary to engineer the central carbon metabolism so that ethanol production is minimized (or eliminated) and acetyl-CoA can be formed from glucose in high yield. Here the perspective of generating yeast platform strains that have such properties is discussed in the context of a major breakthrough with expression of a functional pyruvate dehydrogenase complex in the cytosol.

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PanM, an Acetyl-Coenzyme A Sensor Required for Maturation of l-Aspartate Decarboxylase (PanD)

mBio ◽

10.1128/mbio.00158-12 ◽

2012 ◽

Vol 3 (4) ◽

Cited By ~ 8

Author(s):

Tara N. Stuecker ◽

Alex C. Tucker ◽

Jorge C. Escalante-Semerena

Keyword(s):

Coenzyme A ◽

Lysine Acetylation ◽

Acetyl Coa ◽

Transfer Event ◽

Acetyl Coenzyme A ◽

Content Type ◽

Acetyltransferase Activity ◽

Acetyl Coenzyme

ABSTRACTCoenzyme A (CoA) is essential for cellular chemistry in all forms of life. The pantothenate moiety of CoA is generated from the condensation of pantoate and β-alanine. β-Alanine is formed by decarboxylation ofl-aspartate catalyzed by PanD, a pyruvoyl enzyme that is synthesized by the cell as an inactive precursor (pro-PanD). Maturation of pro-PanD into PanD occurs via a self-cleavage event at residue Ser25, which forms the catalytic pyruvoyl moiety. We recently reported thatSalmonella entericaPanM was necessary for pro-PanD maturation, bothin vitroandin vivo. Notably, PanM is annotated as a Gcn5-likeN-acetyltransferase (GNAT), which suggested that lysine acetylation might be part of the mechanism of maturation. Here we show that PanM lacks acetyltransferase activity and that acetyl-CoA stimulates its activity. Results of experiments with nonhydrolyzable ethyl-CoA and genetically encoded acetyl-lysine-containing PanD support the conclusion that PanM-dependent pro-PanD maturation does not involve an acetyl transfer event. We also show that CoA binding to PanM is needed forin vivoactivity and that disruption of CoA binding prevents PanM from interacting with PanD. We conclude that PanM is a GNAT homologue that lost its acetyltransferase activity and evolved a new function as an acetyl-CoA sensor that can trigger the maturation of pro-PanD.IMPORTANCENε-lysine acetylation is increasingly being recognized as a widespread and important form of posttranslational regulation in bacteria. The acetyltransferases that catalyze these reactions are poorly characterized in bacteria. Based on annotation, most bacterial genomes contain several acetyltransferases, but the physiological roles of only a handful have been determined. Notably, a subset of putative acetyltransferases lack residues that are critical for activity in most biochemically characterized acetyltransferases. We show that one such putative acetyltransferase, PanM (formerly YhhK), lacks acetyltransferase activity but functions instead as an acetyl-coenzyme A (CoA) sensor. This work establishes the possibility that, like PanM, other putative acetyltransferases may have evolved new functions while retaining the ability to sense acetyl-CoA.

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Deconstructing Methanosarcina acetivorans into an acetogenic archaeon

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2113853119 ◽

2022 ◽

Vol 119 (2) ◽

pp. e2113853119

Author(s):

Christian Schöne ◽

Anja Poehlein ◽

Nico Jehmlich ◽

Norman Adlung ◽

Rolf Daniel ◽

...

Keyword(s):

Carbon Monoxide ◽

Energy Conservation ◽

Carbon Fixation ◽

Coenzyme A ◽

Methanogenic Archaea ◽

Methanosarcina Acetivorans ◽

Acetyl Coa ◽

Acetyl Coenzyme A ◽

Acetyl Coenzyme ◽

Acetyl Coa Pathway

The reductive acetyl-coenzyme A (acetyl-CoA) pathway, whereby carbon dioxide is sequentially reduced to acetyl-CoA via coenzyme-bound C1 intermediates, is the only autotrophic pathway that can at the same time be the means for energy conservation. A conceptually similar metabolism and a key process in the global carbon cycle is methanogenesis, the biogenic formation of methane. All known methanogenic archaea depend on methanogenesis to sustain growth and use the reductive acetyl-CoA pathway for autotrophic carbon fixation. Here, we converted a methanogen into an acetogen and show that Methanosarcina acetivorans can dispense with methanogenesis for energy conservation completely. By targeted disruption of the methanogenic pathway, followed by adaptive evolution, a strain was created that sustained growth via carbon monoxide–dependent acetogenesis. A minute flux (less than 0.2% of the carbon monoxide consumed) through the methane-liberating reaction remained essential, indicating that currently living methanogens utilize metabolites of this reaction also for anabolic purposes. These results suggest that the metabolic flexibility of methanogenic archaea might be much greater than currently known. Also, our ability to deconstruct a methanogen into an acetogen by merely removing cellular functions provides experimental support for the notion that methanogenesis could have evolved from the reductive acetyl-coenzyme A pathway.

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Mitochondrial Carnitine-Dependent Acetyl Coenzyme A Transport Is Required for Normal Sexual and Asexual Development of the Ascomycete Gibberella zeae

Eukaryotic Cell ◽

10.1128/ec.00104-12 ◽

2012 ◽

Vol 11 (9) ◽

pp. 1143-1153 ◽

Cited By ~ 17

Author(s):

Hokyoung Son ◽

Kyunghun Min ◽

Jungkwan Lee ◽

Gyung Ja Choi ◽

Jin-Cheol Kim ◽

...

Keyword(s):

Coenzyme A ◽

Developmental Stages ◽

Pathogenic Fungus ◽

Gibberella Zeae ◽

Asexual Development ◽

Acetyl Coa ◽

Alternative Source ◽

Acetyl Coenzyme A ◽

Content Type ◽

Acetyl Coenzyme

ABSTRACTFungi have evolved efficient metabolic mechanisms for the exact temporal (developmental stages) and spatial (organelles) production of acetyl coenzyme A (acetyl-CoA). We previously demonstrated mechanistic roles of several acetyl-CoA synthetic enzymes, namely, ATP citrate lyase and acetyl-CoA synthetases (ACSs), in the plant-pathogenic fungusGibberella zeae. In this study, we characterized two carnitine acetyltransferases (CATs; CAT1 and CAT2) to obtain a better understanding of the metabolic processes occurring inG. zeae. We found that CAT1 functioned as an alternative source of acetyl-CoA required for lipid accumulation in anACS1deletion mutant. Moreover, deletion ofCAT1and/orCAT2resulted in various defects, including changes to vegetative growth, asexual/sexual development, trichothecene production, and virulence. Although CAT1 is associated primarily with peroxisomal CAT function, mislocalization experiments showed that the role of CAT1 in acetyl-CoA transport between the mitochondria and cytosol is important for sexual and asexual development inG. zeae. Taking these data together, we concluded thatG. zeaeCATs are responsible for facilitating the exchange of acetyl-CoA across intracellular membranes, particularly between the mitochondria and the cytosol, during various developmental stages.

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