scholarly journals Phosphoglycolate salvage in a chemolithoautotroph using the Calvin cycle

2020 ◽  
Vol 117 (36) ◽  
pp. 22452-22461 ◽  
Author(s):  
Nico J. Claassens ◽  
Giovanni Scarinci ◽  
Axel Fischer ◽  
Avi I. Flamholz ◽  
William Newell ◽  
...  
Keyword(s):  
Carbon Fixation ◽  
Ralstonia Eutropha ◽  
Calvin Cycle ◽  
General Term ◽  
Oxidative Decarboxylation ◽  
Autotrophic Growth ◽  
Acetyl Coa ◽  
Glycolate Metabolism ◽  
Microbial Carbon ◽  
Chemolithoautotrophic Bacteria

Carbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of phosphoglycolate was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, where it is referred to as photorespiration. While receiving little attention so far, aerobic chemolithoautotrophic bacteria that operate the Calvin cycle independent of light must also recycle phosphoglycolate. As the term photorespiration is inappropriate for describing phosphoglycolate recycling in these nonphotosynthetic autotrophs, we suggest the more general term “phosphoglycolate salvage.” Here, we study phosphoglycolate salvage in the model chemolithoautotrophCupriavidus necatorH16 (Ralstonia eutrophaH16) by characterizing the proxy process of glycolate metabolism, performing comparative transcriptomics of autotrophic growth under low and high CO2concentrations, and testing autotrophic growth phenotypes of gene deletion strains at ambient CO2. We find that the canonical plant-like C2cycle does not operate in this bacterium, and instead, the bacterial-like glycerate pathway is the main route for phosphoglycolate salvage. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports phosphoglycolate salvage. In this cycle, glyoxylate is condensed with acetyl coenzyme A (acetyl-CoA) to give malate, which undergoes two oxidative decarboxylation steps to regenerate acetyl-CoA. When both pathways are disrupted, autotrophic growth is abolished at ambient CO2. We present bioinformatic data suggesting that the malate cycle may support phosphoglycolate salvage in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so far unknown phosphoglycolate salvage pathway, highlighting important diversity in microbial carbon fixation metabolism.

scholarly journals Photorespiration pathways in a chemolithoautotroph

2020 ◽  
Author(s):  
Nico J. Claassens ◽  
Giovanni Scarinci ◽  
Axel Fischer ◽  
Avi I. Flamholz ◽  
William Newell ◽  
...  
Keyword(s):  
Carbon Fixation ◽  
Ralstonia Eutropha ◽  
Calvin Cycle ◽  
Oxidative Decarboxylation ◽  
Autotrophic Growth ◽  
Acetyl Coa ◽  
Oxidative Pathway ◽  
Glycolate Metabolism ◽  
Microbial Carbon ◽  
Chemolithoautotrophic Bacteria

AbstractCarbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of 2-phosphoglycolate, an essential process termed photorespiration, was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, but remains uncharacterized in chemolithoautotrophic bacteria. Here, we study photorespiration in the model chemolithoautotroph Cupriavidus necator (Ralstonia eutropha) by characterizing the proxy-process of glycolate metabolism, performing comparative transcriptomics of autotrophic growth under low and high CO2 concentrations, and testing autotrophic growth phenotypes of gene deletion strains at ambient CO2. We find that the canonical plant-like C2 cycle does not operate in this bacterium and instead the bacterial-like glycerate pathway is the main photorespiratory pathway. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports photorespiration. In this cycle, glyoxylate is condensed with acetyl-CoA to give malate, which undergoes two oxidative decarboxylation steps to regenerate acetyl-CoA. When both pathways are disrupted, autotrophic growth is abolished at ambient CO2. We present bioinformatic data suggesting that the malate cycle may support photorespiration in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so-far unknown photorespiration pathway, highlighting important diversity in microbial carbon fixation metabolism.

Engineering the CBB cycle and hydrogen utilization pathway of Ralstonia eutropha H16 for improved autotrophic growth and PHB production

2020 ◽  
Author(s):  
Zhongkang Li ◽  
Muzi Hu ◽  
Bin Xiong ◽  
Dongdong Zhao ◽  
Chunzhi Zhang ◽  
...  
Keyword(s):  
Carbon Fixation ◽  
Ralstonia Eutropha ◽  
Biological Research ◽  
Industrial Biotechnology ◽  
Cell Factory ◽  
Autotrophic Growth ◽  
Microbial Cell Factory ◽  
Knock Out ◽  
Ralstonia Eutropha H16 ◽  
Living Organisms

Abstract CO 2 is fixed by all living organisms with an autotrophic metabolism, among which the Calvin-Benson-Bassham ( CBB) cycle is the most important and widespread carbon fixation pathway. Thus, studying and engineering the CBB cycle with the associated energy providing pathways to increase the CO 2 fixation efficiency of cells is an important subject of biological research with significant application potential. In this work, the autotrophic microbe Ralstonia eutropha H16 was selected as a research platform for CBB cycle optimization engineering. By knocking out either CBB operon genes on the operon or mega-plasmid of R. eutropha , we found that both CBB operons were active and contributed almost equally to the carbon fixation process. With similar knock-out experiments, we found while both soluble and membrane-bound hydrogenases (SH and MBH), belonging to the energy providing hydrogenase module, were f unctional d uring autotrophic growth of R. eutropha. And SH played a more significant role. By introducing a heterologous cyanobacterial RuBisCO with the endogenous GroES/EL chaperone system and RbcX, the culture OD 600 of engineered strain increased 89.15% after 72 hours of autotrophic growth, indicating cyanobacterial RuBisCO with a higher activity was functional in R. eutropha and improved upon original CBB pathway. Meanwhile, expression of hydrogenases were optimized by modulating the expression of MBH and SH, which could further increase the R. eutropha H16 culture OD 600 to 93.4% at 72 hours. Moreover, the autotrophic yield of its major industrially relevant product, polyhydroxybutyrate (PHB), was increased by 99.71%. To our best knowledge, this is the first report of successfully engineering the CBB pathway of R. eutropha for improved activity , and is one of only a few cases where the efficiency of CO 2 assimilation pathway was improved. Our work demonstrates that R. eutropha is an extremely useful platform for studying and engineering the CBB for applications in more important organisms, such as agricultural crops, and a potential microbial cell factory to develop industrial biotechnology for sequestrating CO 2 .

scholarly journals New Insight into the Role of the Calvin Cycle: Reutilization of CO2 Emitted through Sugar Degradation

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Rie Shimizu ◽  
Yudai Dempo ◽  
Yasumune Nakayama ◽  
Satoshi Nakamura ◽  
Takeshi Bamba ◽  
...  
Keyword(s):  
Ralstonia Eutropha ◽  
Calvin Cycle ◽  
Oxidative Decarboxylation ◽  
Carbon Yield ◽  
Efficient Utilization ◽  
Sugar Degradation ◽  
Biomass Resources ◽  
Insight Into ◽  
Reducing Equivalents

Abstract Ralstonia eutropha is a facultative chemolithoautotrophic bacterium that uses the Calvin–Benson–Bassham (CBB) cycle for CO2 fixation. This study showed that R. eutropha strain H16G incorporated 13CO2, emitted by the oxidative decarboxylation of [1-13C1]-glucose, into key metabolites of the CBB cycle and finally into poly(3-hydroxybutyrate) [P(3HB)] with up to 5.6% 13C abundance. The carbon yield of P(3HB) produced from glucose by the strain H16G was 1.2 times higher than that by the CBB cycle-inactivated mutants, in agreement with the possible fixation of CO2 estimated from the balance of energy and reducing equivalents through sugar degradation integrated with the CBB cycle. The results proved that the ‘gratuitously’ functional CBB cycle in R. eutropha under aerobic heterotrophic conditions participated in the reutilization of CO2 emitted during sugar degradation, leading to an advantage expressed as increased carbon yield of the storage compound. This is a new insight into the role of the CBB cycle and may be applicable for more efficient utilization of biomass resources.

scholarly journals 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

1999 ◽  
Vol 181 (4) ◽  
pp. 1088-1098 ◽  
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.

scholarly journals Engineering the Calvin–Benson–Bassham cycle and hydrogen utilization pathway of Ralstonia eutropha for improved autotrophic growth and polyhydroxybutyrate production

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Zhongkang Li ◽  
Xiuqing Xin ◽  
Bin Xiong ◽  
Dongdong Zhao ◽  
Xueli Zhang ◽  
...  
Keyword(s):  
Carbon Fixation ◽  
Ralstonia Eutropha ◽  
Biological Research ◽  
Autotrophic Growth ◽  
Knock Out ◽  
System A ◽  
Autotrophic Metabolism ◽  
The Difference ◽  
Living Organisms ◽  
Utilization Pathway

Abstract Background CO2 is fixed by all living organisms with an autotrophic metabolism, among which the Calvin–Benson–Bassham (CBB) cycle is the most important and widespread carbon fixation pathway. Thus, studying and engineering the CBB cycle with the associated energy providing pathways to increase the CO2 fixation efficiency of cells is an important subject of biological research with significant application potential. Results In this work, the autotrophic microbe Ralstonia eutropha (Cupriavidus necator) was selected as a research platform for CBB cycle optimization engineering. By knocking out either CBB operon genes on the operon or mega-plasmid of R. eutropha, we found that both CBB operons were active and contributed almost equally to the carbon fixation process. With similar knock-out experiments, we found both soluble and membrane-bound hydrogenases (SH and MBH), belonging to the energy providing hydrogenase module, were functional during autotrophic growth of R. eutropha. SH played a more significant role. By introducing a heterologous cyanobacterial RuBisCO with the endogenous GroES/EL chaperone system(A quality control systems for proteins consisting of molecular chaperones and proteases, which prevent protein aggregation by either refolding or degrading misfolded proteins) and RbcX(A chaperone in the folding of Rubisco), the culture OD600 of engineered strain increased 89.2% after 72 h of autotrophic growth, although the difference was decreased at 96 h, indicating cyanobacterial RuBisCO with a higher activity was functional in R. eutropha and lead to improved growth in comparison to the host specific enzyme. Meanwhile, expression of hydrogenases was optimized by modulating the expression of MBH and SH, which could further increase the R. eutropha H16 culture OD600 to 93.4% at 72 h. Moreover, the autotrophic yield of its major industrially relevant product, polyhydroxybutyrate (PHB), was increased by 99.7%. Conclusions To our best knowledge, this is the first report of successfully engineering the CBB pathway and hydrogenases of R. eutropha for improved activity, and is one of only a few cases where the efficiency of CO2 assimilation pathway was improved. Our work demonstrates that R. eutropha is a useful platform for studying and engineering the CBB for applications.

scholarly journals Autotrophic CO2 Fixation by Chloroflexus aurantiacus: Study of Glyoxylate Formation and Assimilation via the 3-Hydroxypropionate Cycle

2001 ◽  
Vol 183 (14) ◽  
pp. 4305-4316 ◽  
Author(s):  
Sylvia Herter ◽  
Jan Farfsing ◽  
Nasser Gad'On ◽  
Christoph Rieder ◽  
Wolfgang Eisenreich ◽  
...  
Keyword(s):  
Carbon Fixation ◽  
Calvin Cycle ◽  
Carbon Assimilation ◽  
Growth Conditions ◽  
Chloroflexus Aurantiacus ◽  
Acetyl Coa ◽  
Autotrophic Co2 Fixation ◽  
Whole Cells ◽  
Coa Transferase ◽  
Malonyl Coa

ABSTRACT In the facultative autotrophic organism Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium, the Calvin cycle does not appear to be operative in autotrophic carbon assimilation. An alternative cyclic pathway, the 3-hydroxypropionate cycle, has been proposed. In this pathway, acetyl coenzyme A (acetyl-CoA) is assumed to be converted to malate, and two CO2 molecules are thereby fixed. Malyl-CoA is supposed to be cleaved to acetyl-CoA, the starting molecule, and glyoxylate, the carbon fixation product. Malyl-CoA cleavage is shown here to be catalyzed by malyl-CoA lyase; this enzyme activity is induced severalfold in autotrophically grown cells. Malate is converted to malyl-CoA via an inducible CoA transferase with succinyl-CoA as a CoA donor. Some enzyme activities involved in the conversion of malonyl-CoA via 3-hydroxypropionate to propionyl-CoA are also induced under autotrophic growth conditions. So far, no clue as to the first step in glyoxylate assimilation has been obtained. One possibility for the assimilation of glyoxylate involves the conversion of glyoxylate to glycine and the subsequent assimilation of glycine. However, such a pathway does not occur, as shown by labeling of whole cells with [1,2-13C2]glycine. Glycine carbon was incorporated only into glycine, serine, and compounds that contained C1 units derived therefrom and not into other cell compounds.

scholarly journals Insights into Autotrophic Activities and Carbon Flow in Iron-Rich Pelagic Aggregates (Iron Snow)

2021 ◽  
Vol 9 (7) ◽  
pp. 1368
Author(s):  
Qianqian Li ◽  
Rebecca E. Cooper ◽  
Carl-Eric Wegner ◽  
Martin Taubert ◽  
Nico Jehmlich ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Co2 Fixation ◽  
Carbon Fixation ◽  
Stable Isotope Probing ◽  
Acidic Lake ◽  
General Role ◽  
Polysaccharide Biosynthesis ◽  
Iron Oxidizers ◽  
Metabolic Activities ◽  
Chemolithoautotrophic Bacteria

Pelagic aggregates function as biological carbon pumps for transporting fixed organic carbon to sediments. In iron-rich (ferruginous) lakes, photoferrotrophic and chemolithoautotrophic bacteria contribute to CO2 fixation by oxidizing reduced iron, leading to the formation of iron-rich pelagic aggregates (iron snow). The significance of iron oxidizers in carbon fixation, their general role in iron snow functioning and the flow of carbon within iron snow is still unclear. Here, we combined a two-year metatranscriptome analysis of iron snow collected from an acidic lake with protein-based stable isotope probing to determine general metabolic activities and to trace 13CO2 incorporation in iron snow over time under oxic and anoxic conditions. mRNA-derived metatranscriptome of iron snow identified four key players (Leptospirillum, Ferrovum, Acidithrix, Acidiphilium) with relative abundances (59.6–85.7%) encoding ecologically relevant pathways, including carbon fixation and polysaccharide biosynthesis. No transcriptional activity for carbon fixation from archaea or eukaryotes was detected. 13CO2 incorporation studies identified active chemolithoautotroph Ferrovum under both conditions. Only 1.0–5.3% relative 13C abundances were found in heterotrophic Acidiphilium and Acidocella under oxic conditions. These data show that iron oxidizers play an important role in CO2 fixation, but the majority of fixed C will be directly transported to the sediment without feeding heterotrophs in the water column in acidic ferruginous lakes.

scholarly journals Metagenomics-guided analysis of microbial chemolithoautotrophic phosphite oxidation yields evidence of a seventh natural CO2fixation pathway

2017 ◽  
Vol 115 (1) ◽  
pp. E92-E101 ◽  
Author(s):  
Israel A. Figueroa ◽  
Tyler P. Barnum ◽  
Pranav Y. Somasekhar ◽  
Charlotte I. Carlström ◽  
Anna L. Engelbrektson ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
16S Rrna ◽  
Carbon Fixation ◽  
Community Analysis ◽  
Metabolic Model ◽  
Rrna Gene ◽  
Autotrophic Growth ◽  
Anaerobic Wastewater Treatment ◽  
Natural Carbon ◽  
Environmental Relevance

Dissimilatory phosphite oxidation (DPO), a microbial metabolism by which phosphite (HPO32−) is oxidized to phosphate (PO43−), is the most energetically favorable chemotrophic electron-donating process known. Only one DPO organism has been described to date, and little is known about the environmental relevance of this metabolism. In this study, we used 16S rRNA gene community analysis and genome-resolved metagenomics to characterize anaerobic wastewater treatment sludge enrichments performing DPO coupled to CO2reduction. We identified an uncultivated DPO bacterium,CandidatusPhosphitivorax (Ca.P.) anaerolimi strain Phox-21, that belongs to candidate order GW-28 within theDeltaproteobacteria, which has no known cultured isolates. Genes for phosphite oxidation and for CO2reduction to formate were found in the genome ofCa.P. anaerolimi, but it appears to lack any of the known natural carbon fixation pathways. These observations led us to propose a metabolic model for autotrophic growth byCa.P. anaerolimi whereby DPO drives CO2reduction to formate, which is then assimilated into biomass via the reductive glycine pathway.

scholarly journals Design and in vitro realization of carbon-conserving photorespiration

2018 ◽  
Vol 115 (49) ◽  
pp. E11455-E11464 ◽  
Author(s):  
Devin L. Trudeau ◽  
Christian Edlich-Muth ◽  
Jan Zarzycki ◽  
Marieke Scheffen ◽  
Moshe Goldsmith ◽  
...  
Keyword(s):  
Carbon Fixation ◽  
Calvin Cycle ◽  
Computational Design ◽  
Carbon Loss ◽  
Fixation Rate ◽  
Bisphosphate Carboxylase ◽  
Stoichiometric Model ◽  
Synthetic Routes ◽  
Coa Reductase

Photorespiration recycles ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenation product, 2-phosphoglycolate, back into the Calvin Cycle. Natural photorespiration, however, limits agricultural productivity by dissipating energy and releasing CO2. Several photorespiration bypasses have been previously suggested but were limited to existing enzymes and pathways that release CO2. Here, we harness the power of enzyme and metabolic engineering to establish synthetic routes that bypass photorespiration without CO2 release. By defining specific reaction rules, we systematically identified promising routes that assimilate 2-phosphoglycolate into the Calvin Cycle without carbon loss. We further developed a kinetic–stoichiometric model that indicates that the identified synthetic shunts could potentially enhance carbon fixation rate across the physiological range of irradiation and CO2, even if most of their enzymes operate at a tenth of Rubisco’s maximal carboxylation activity. Glycolate reduction to glycolaldehyde is essential for several of the synthetic shunts but is not known to occur naturally. We, therefore, used computational design and directed evolution to establish this activity in two sequential reactions. An acetyl-CoA synthetase was engineered for higher stability and glycolyl-CoA synthesis. A propionyl-CoA reductase was engineered for higher selectivity for glycolyl-CoA and for use of NADPH over NAD+, thereby favoring reduction over oxidation. The engineered glycolate reduction module was then combined with downstream condensation and assimilation of glycolaldehyde to ribulose 1,5-bisphosphate, thus providing proof of principle for a carbon-conserving photorespiration pathway.

A simple dynamic model of photosynthesis in oak leaves: coupling leaf conductance and photosynthetic carbon fixation by a variable intracellular CO2 pool

2004 ◽  
Vol 31 (12) ◽  
pp. 1195 ◽  
Author(s):  
Steffen M. Noe ◽  
Christoph Giersch
Keyword(s):  
Carbon Fixation ◽  
Calvin Cycle ◽  
Target Function ◽  
Vapour Pressure Deficit ◽  
Co2 Assimilation ◽  
Leaf Conductance ◽  
Co2 Partial Pressure ◽  
Sink Term ◽  
Essential Components ◽  
Oak Leaves

Modelling the diurnal course of photosynthesis in oak leaves (Quercus robur L.) requires appropriate description of the dynamics of leaf photosynthesis of which diurnal variations in leaf conductance and in CO2 assimilation are essential components. We propose and analyse a simple photosynthesis model with three variables: leaf conductance (gs), the CO2 partial pressure inside the leaf (pi), and a pool of Calvin cycle intermediates (aps). The environmental factors light (I) and vapour pressure deficit (VPD) are used to formulate a target function G(I, VPD) from which the actual leaf conductance is calculated. Using this gs value and a CO2 consumption term representing CO2 fixation, a differential equation for pi is derived. Carboxylation corresponds to the sink term of the pi pool and is assumed to be feedback-inhibited by aps. This simple model is shown to produce reasonable to excellent fits to data on the diurnal time courses of photosythesis, pi and gs sampled for oak leaves.

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