Fig 1.
A. An abstraction of the cell acting as a dissipative structure looking at the overall chemical reaction in which the nutrients C4H6O5 (malate) and NH3 are turned into biomass, C4H7O2N.
H2 simply represents an environmental reductant. The cycle can operate in either in the reductive (blue) direction and consume reductants such as H2 or in the oxidative direction (green) direction and produce reduced compounds. Whether net oxidation or net reduction occurs in the cell determines the redox poise, represented here by the cell’s ratio of NAD(P)H/NAD(P)+. In net reductive conditions, the odds of NAD(P)H/NAD(P)+ >1, while in net oxidative conditions the odds of NAD(P)H/NAD(P)+ <1. B. Examples of balanced chemical reactions for the reductive process (blue) and the oxidative process (green). Here, the biomass elemental composition, C4H7.07O2.04N0.63, is the average value found in this study.
Table 1.
Carbon oxidation states of compounds used for photoheterotrophic growth compared to the average carbon oxidation state and average biomass formula calculated from across multiple microbes where the carbon oxidation state ZC = (-nH + 2nO + 3nN)/nC [17].
Table 2.
Model redox scenarios for growth on malate (top) and acetate (bottom) with H2 and CO2 production or consumption resulting in biomass of the formula C4H7.07O2.04N0.63.
Whether H2 and CO2 are produced or consumed determines whether the oxidative (green) direction or the reductive (blue) direction is taken in the dissipative cell cycles of Fig 1. In the third column, negative values indicate consumption of malate/acetate or CO2 and positive values indicate production. Redox Condition refers to the net oxidation or reductive environment the cell is in due to available growth substrate and electron donors (e.g. H2) relative to biomass carbon oxidation state.
Table 3.
Primary CO2 assimilation reactions and enzyme catalysts. These reactions are each shown in their metabolic context of the larger model in Fig 2, where they are highlighted in orange boxes.
Fig 2.
Overview of cyclic photophosphorylation by the branched-cyclic Q cycle, hydrogen uptake, and primary and secondary biosynthetic pathways of Rhodospirillum rubrum included in the thermo-kinetic model.
Red dashed arrows indicate the flow of single electrons in the Q cycle unless otherwise noted, and each cytochrome C (Cyt_C) carries each, so two (2) Cyt−C proteins are required. For hydrogen uptake, note the specific redox carrier that interacts with the HupSLM [Ni-Fe] hydrogenase is not known and thus indicated by X/H2X for a general thermodynamically acceptable redox carrier, e.g., NAD(P)+/NAD(P)H. Both the oxidative and reductive versions of the TCA cycle were tested in the modeling. The reductive TCA cycle was used in the final model. The main pathways for CO2 uptake or release are highlighted in orange blocks. Metabolites (alphabetical order): 2PG - 2-phospho-glycerate, 3PG - 3-phospho-glycerate (2 indicates the formation of 2 molecules from RuBP), Ac-CoA - acetyl-CoA,
KG - 2-keto-glutarate, BPG - 1,3-bisphospho-glycerate, Cit - citrate, CCA - citryl-CoA (intermediate of Citrate Synthase), CoA-SH - Coenzyme A, DHAP - dihydroxyacetone phosphate, E4P - erythrose-4-phosphate, F6P - fructose-6-phosphate, Fum - fumarate, G3P - glyceraldehyde-3-phosphate, G6P - glucose-6-phosphate, H4THF – Tetrahydrofolate, Hcy – homocysteine, Isc - isocitrate, LPS - lipopolysaccharide, Mal - malate, MCA - malyl-CoA, MmCA - methylmalyl-CoA, Oxa - oxaloacetate, PCA - propanoyl-CoA, PEP - phosphoenol-pyruvate, PHB - polyhydroxybutyrate, Pyr - pyruvate, R-CH3 - methylated methyl acceptor (R), Q/QH2 - oxidized and
- reduced quinone, R5P - ribose-5-phosphate, Ru5P - ribulose-5-phosphate, RuBP - ribulose-1,5-bisphosphate, SAM – S-adenosyl-methionine, SAH – S-adenosyl-homocysteine, S7P - sedoheptulose-7-phosphate, SBP - sedoheptulose-1,7-bisphosphate, SCA - succinyl-CoA, Suc - succinate, Xu5P - xylulose-5-phosphate,
- a redox carrier. All amino acids are indicated by standard 3-letter code. Enzymes (alphabetical order): ACL - acetate:CoA ligase (AMP-forming), ACON – aconitase, CLY – ATP-independent citrate lyase, CS - Citrate Synthase (with CCA intermediate) Cyt_bc1-ISP - complex of cytchrome b (heme b560 and b566 containing) cytochrome c1 (heme c containing) and Rieske iron sulfur protein (ISP, yellow), Cyt_C - bacterial cytochrome C2 (heme c containing), Eno – enolase, F0F1 - ATP synthasae, FBPA – fructose-1,6-bisphosphate aldolase, FBPase – fructose-1,6-bisphosphotase, FRD – fumarate reductase, FUM – fumarase, GAPDH – glyceraldehyde-3-phosphate dehydrogenase, GPI – glucose-6-phosphate isomerase, HupSLM - Uptake hydrogenase complex of small, large, and medium subunits, IDH – isocitrate dehydrogenase, KGOR – 2-keto-glutarate:ferredoxin oxidoreductase (
-KG synthase), MDH – malate dehydrogenase, NAD-ME – NAD-dependent malic enzyme, ODC – oxaloacetate decarboxylase, PEP-CK – phosphoenol-pyruvate carboxykinase, PFK – phosphofructokinase, PFOR – pyruvate-ferredoxin oxidoreductase (pyruvate synthase), PGK – 3-phosphoglycerate kinase, PGM – 3-phosphoglycerate mutase, PK – pyruvate kinase, PRK – phosphoribulokinase, RC(P870) - Type II photosynthetic reaction center with characteristic P870 pigment for photo-oxidation of
, RPE – ribulose-5-phosphate epimerase, RPI – ribose-5-phosphate isomerase, RubisCO – ribulose-1,5-bisphosphate oxygenase/carboxylase, SBPase – sedoheptulose-1,7-bisphosphate phosphatase, SCS – succinyl-CoA synthetase, TKT – transketolase, TPI – triose-phosphate isomerase.
Table 4.
The top two rows show the correspondence between thermodynamic odds of oxidation of substrate (H2, Eq (24) and the ratio of concentrations of NADP+ to NADPH. The second row shows the actual ratio of concentrations used. These values are held fixed in each simulation. The lower rows show the observed ratio of various redox pairs during each simulation.
Table 5.
(Left) Experimentally measured elemental composition of cell biomass and estimated oxidation state of carbon from the elemental formula.
(Right) Experimentally measured mass ratios of macromolecules relative to DNA. Elemental analysis sources: a elemental composition of R. palustris biomass when grown on malate from Carlozzi and Sacchi [67]; b average elemental composition of Rhodobacter sphaeroides from Waligórska et al. [68], c average elemental composition of Rhodospirillum rubrum growth on acetate from Favier-Teodorescu, et al. [51]; d measurements for R. rubrum in this study.
Table 6.
Predicted elemental biomass composition (bold) estimated from overall chemical reactions for growth on malate.
The color of blue or green for the overall cellular reaction indicates that the cell operates in a reductive or oxidative cycle, respectively, as shown in Fig 1. Estimates were obtained from simulations under oxidizing, neutral and reducing redox conditions, according to the concentrations of NADP+:NADPH being held at the thermodynamic odds (Eq 24) of 1010, and 103, respectively. The rows labeled Simulation are those from the optimization for growth of the metabolic model while those labeled Estimate are estimates of the biomass elemental composition using the stoichiometry found in the model. The grey highlighted rows are for those compounds in the overall chemical equation for the metabolic model that are not used in estimating the elemental composition of the biomass. Abbreviations: ABP: adenine-3,5-bisphosphate; THF: tetrahydrofolate; 5,10-MTHF: 5, 10 methylenetetrahydrofolate; N10-fTHF: N10-formyltetrahydrofolate; Pi,2: diphosphate.
Fig 3.
Plots of growth rate, palmitate (fatty acid synthesis) rate, and rates of CO2 flux at the Ethylmalonyl-CoA pathway, RubisCO, 2-oxoglutarate synthase, isocitrate dehydrogenase, pyruvate ferredoxin oxidoreductase, phosphoenolpyruvate carboxykinase (PEPCK) as a function of the thermodynamic odds () of NADP+:NADPH.
A thermodynamic odds (, Eq (24) of 10−3 indicates that ratio of products to reactants (Q) in the chemical reaction NADPH
NADP+ + H2 is 1000-fold higher compared to the equilibrium ratio (K), assuming that the hydrogen concentration is at the reference value of 1 M in Eq (24).
Table 7.
Predicted elemental biomass composition (bold) estimated from overall chemical reactions for growth on acetate.
The color of blue for the overall cellular reaction indicates that the cell operates in a reductive cycle, as shown in Fig 1. Estimates were obtained from simulations under oxidizing, neutral and reducing redox conditions, according to the concentrations of NADP+:NADPH being held at the thermodynamic odds (Eq 24) of 1010, 107 and 103, respectively. The rows labeled Simulation are those from the optimization for growth of the metabolic model while those labeled Estimate are estimates of the biomass elemental composition using the stoichiometry found in the model. The grey highlighted rows are for those compounds in the overall chemical equation for the metabolic model that are not used in estimating the elemental composition of the biomass. Abbreviations: ABP: adenine-3,5-bisphosphate; THF: tetrahydrofolate; 5,10-MTHF: 5, 10 methylenetetrahydrofolate; N10-fTHF: N10-formyltetrahydrofolate; Pi,2: diphosphate.
Table 8.
Elemental biomass composition (bold) estimated from overall chemical reactions for growth on acetate under varying levels of DNA:RNA:protein (P):fatty acid (FA).
Estimates were obtained from simulations under redox conditions corresponding to the concentrations of NADP+:NADPH being held at the thermodynamic odds (Eq 24) of 107. The rows labeled with DNA:RNA:protein:lipid values are those from the optimization for growth of the metabolic model while those labeled estimate are estimates of the biomass elemental composition using the stoichiometry found in the model, and can be compared to the similar condition shown for acetate growth shown in Table 7 in which the respective levels were 1.0: 2.9: 44.1: 8.5. The grey rows are for those compounds in the overall chemical equation for the metabolic model that are not used in estimating the elemental composition of the biomass. Abbreviations: ABP: adenine-3,5-bisphosphate; THF: tetrahydrofolate; 5,10-MTHF: 5, 10 methylenetetrahydrofolate; N10-fTHF: N10-formyltetrahydrofolate; Pi,2: diphosphate.
Table 9.
Redox states of amino acids and nucleotides. Charge states of atoms were calculated with the Python module OxidationNumberCalculator (https://github.com/Hiwen-STEM/OxidationNumberCalculator). The formula for Nucleotides, RNA, and DNA is for the average 4-mer A(T/U)GC.