CO2 Assimilation Pathway Research Articles

Bacterial conversion of CO2 to organic compounds

Utilization of CO2 by converting it to useful organic compounds is an important strategy to move toward carbon neutrality and sustainable societies. Biological systems including plants and algae have driven conversion of CO2 to diverse organic compounds using their CO2 assimilation pathways and reducing powers. In this article, metabolic pathways and reactions assimilating CO2 and corresponding enzymes are reviewed. In addition, metabolic systems and the sources of energy and electrons for regenerating reducing powers are revisited. Finally, examples of exploiting native and heterologous metabolisms of bacteria for biological conversion of CO2 to food resources and value-added organic compounds are discussed.

Chemical Basis of Carbon Fixation Autotrophic Paleometabolism

On the basis of biomimetic, phylometabolic, and thermodynamic analysis of modern CO2 assimilation pathways, a paleophenotypic reconstruction of ancient autotrophic metabolism systems was carried out. As a chemical basis for CO2 fixation paleometabolism, metabolic networks capable of self-reproduction and evolution are considered, and the reversibility of the transformation reactions of its intermediates is the most important factor in self-development of this network. The substances of the C–H–O system, paragenetically associated with hydrocarbons, create a phase space, which is a set of universal intermediates of the autotrophic paleometabolism chemical network. The concept of two strategies for the origin and development of autotrophic carbon fixation paleometabolism in the oxidized (CO2) and reduced (CH4) redox regimes of degassing of the ancient Earth is proposed. It was shown that P, T, and the redox conditions of hydrothermal systems of the early Archean were favorable for the development of primary methanotrophic metabolism.

Synthetic Formatotrophs for One-Carbon Biorefinery.

The use of CO2 as a carbon source in biorefinery is of great interest, but the low solubility of CO2 in water and the lack of efficient CO2 assimilation pathways are challenges to overcome. Formic acid (FA), which can be easily produced from CO2 and more conveniently stored and transported than CO2, is an attractive CO2‐equivalent carbon source as it can be assimilated more efficiently than CO2 by microorganisms and also provides reducing power. Although there are native formatotrophs, they grow slowly and are difficult to metabolically engineer due to the lack of genetic manipulation tools. Thus, much effort is exerted to develop efficient FA assimilation pathways and synthetic microorganisms capable of growing solely on FA (and CO2). Several innovative strategies are suggested to develop synthetic formatotrophs through rational metabolic engineering involving new enzymes and reconstructed FA assimilation pathways, and/or adaptive laboratory evolution (ALE). In this paper, recent advances in development of synthetic formatotrophs are reviewed, focusing on biological FA and CO2 utilization pathways, enzymes involved and newly developed, and metabolic engineering and ALE strategies employed. Also, future challenges in cultivating formatotrophs to higher cell densities and producing chemicals from FA and CO2 are discussed.

Potassium Alleviates Post-anthesis Photosynthetic Reductions in Winter Wheat Caused by Waterlogging at the Stem Elongation Stage.

Waterlogging occurs frequently at the stem elongation stage of wheat in southern China, decreasing post-anthesis photosynthetic rates and constraining grain filling. This phenomenon, and the mitigating effect of nutrient application, should be investigated as it could lead to improved agronomic guidelines. We exposed pot-cultured wheat plants at the stem elongation stage to waterlogging treatment in combination with two rates of potassium (K) application. Waterlogging treatment resulted in grain yield losses, which we attributed to a reduction in the 1,000-grain weight caused by an early decline in the net photosynthetic rate (Pn) post-anthesis. These decreases were offset by increasing K application. Stomatal conductance (Gs) and the intercellular CO2 concentration (Ci) decreased in the period 7–21 days after anthesis (DAA), and these reductions were exacerbated by waterlogging. However, in the period 21–28 DAA, Gs and Ci increased, while Pn decreased continuously, suggesting that non-stomatal factors constrained photosynthesis. On DAA 21, Pn was reduced by waterlogging, but photochemical efficiency (ΦPSII) remained unchanged, indicating a reduction in the dissipation of energy captured by photosystem II (PSII) through the CO2 assimilation pathway. This reduction in energy dissipation increased the risk of photodamage, as shown by early reductions in ΦPSII in waterlogged plants on DAA 28. However, increased K application promoted root growth and nutrient status under waterlogging, thereby improving photosynthesis post-anthesis. In conclusion, the decrease in Pn caused by waterlogging was attributable to stomatal closure during early senescence; during later senescence, a reduction in CO2 assimilation accounted for the reduced Pn and elevated the risk of photodamage. However, K application mitigated waterlogging-accelerated photosynthetic reductions and reduced yield losses.

Engineering the Calvin\u2013Benson\u2013Bassham cycle and hydrogen utilization pathway of Ralstonia eutropha for improved autotrophic growth and polyhydroxybutyrate production

BackgroundCO2 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.ResultsIn 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%.ConclusionsTo 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.

Escherichia coli is engineered to grow on CO2 and formic acid.

We engineered Escherichia coli to grow on CO2 and formic acid alone by introducing the synthetic CO2 and formic acid assimilation pathway, expressing two formate dehydrogenase genes, fine-tuning metabolic fluxes and optimizing the levels of cytochrome bo3 and bd-I ubiquinol oxidase. Our engineered strain can grow to an optical density at 600 nm of 7.38 in 450 h, and shows promise as a platform strain growing on CO2 and formic acid alone.

Spatiotemporal dynamics of different CO2 fixation strategies used by prokaryotes in a dimictic lake

The Calvin-Benson-Bassham (CBB) cycle and the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle are two inorganic carbon assimilation pathways widely used by prokaryotic autotrophs in lakes. We investigated the effect of mixing periods and stable water stratification patterns on the trajectories of both CO2 fixation strategies in a dimictic lake (Piburger See), because information on the spatiotemporal dynamics of prokaryotes using these pathways in freshwater ecosystems is far from complete. Based on a quantitative approach (droplet digital PCR) of genes coding for key enzymes in different CO2 assimilation pathways, nine depths covering the entire water column were investigated on a monthly basis for one year. Our data show that the abundance of photoautotrophs and obligate chemolithoautotrophs preferentially using form IA RubisCO was determined by seasonal variations. Highest numbers were observed in summer, while a strong decline of prokrayotes using RubisCO form IA was measured between December and May, the period where the lake was mostly covered by ice. The spatiotemporal distribution patterns of genes coding for RubisCO form IC genes, an enzyme usually used by facultative autotrophs for CO2 assimilation, were less pronounced. Bacteria harboring RubisCO form II were dominating the oxygen limited hypolimnion, while nitrifying Thaumarchaeota using the HP/HB cycle were of minor importance in the lake. Our data reveal that the seasonal heterogeneity, which is determined by the dimictic thermal regime of the lake, results in pronounced spatiotemporal changes of different CO2 assimilation pathways with depth-dependent environmental parameters as key factors for their distribution.

Long-term drought resistance in rice (Oryza sativa L.) during leaf senescence: a photosynthetic view

Drought is one of the major abiotic stress that limits crops yield. Here we investigated photosynthetic performance in flag leaves of a hybrid rice (Oryza sativa. L) LYPJ exposed to prolonged drought stress during leaf senescence. Our results highlighted an architecture remodeling of thylakoid membrane proteins characterizing by amplified PSI-LHCII and PSII dimers supercomplexes and LHCII assemblies played crucial role in drought resistance. Coordination of PSII and LHCII protein phosphorylation, thermal dissipation, and CEF around PSI sustained photo-equilibrium when LEF was suppressed by decreased Cytb6f complex. CO2 limitation resulted in degradation of Rubisco content and loss of Rubisco activity, but up-regulated the enzymes responsible for C4 photosynthesis pathway. It was collectively pointed to that redox poise was a pivotal and ATP/NADPH ratio was probably a linker of light reaction, CO2 assimilation and other metabolism pathways. In general, our results provided additional insights into the role of thylakoid membrane plasticity in long-term ambient acclimation and complex coordination of drought responses in rice LYPJ during the senescence stage.

Photosynthesis—European Congress on Photosynthesis Research

Photosynthesis—European Congress on Photosynthesis Research

Autotrophic carbon fixation strategies used by nitrifying prokaryotes in freshwater lakes.

ABSTRACTNiche specialization of nitrifying prokaryotes is usually studied with tools targeting molecules involved in the oxidation of ammonia and nitrite. The ecological significance of diverse CO2 fixation strategies used by nitrifiers is, however, mostly unexplored. By analyzing autotrophy-related genes in combination with amoA marker genes based on droplet digitial PCR and CARD-FISH counts targeting rRNA, we quantified the distribution of nitrifiers in eight stratified lakes. Ammonia oxidizing (AO) Thaumarchaeota using the 3-hydroxypropionate/4-hydroxybutyrate pathway dominated deep and oligotrophic lakes, whereas Nitrosomonas-related taxa employing the Calvin cycle were important AO bacteria in smaller lakes. The occurrence of nitrite oxidizing Nitrospira, assimilating CO2 with the reductive TCA cycle, was strongly correlated with the distribution of Thaumarchaeota. Recently discovered complete ammonia-oxidizing bacteria (comammox) belonging to Nitrospira accounted only for a very small fraction of ammonia oxidizers (AOs) present at the study sites. Altogether, this study gives a first insight on how physicochemical characteristics in lakes are associated to the distribution of nitrifying prokaryotes with different CO2 fixation strategies. Our investigations also evaluate the suitability of functional genes associated with individual CO2 assimilation pathways to study niche preferences of different guilds of nitrifying microorganisms based on an autotrophic perspective.

Improving Succinate Productivity by Engineering a Cyanobacterial CO2 Concentrating System (CCM) in Escherichia coli

Biologically fixation of CO2 has great potential as a significant carbon source for biosynthesis, which is also a major way to reduce CO2 accumulation in atmosphere. Phosphoenolpyruvate (PEP) carboxylation is the key step of anaerobic succinate production in Escherichia coli. In this reaction, one mole CO2 is assimilated with PEP to form oxaloacetate by PEP carboxykinase (PCK). The preferred substrate of PCK is CO2 , which is very limited in cytoplasm. In this study, the carbon concentration mechanism (CCM) of cyanobacteria was introduced into Escherichia coli to enhance the intracellular inorganic carbon concentration for improving carboxylation velocity. Overexpression of the bicarbonate transporter (BT) or carbonic anhydrase (CA) gene from Synechococcus sp. PCC7002 led to a 22 or 35% increase in succinate titer at 36 h, respectively. The carboxylation rate of PCK increased from 2.46 to 3.92 µmol min-1 mg-1 protein by overexpression of the CA gene. In addition, co-overexpression of BT and CA genes had a synergetic effect, leading to a 44% increase in succinate titer at 36 h. This work is the first attempt to increase carbon fixation involved in microbial biosynthesis by engineering a biological CO2 delivery system, which provides new direction and strategies for improving industrial fermentations based on biological CO2 assimilation pathways.

Biochemical fossils of the ancient transition from geoenergetics to bioenergetics in prokaryotic one carbon compound metabolism

The deep dichotomy of archaea and bacteria is evident in many basic traits including ribosomal protein composition, membrane lipid synthesis, cell wall constituents, and flagellar composition. Here we explore that deep dichotomy further by examining the distribution of genes for the synthesis of the central carriers of one carbon units, tetrahydrofolate (H4F) and tetrahydromethanopterin (H4MPT), in bacteria and archaea. The enzymes underlying those distinct biosynthetic routes are broadly unrelated across the bacterial–archaeal divide, indicating that the corresponding pathways arose independently. That deep divergence in one carbon metabolism is mirrored in the structurally unrelated enzymes and different organic cofactors that methanogens (archaea) and acetogens (bacteria) use to perform methyl synthesis in their H4F- and H4MPT-dependent versions, respectively, of the acetyl-CoA pathway. By contrast, acetyl synthesis in the acetyl-CoA pathway — from a methyl group, CO2 and reduced ferredoxin — is simpler, uniform and conserved across acetogens and methanogens, and involves only transition metals as catalysts. The data suggest that the acetyl-CoA pathway, while being the most ancient of known CO2 assimilation pathways, reflects two phases in early evolution: an ancient phase in a geochemically confined and non-free-living universal common ancestor, in which acetyl thioester synthesis proceeded spontaneously with the help of geochemically supplied methyl groups, and a later phase that reflects the primordial divergence of the bacterial and archaeal stem groups, which independently invented genetically-encoded means to synthesize methyl groups via enzymatic reactions. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Carbon metabolic pathways in phototrophic bacteria and their broader evolutionary implications.

Photosynthesis is the biological process that converts solar energy to biomass, bio-products, and biofuel. It is the only major natural solar energy storage mechanism on Earth. To satisfy the increased demand for sustainable energy sources and identify the mechanism of photosynthetic carbon assimilation, which is one of the bottlenecks in photosynthesis, it is essential to understand the process of solar energy storage and associated carbon metabolism in photosynthetic organisms. Researchers have employed physiological studies, microbiological chemistry, enzyme assays, genome sequencing, transcriptomics, and 13C-based metabolomics/fluxomics to investigate central carbon metabolism and enzymes that operate in phototrophs. In this report, we review diverse CO2 assimilation pathways, acetate assimilation, carbohydrate catabolism, the tricarboxylic acid cycle and some key, and/or unconventional enzymes in central carbon metabolism of phototrophic microorganisms. We also discuss the reducing equivalent flow during photoautotrophic and photoheterotrophic growth, evolutionary links in the central carbon metabolic network, and correlations between photosynthetic and non-photosynthetic organisms. Considering the metabolic versatility in these fascinating and diverse photosynthetic bacteria, many essential questions in their central carbon metabolism still remain to be addressed.

Remodeling of Light-Harvesting Protein Complexes in Chlamydomonas in Response to Environmental Changes

Photosynthesis is dependent on light. Photosynthetic organisms struggle their entire lives to optimize photosynthetic function and minimize photooxidative damage in response to light quantity and quality. When the absorbed light energy exceeds the capacity of photosynthetic energy consumption, overreduction of electron transport carriers and accumulation of excitation energy in the light-harvesting-antennae may occur. The latter favors the production of excited triplet-state chlorophyll molecules ( 3 Chl) that can interact with O2 to cause the formation of reactive singlet oxygen ( 1 O2). The former favors the direct reduction of O2 by photosystem I (PSI) and the subsequent generation of reactive oxygen species, such as superoxide (O2 ), hydrogen peroxide (H2O2), and the hydroxyl radical (OH) (5, 6, 75). These reactive oxygen species are able to cause photo-oxidative damage to photosystem II (PSII), a primary target for photoinhibition (2, 4, 9), but also to PSI (59, 60), in particular under weak light at chilled temperatures (111, 121). Thus, adaptation mechanisms that balance the energy input, through photochemistry via the photosynthetic machinery, with the energy output through CO2 assimilation and other metabolic pathways, are essential for plant survival. The function of the light-harvesting complexes (LHCs) is, as the name implies, to increase the effective absorption crosssection of the photosystems and to supply them with excitation energy. The LHCs bind chlorophyll a and b but lack any photochemical activity of their own. Both PSI and PSII have their own core antenna pigments, but the addition of the LHCs increases the number of antenna pigments connected to each reaction center by a factor of 2 to 4, depending upon the conditions (47). Energy transfer operates in a time scale of femtoseconds to picoseconds. The transfer time of excitation energy between neighboring Chl molecules in the antenna has been estimated to be 100 to 300 fs (45). It had long been known that the pigment molecules must be closely packed to allow such fast and efficient transfer, a supposition beautifully confirmed by the emergence of the X-ray crystal structures of PSI (65), PSII (42, 66, 135), and LHCI-PSI (12) in the last several years (see discussion below). On average, excitation energy makes 100 to 1,000 such hops between antenna chlorophylls before it is trapped at a reaction center. Depending upon the arrangement of pigment molecules within LHCs and of LHCs vis-a `-vis the photosystems, excitation energy can be directed to specific photosystems, temporarily trapped on low-energy chlorophylls, or even converted to heat by “nonphotochemical quenching.” It does not require a great stretch of imagination to see how evolution might have taken advantage of such possibilities for the development of regulatory mechanisms to deal with variable illumination. The regulation of light energy input and distribution, by the dynamic regulation of the light-harvesting system, probably plays the most important role in balancing the light and dark reactions of photosynthesis. Light-harvesting systems and their functioning in algae and vascular plants have been addressed

Physiological implications of the kinetics of maize leaf phosphoenolpyruvate carboxylase.

It has been a common practice to assay phosphoenolpyruvate carboxylase (PEPC) under high, nonphysiological concentrations of Mg(2+) and bicarbonate. We have performed kinetic studies on the enzyme from maize (Zea mays) leaves at near physiological levels of free Mg(2+) (0.4 mM) and bicarbonate (0.1 mM), and found that both the nonphosphorylated and phosphorylated enzymes exhibited a high degree of cooperativity in the binding of phosphoenolpyruvate, a much lower affinity for this substrate and for activators, and a greater affinity for malate than at high concentrations of these ions. Inhibition of the phosphorylated enzyme by malate was overcome by glycine or alanine but not by glucose-6-phosphate, either in the absence or presence of high concentrations of glycerol, a compatible solute. Alanine caused significant activation at physiological concentrations, suggesting a pivotal role for this amino acid in regulating maize leaf PEPC activity. Our results showed that the maximum enzyme activity attainable in vivo would be less than 50% of that attainable in vitro under optimum conditions. Therefore, the high levels of PEPC protein in the cytosol of C(4) mesophyll cells might be an adaptation for sustaining the steady-state rate of flux through the photosynthetic CO(2) assimilation pathway despite the limitations imposed by the PEPC kinetic properties and the conditions of its environment.

A reverse KREBS cycle in photosynthesis: consensus at last

The Krebs cycle (citric acid or tricarboxylic acid cycle), the final common pathway in aerobic metabolism for the oxidation of carbohydrates, fatty acids and amino acids, is known to be irreversible. It liberates CO2 and generates NADH whose aerobic oxidation yields ATP but it does not operate in reverse as a biosynthetic pathway for CO2 assimilation. In 1966, our laboratory described a cyclic pathway for CO2 assimilation (Evans, Buchanan and Arnon 1966) that was unusual in two respects: (i) it provided the first instance of an obligate photoautotroph that assimilated CO2 by a pathway different from Calvin's reductive pentose phosphate cycle (Calvin 1962) and (ii) in its overall effect the new cycle was a reversal of the Krebs cycle. Named the 'reductive carboxylic acid cycle' (sometimes also called the reductive tricarboxylic acid cycle) the new cycle appeared to be the sole CO2 assimilation pathway in Chlorobium thiosulfatophilum (Evans et al. 1966) (now known as Chlorobium limicola forma thiosulfatophilum). Chlorobium is a photosynthetic green sulfur bacterium that grows anaerobically in an inorganic medium with sulfide and thiosulfate as electron donors and CO2 as an obligatory carbon source. In the ensuing years, the new cycle was viewed with skepticism. Not only was it in conflict with the prevailing doctrine that the 'one important property ... shared by all (our emphasis) autotrophic species is the assimilation of CO2 via the Calvin cycle' (McFadden 1973) but also some of its experimental underpinnings were challenged. It is only now that in the words of one of its early skeptics (Tabita 1988) 'a long and tortuous controversy' has ended with general acceptance of the reductive carboxylic acid cycle as a photosynthetic CO2 assimilation pathway distinct from the pentose cycle. (Henceforth, to minimize repetitiveness, the reductive pentose phosphate cycle will often be referred to as the pentose cycle and the reductive carboxylic acid cycle as the carboxylic acid cycle.) Aside from photosynthetic pathways which are the focus of this article, CO2 assimilation is also known to sustain autotrophic growth via the acetyl-CoA pathway (Wood et al. 1986). Our aim here is to discuss (i) the findings that led our group to the discovery of the reductive carboxylic acid cycle, (ii) the nature and resolution of the controversy that followed, and (iii) the possible evolutionary implications of the cycle as an ancient mechanism for photosynthetic CO2 assimilation that preceded the pentose cycle and served as a precursor of the Krebs cycle in aerobic metabolism.

Carbon assimilation pathways in sulfate-reducing bacteria II. Enzymes of a reductive citric acid cycle in the autotrophic Desulfobacter hydrogenophilus

The strict anaerobe Desulfobacter hydrogenophilus is able to grow autotrophically with CO2, H2, and sulfate as sole carbon and energy sources. The generation time at 30°C under autotrophic conditions in a pure mineral medium was 15 h, the growth yield was 8 g cell dry mass per mol sulfate reduced to H2S. Enzymes of the autotrophic CO2 assimilation pathway were investigated. Key enzymes of the Calvin cycle and of the acetyl CoA pathway could not be found. All enzymes of a reductive citric acid cycle were present at specific activities sufficient to account for the observed growth rate. Notably, an ATP-citrate lyase (1.3 μmol · min-1 · mg cell protein-1) was present both in autotrophically and in heterotrophically grown cells, which was rapidly inactivated in the absence of ATP. The data indicate that in D. hydrogenophilus a reductive citric acid cycle is operating in autotrophic CO2 fixation. Since other autotrophic sulfate reducers possess an acetyl CoA pathway for CO2 fixation, two different autotrophic pathways occur in the same physiological group.

The inhibition of photosynthesis and photorespiration in isolated mesophyll cells of Phaseolus and Lycopersicon by reduced osmotic potentials

Mesophyll cells isolated from Phaseolus vulgaris and Lycopersicon esculentum show decreasing photosynthetic rates when suspended in media containing increasing concentrations of osmoticum. The photosynthetic activity was sensitive to small changes in osmotic potential over a range of sorbitol concentrations from 0.44 M (−1.08 MPa) to 0.77 M (−1.88 MPa). Photorespiration assayed by 14CO2 release in CO2‐free air and by 14CO2 release from the oxidation of [1–14C] glycolate also decreased as the osmotic potential of the incubation medium was reduced. The CO2 compensation points of the cells increased with increasing concentration of osmoticum from approximately 60 μ I−11 at −1.08 MPa to 130 μl 1−1 for cells stressed at −1.88 MPa. Changes in photosynthetic and photorespiratory activities occurred at moderate osmotic potentials in these cells suggesting that in whole leaves during a reduction in water potential, non‐ stomatal inhibition of CO2 assimilation and glycolate pathway metabolism occurs simultaneously with stomatal closure.

Phosphoenolpyruvate synthetase inMethanobacterium thermoautotrophicum

The thermophilic autotrophMethanobacterium thermoautotrophicum assimilates CO2 via a novel pathway rather than via the Calvin cycle. The central intermediate of this pathway is acetyl CoA which is reductively carboxylated to pyruvate. Cell extracts of the organism contained phosphoenolpyruvate synthetase with a specific activity of 100 nmol min-1 mg-1 protein (65°C). Pyruvate kinase and pyruvate, phosphate dikinase were not detected. Phosphoenolpyruvate synthetase was partially purified (50-fold) and the following reaction stoichiometry was established: $${\text{Pyruvate + ATP + H}}_{\text{2}} {\text{O }} \to {\text{ Phosphoenolpyruvate + AMP + P}}_{\text{i}} $$ The enzyme activity was depedent on free Mg2+ ions, NH 4 + or K+ ions, and SH-groups. Mn2+, but not Ca2+, could partially substitute for Mg2+; Na+ could not substitute for K+ or NH 4 + . The pH-optima,V max-values and the apparentK M-values for the substrates of the enzyme in both directions were determined. Thermodynamic, kinetic and regulatory features indicate that, in vivo, the enzyme functions in the direction of phosphoenolpyruvate synthesis from pyruvate. Not only is the synthesis of phosphoenolpyruvate via the PEP synthetase reaction energetically favorable; the enzyme also catalyzed this synthesis 100 times faster than the reverse reaction, the apparentK M value for pyruvate (40 μM) being low and the apparentK M value for phosphate (100 mM) being high. Furthermore, AMP, ADP, PP and α-ketoglutarate were inhibitors of PEP synthesis, indicating that the enzyme activity may be controlled in vivo. The role of phosphoenolpyruvate synthetase in autotrophic CO2 assimilation pathway ofMethanobacterium, as expected from previous labelling studies, is confirmed.

Photosynthetic Capacities and Growth Characteristics of Nicotiana tabacum (cv. Xanthi) Cell Suspension Cultures

AbstractPhotoheterotrophic growth of cell suspensions of Nicotiana tabacum L. (cv. Xanthi) in organic culture medium enriched in sucrose (30 g per liter) showed a classical sigmoid growth curve. The cells developed functional chloroplast structures during the exponential growth phase, when their chlorophyll content increased steadily. A limited drop (30%) in the chlorophyll amount and structural changes of the plastids (starch accumulation) were observed during the lag phase. The measurements of photosynthetic capacities (O2 evolution and CO2 fixation) during the growth cycle revealed changes in the photosynthetic ratio (O2/CO2), which was near 1 during the lag and stationary phases and near 2 during exponential growth. During exponential growth there was also a rapid NO3− uptake. Analysis of label distribution among the products of 14CO2 fixation showed that both CO2 assimilation pathways, linked to the ribulose‐biphosphate carboxylase (the autotrophic pathway) and to phosphoenolpyruvate carboxylase (the non‐autotrophic pathway) were operative with an important increase of the capacity of the latter during the exponential growth phase. Maximum rate of oxygen evolution, either endogenous or with p‐benzoquinone as Hill reagent, as well as the increased CO2 Fixation capacity via the non‐autotrophic pathway during the exponential phase were concomitant with a high cyanide inhibited O2 uptake.