Flux balance analysis of photoautotrophic metabolism

Photosynthesis is the principal process responsible for fixation of inorganic carbon dioxide into organic molecules with sunlight as the energy source. Potentially, many chemicals could be inexpensively produced by photosynthetic organisms. Mathematical modeling of photoautotrophic metabolism is therefore important to evaluate maximum theoretical product yields and to deeply understand the interactions between biochemical energy, carbon fixation, and assimilation pathways. Flux balance analysis based on linear programming is applied to photoautotrophic metabolism. The stoichiometric network of a model photosynthetic prokaryote, Synechocystis sp. PCC 6803, has been reconstructed from genomic data and biochemical literature and coupled with a model of the photophosphorylation processes. Flux map topologies for the hetero-, auto-, and mixotrophic modes of metabolism under conditions of optimal growth were determined and compared. The roles of important metabolic reactions such as the glyoxylate shunt and the transhydrogenase reaction were analyzed. We also theoretically evaluated the effect of gene deletions or additions on biomass yield and metabolic flux distributions.     

A survey of carbon fixation pathways through a quantitative lens

While the reductive pentose phosphate cycle is responsible for the fixation of most of the carbon in the biosphere, it has several natural substitutes. In fact, due to the characterization of three new carbon fixation pathways in the last decade, the diversity of known metabolic solutions for autotrophic growth has doubled. In this review, the different pathways are analysed and compared according to various criteria, trying to connect each of the different metabolic alternatives to suitable environments or metabolic goals. The different roles of carbon fixation are discussed; in addition to sustaining autotrophic growth it can also be used for energy conservation and as an electron sink for the recycling of reduced electron carriers. Our main focus in this review is on thermodynamic and kinetic aspects, including thermodynamically challenging reactions, the ATP requirement of each pathway, energetic constraints on carbon fixation, and factors that are expected to limit the rate of the pathways. Finally, possible metabolic structures of yet unknown carbon fixation pathways are suggested and discussed.     

Energetics and carbon metabolism during growth of microalgal cells under photoautotrophic, mixotrophic and cyclic light-autotrophic/dark-heterotrophic conditions

Chlorella pyrenoidosa was cultivated under photoautotrophic, mixotrophic and cyclic light-autotrophic/dark-heterotrophic conditions. The influence of light on the carbon and energy metabolism of microalgae was investigated by the use of metabolic flux analysis. The respiratory activity of microalgae in the light was assessed from the autotrophic flux distribution. Results showed that the glycolytic pathway, tricarboxylic acid cycle and mitochondrial oxidative phosphorylation maintained high activities during illumination, indicating little effect of light on these pathways, while the flux through the pentose phosphate pathway during illumination was very small due to the light-mediated regulation. The theoretical yields of biomass on ATP decreased in the following order: heterotrophic culture>mixotrophic culture>autotrophic culture, and a significant amount of the available ATP was required for maintenance processes in microalgal cells. The energy conversion efficiency between the supplied energy to culture, the absorbed energy by cells and the free energy conserved in ATP were analyzed for the different cultures. Analysis showed that the heterotrophic culture generated more ATP from the supplied energy than the autotrophic and mixotrophic cultures. The maximum thermodynamic efficiency of ATP production from the absorbed energy, which was calculated from the metabolic fluxes at zero growth rate, was the highest in the heterotrophic culture and as low as 16% in the autotrophic culture. By evaluating the energy economy through the energy utilization efficiency, it was found that the biomass yield on the supplied energy was the lowest in the autotrophic cultivation, and the cyclic culture gave the most efficient utilization of energy for biomass production.     

Thermodynamic constraints shape the structure of carbon fixation pathways

Thermodynamics impose a major constraint on the structure of metabolic pathways. Here, we use carbon fixation pathways to demonstrate how thermodynamics shape the structure of pathways and determine the cellular resources they consume. We analyze the energetic profile of prototypical reactions and show that each reaction type displays a characteristic change in Gibbs energy. Specifically, although carbon fixation pathways display a considerable structural variability, they are all energetically constrained by two types of reactions: carboxylation and carboxyl reduction. In fact, all adenosine triphosphate (ATP) molecules consumed by carbon fixation pathways - with a single exception - are used, directly or indirectly, to power one of these unfavorable reactions. When an indirect coupling is employed, the energy released by ATP hydrolysis is used to establish another chemical bond with high energy of hydrolysis, e.g. a thioester. This bond is cleaved by a downstream enzyme to energize an unfavorable reaction. Notably, many pathways exhibit reduced ATP requirement as they couple unfavorable carboxylation or carboxyl reduction reactions to exergonic reactions other than ATP hydrolysis. In the most extreme example, the reductive acetyl coenzyme A (acetyl-CoA) pathway bypasses almost all ATP-consuming reactions. On the other hand, the reductive pentose phosphate pathway appears to be the least ATP-efficient because it is the only carbon fixation pathway that invests ATP in metabolic aims other than carboxylation and carboxyl reduction. Altogether, our analysis indicates that basic thermodynamic considerations accurately predict the resource investment required to support a metabolic pathway and further identifies biochemical mechanisms that can decrease this requirement.     

The emergence and early evolution of biological carbon-fixation

The fixation of into living matter sustains all life on Earth, and embeds the biosphere within geochemistry. The six known chemical pathways used by extant organisms for this function are recognized to have overlaps, but their evolution is incompletely understood. Here we reconstruct the complete early evolutionary history of biological carbon-fixation, relating all modern pathways to a single ancestral form. We find that innovations in carbon-fixation were the foundation for most major early divergences in the tree of life. These findings are based on a novel method that fully integrates metabolic and phylogenetic constraints. Comparing gene-profiles across the metabolic cores of deep-branching organisms and requiring that they are capable of synthesizing all their biomass components leads to the surprising conclusion that the most common form for deep-branching autotrophic carbon-fixation combines two disconnected sub-networks, each supplying carbon to distinct biomass components. One of these is a linear folate-based pathway of reduction previously only recognized as a fixation route in the complete Wood-Ljungdahl pathway, but which more generally may exclude the final step of synthesizing acetyl-CoA. Using metabolic constraints we then reconstruct a “phylometabolic” tree with a high degree of parsimony that traces the evolution of complete carbon-fixation pathways, and has a clear structure down to the root. This tree requires few instances of lateral gene transfer or convergence, and instead suggests a simple evolutionary dynamic in which all divergences have primary environmental causes. Energy optimization and oxygen toxicity are the two strongest forces of selection. The root of this tree combines the reductive citric acid cycle and the Wood-Ljungdahl pathway into a single connected network. This linked network lacks the selective optimization of modern fixation pathways but its redundancy leads to a more robust topology, making it more plausible than any modern pathway as a primitive universal ancestral form.     

聚球藻7942光自养培养的碳代谢和能量代谢

代谢通量分析是研究微藻光自养培养过程中CO2和光能利用的一个非常有用的工具。本研究建立了聚球藻7942光自养培养代谢网络,并通过代谢通量方法分析了不同入射光强下的碳代谢流分布和能量代谢。研究结果表明,CO2固定是代谢能量和还原力消耗的主要途径,分别约占总消耗能量的85%和70%。研究还发现在一定光强范围,基于ATP生成的细胞得率和最大细胞得率基本维持不变,分别约为2.80g/molATP和2.95g/molATP,但基于总吸收光能的细胞得率和对应的光能转换效率则随着光强的增加而降低。     

Evidence for autotrophic CO2 fixation via the reductive tricarboxylic acid cycle by members of the epsilon subdivision of proteobacteria

Based on 16S rRNA gene surveys, bacteria of the epsilon subdivision of proteobacteria have been identified to be important members of microbial communities in a variety of environments, and quite a few have been demonstrated to grow autotrophically. However, no information exists on what pathway of autotrophic carbon fixation these bacteria might use. In this study, Thiomicrospira denitrificans and Candidatus Arcobacter sulfidicus, two chemolithoautotrophic sulfur oxidizers of the epsilon subdivision of proteobacteria, were examined for activities of the key enzymes of the known autotrophic CO(2) fixation pathways. Both organisms contained activities of the key enzymes of the reductive tricarboxylic acid cycle, ATP citrate lyase, 2-oxoglutarate:ferredoxin oxidoreductase, and pyruvate:ferredoxin oxidoreductase. Furthermore, no activities of key enzymes of other CO(2) fixation pathways, such as the Calvin cycle, the reductive acetyl coenzyme A pathway, and the 3-hydroxypropionate cycle, could be detected. In addition to the key enzymes, the activities of the other enzymes involved in the reductive tricarboxylic acid cycle could be measured. Sections of the genes encoding the alpha- and beta-subunits of ATP citrate lyase could be amplified from both organisms. These findings represent the first direct evidence for the operation of the reductive tricarboxylic acid cycle for autotrophic CO(2) fixation in epsilon-proteobacteria. Since epsilon-proteobacteria closely related to these two organisms are important in many habitats, such as hydrothermal vents, oxic-sulfidic interfaces, or oilfields, these results suggest that autotrophic CO(2) fixation via the reductive tricarboxylic acid cycle might be more important than previously considered.     

Recreating ancient metabolic pathways before enzymes

The biochemistry of all living organisms uses complex, enzyme-catalyzed metabolic reaction networks. Yet, at life’s origins, enzymes had not yet evolved. Therefore, it has been postulated that non-enzymatic metabolic pathways predated their enzymatic counterparts. In this account article, we describe our recent work to evaluate whether two ancient carbon fixation pathways, the rTCA (reductive tricarboxylic acid) cycle and the reductive AcCoA (Wood-Ljungdahl) pathway, could have operated without enzymes and therefore have originated as prebiotic chemistry. We also describe the discovery of an Fe²⁺-promoted complex reaction network that may represent a prebiotic predecessor to the TCA and glyoxylate cycles. The collective results support the idea that most central metabolic pathways could have roots in prebiotic chemistry.     

蓝细菌固碳合成乳酸技术的发展与展望

乳酸是一种重要的工业化学品,被广泛应用于各个行业。近年来,随着聚乳酸(PLA)市场的兴起,乳酸原料的需求也在不断增加。糖基异养生产乳酸所带来的高昂成本与市场需求的矛盾吸引着研究人员积极寻找其他有利的解决方案。蓝细菌光合固碳生产乳酸是一种潜力巨大的新型原料供应策略,基于光合自养的细胞工厂,可以在单一平台上以太阳能为驱动力,从二氧化碳中直接生产出高光学纯度的乳酸。该方法原料廉价易得、过程简单可控、产物明确且易分离,同时达到节能减排和高附加值产品生产的双重效果,具有重要的研究与应用价值。文中回顾了蓝细菌固碳产乳酸技术的发展历程,从代谢基础、代谢工程策略、代谢动力学分析与技术应用等方面,梳理其研究进展和所遇到的技术难点,并对该技术的未来进行展望。     

Developments and perspectives of photobioreactors for biofuel production

The production of biofuels from microalgae requires efficient photobioreactors in order to meet the tight constraints of energy efficiency and economic profitability. Current cultivation systems are designed for high-value products rather than for mass production of cheap energy carriers. Future bioreactors will imply innovative solutions in terms of energy efficiency, light and gas transfer or attainable biomass concentration to lower the energy demand and cut down production costs. A new generation of highly developed reactor designs demonstrates the enormous potential of photobioreactors. However, a net energy production with microalgae remains challenging. Therefore, it is essential to review all aspects and production steps for optimization potential. This includes a custom process design according to production organism, desired product and production site. Moreover, the potential of microalgae to synthesize valuable products additionally to the energetic use can be integrated into a production concept as well as waste streams for carbon supply or temperature control.     

Photosynthesis in Rhodospirillum rubrum. II. Photoheterotrophic Carbon Dioxide Fixation

The contribution of the reductive pentose phosphate cycle to the photometabolism of carbon dioxide and to carbon metabolism in Rhodospirillum rubrum grown photoheterotrophically with l-malate as the carbon source is nil, unlike autotrophically grown R. rubrum. Glycolic acid appears to be the first stable product of CO(2) fixation in R. rubrum cultured photoheterotrophically on l-malate. The results obtained in (14)CO(2) fixation experiments suggest that the photometabolism of CO(2) through glycolate into malate is a major pathway of CO(2) fixation in such cells. However, l-malate was a much more efficient precursor of phosphate esters, and of glutamic acid, than was carbon dioxide; l-malate is therefore, in this case, a far more important source of cell carbon than is carbon dioxide.The products of the light-dependent incorporation of CO(2) and of acetate were investigated in R. rubrum grown photoheterotrophically on acetate. Carboxylation reactions and the reductive pentose phosphate cycle are apparently of greater significance in the photometabolism of acetate heterotrophs than in malate heterotrophs; the photometabolism of the acetate photoheterotrophs seems to be intermediate between the photoheterotrophy of malate heterotrophs and strict autotrophy.     

Flux balance analysis of primary metabolism in Chlamydomonas reinhardtii

Background  

Photosynthetic organisms convert atmospheric carbon dioxide into numerous metabolites along the pathways to make new biomass. Aquatic photosynthetic organisms, which fix almost half of global inorganic carbon, have great potential: as a carbon dioxide fixation method, for the economical production of chemicals, or as a source for lipids and starch which can then be converted to biofuels. To harness this potential through metabolic engineering and to maximize production, a more thorough understanding of photosynthetic metabolism must first be achieved. A model algal species, C. reinhardtii, was chosen and the metabolic network reconstructed. Intracellular fluxes were then calculated using flux balance analysis (FBA).     

代谢工程改造异养微生物固定CO2研究进展

环境保护和能源供应是人类关心的两大问题。能源消耗释放出的温室气体对环境造成了严重影响。利用CO_2固定途径可将CO_2转化成燃料或化学品。天然固碳生物通常存在生长缓慢、固碳效率低等问题。通过在模式微生物中增强或重构CO_2固定途径,实现CO_2的再循环,可提高燃料或化学品的产量,减少温室气体排放。文中详细介绍了通过代谢工程手段改造CO_2固定途径改善化学品生产以及糖合成,阐述了相关代谢途径及其中的关键酶在CO_2固定中的作用,介绍了电生化合成系统的应用,显示出CO_2固定的巨大潜力,并展望了未来CO_2固定的研究方向。     

Biosynthetic controls on the 13C contents of organic components in the photoautotrophic bacterium Chloroflexus aurantiacus.

To assess the effects related to known and proposed biosynthetic pathways on the (13)C content of lipids and storage products of the photoautotrophic bacterium Chloroflexus aurantiacus, the isotopic compositions of bulk cell material, alkyl and isoprenoid lipids, and storage products such as glycogen and polyhydroxyalkanoic acids have been investigated. The bulk cell material was 13 per thousand depleted in (13)C relative to the dissolved inorganic carbon. Evidently, inorganic carbon fixation by the main carboxylating enzymes used by C. aurantiacus, which are assumed to use bicarbonate rather than CO(2), results in a relatively small carbon isotopic fractionation compared with CO(2) fixation by the Calvin cycle. Even carbon numbered fatty acids, odd carbon numbered fatty acids, and isoprenoid lipids were 14, 15, and 17-18 per thousand depleted in (13)C relative to the carbon source, respectively. Based on the (13)C contents of alkyl and isoprenoid lipids, a 40 per thousand difference in (13)C content between the carboxyl and methyl carbon from acetyl-coenzyme A has been calculated. Both sugars and polyhydroxyalkanoic acid were enriched in (13)C relative to the alkyl and isoprenoid lipids. To the best of our knowledge this is the first report in which the stable carbon isotopic composition of a large range of biosynthetic products in a photoautotrophic organism has been investigated and interpreted based on previously proposed inorganic carbon fixation and biosynthetic pathways. Our results indicate that compound-specific stable carbon isotope analysis may provide a rapid screening tool for carbon fixation pathways.     

Biosynthetic controls on the 13C contents of organic components in the photoautotrophic bacterium Chloroflexus aurantiacus

To assess the effects related to known and proposed biosynthetic pathways on the (13)C content of lipids and storage products of the photoautotrophic bacterium Chloroflexus aurantiacus, the isotopic compositions of bulk cell material, alkyl and isoprenoid lipids, and storage products such as glycogen and polyhydroxyalkanoic acids have been investigated. The bulk cell material was 13 per thousand depleted in (13)C relative to the dissolved inorganic carbon. Evidently, inorganic carbon fixation by the main carboxylating enzymes used by C. aurantiacus, which are assumed to use bicarbonate rather than CO(2), results in a relatively small carbon isotopic fractionation compared with CO(2) fixation by the Calvin cycle. Even carbon numbered fatty acids, odd carbon numbered fatty acids, and isoprenoid lipids were 14, 15, and 17-18 per thousand depleted in (13)C relative to the carbon source, respectively. Based on the (13)C contents of alkyl and isoprenoid lipids, a 40 per thousand difference in (13)C content between the carboxyl and methyl carbon from acetyl-coenzyme A has been calculated. Both sugars and polyhydroxyalkanoic acid were enriched in (13)C relative to the alkyl and isoprenoid lipids. To the best of our knowledge this is the first report in which the stable carbon isotopic composition of a large range of biosynthetic products in a photoautotrophic organism has been investigated and interpreted based on previously proposed inorganic carbon fixation and biosynthetic pathways. Our results indicate that compound-specific stable carbon isotope analysis may provide a rapid screening tool for carbon fixation pathways.     

Photolithoautotrophic growth and control of CO2 fixation in Rhodobacter sphaeroides and Rhodospirillum rubrum in the absence of ribulose bisphosphate carboxylase-oxygenase.

Rhodospirillum rubrum and Rhodobacter sphaeroides were shown to be capable of photolithoautotrophic growth in the absence of the reductive pentose phosphate (Calvin) cycle. Ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion strains were incapable of photolithoautotrophic growth using hydrogen as an electron donor but were able to grow in the absence of organic carbon using less reduced inorganic electron donors, i.e., thiosulfate or sulfide. Wild-type R. rubrum grown in the presence of thiosulfate contained RubisCO levels that were 50-fold lower compared with those in cells growth with hydrogen as an electron donor without substantially influencing rates of photolithoautotrophic growth. These results suggest there are two independent CO2 fixation pathways that support photolithoautotrophic growth in purple nonsulfur photosynthetic bacteria, indicating that these organisms have developed sophisticated control mechanisms to regulate the flow of carbon from CO2 through these separate pathways.     

A state of the art of metabolic networks of unicellular microalgae and cyanobacteria for biofuel production

The most promising and yet challenging application of microalgae and cyanobacteria is the production of renewable energy: biodiesel from microalgae triacylglycerols and bioethanol from cyanobacteria carbohydrates. A thorough understanding of microalgal and cyanobacterial metabolism is necessary to master and optimize biofuel production yields. To this end, systems biology and metabolic modeling have proven to be very efficient tools if supported by an accurate knowledge of the metabolic network. However, unlike heterotrophic microorganisms that utilize the same substrate for energy and as carbon source, microalgae and cyanobacteria require light for energy and inorganic carbon (CO₂ or bicarbonate) as carbon source. This double specificity, together with the complex mechanisms of light capture, makes the representation of metabolic network nonstandard. Here, we review the existing metabolic networks of photoautotrophic microalgae and cyanobacteria. We highlight how these networks have been useful for gaining insight on photoautotrophic metabolism.     

Flux balance analysis of Chlorella sp. FC2 IITG under photoautotrophic and heterotrophic growth conditions

Quantification of carbon flux distribution in the metabolic network of microalgae remains important to understand the complex interplay between energy metabolism, carbon fixation, and assimilation pathways. This is even more relevant with respect to cyclic metabolism of microalgae under light–dark cycle. In the present study, flux balance analysis (FBA) was carried out for an indigenous isolate Chlorella sp. FC2 IITG under photoautotrophic and heterotrophic growth conditions. A shift in intracellular flux distribution was predicted during transition from nutrient sufficient phase to nutrient starvation phase of growth. Further, dynamic flux analysis (dFBA) was carried out to capture light–dark metabolism over discretized pseudo steady state time intervals. Our key findings include the following: (i) unlike heterotrophic condition, oxidative pentose phosphate (PP) pathway, and Krebs cycle were relatively inactive under photoautotrophic growth; (ii) in both growth conditions, while transhydrogenation reaction was highly active, glyoxalate shunt was found to be nonoperative; (iii) flux distribution during transition period was marked with up regulation of carbon flux toward nongrowth associated (NGA) maintenance energy, oxidative phosphorylation, and photophosphorylation; (iv) redirection of carbon flux from polysaccharide and neutral lipid resulted in up regulation of Krebs cycle flux in the dark phase; (v) elevated glycolytic and acetyl-CoA flux were coupled with induction of neutral lipid during light cycle of the growth; (vi) significantly active photophosphorylation in the light phase was able to satisfy cellular energy requirement without need of oxidative PP pathway; and (vi) unlike static FBA, dFBA predicted an unaltered NGA maintenance energy of 1.5 mmol g^?1 DCW h^?1.     

Roles of microorganisms other than Clostridium and Enterobacter in anaerobic fermentative biohydrogen production systems--a review

Anaerobic fermentative biohydrogen production, the conversion of organic substances especially from organic wastes to hydrogen gas, has become a viable and promising means of producing sustainable energy. Successful biological hydrogen production depends on the overall performance (results of interactions) of bacterial communities, i.e., mixed cultures in reactors. Mixed cultures might provide useful combinations of metabolic pathways for the processing of complex waste material ingredients, thereby supporting the more efficient decomposition and hydrogenation of biomass than pure bacteria species would. Therefore, understanding the relationships between variations in microbial composition and hydrogen production efficiency is the first step in constructing more efficient hydrogen-producing consortia, especially when complex and non-sterilized organic wastes are used as feeding substrates. In this review, we describe recent discoveries on bacterial community composition obtained from dark fermentation biohydrogen production systems, with emphasis on the possible roles of microorganisms that co-exist with common hydrogen producers.     

Metabolic Features of Methane- and Methanol-Utilizing Bacteria

Investigations of a wide range of methane- and methanol-utilizers showed a striking versatility of their metabolism dependent on the genotype and growth conditions. A correlation between pathways of carbon and nitrogen metabolism was found. It was most stringent in obligate methane-utilizers: the hexulosephosphate pathway bacteria assimilated NH₃ by the reductive amination of α-ketoglutarate or pyruvate whereas the serine pathway bacteria used the glutamate cycle (glutamine synthetase + glutamate-oxoglutarate aminotransferase). Multiple enzymic lesions were found in central metabolism of obligate methylotrophs, i.e. the absence of the enzymes of glycolytic and pentosephosphate pathways, gluconeogenesis, citric acid cycle and glyoxylate shunt. These metabolic blocks were not so profound and could be compensated in restricted and facultative methylotrophs during heterotrophic growth. The average levels of exogenous CO₂ fixation in methylotrophic bacteria with the hexulosephosphate, serine and ribulosebisphosphate pathways were found to be 10, 30 and 80% of their total cell carbon, respectively. These results served as a basis for biotechnological applications of metabolic potential of methylotrophs (production of biomass, polysaccharides and enzymes as well as for microbiological treatment of industrial waters containing toxic C₁- and C_n-compounds).