Genome-scale metabolic modeling of cyanbacteria: network structure, interactions, reconstruction and dynamics

2016 Fall.Includes bibliographical references.Metabolic network modeling, a field of systems biology and bioengineering, enhances the quantitative predictive understanding of cellular metabolism and thereby assists in the development of model-guided metabolic engineering strategies. Metabolic models use genome-scale network reconstructions, and combine it with mathematical methods for quantitative prediction. Metabolic system reconstructions, contain information on genes, enzymes, reactions, and metabolites, and are converted into two types of networks: (i) gene-enzyme-reaction, and (ii) reaction-metabolite. The former details the links between the genes that are known to code for metabolic enzymes, and the reaction pathways that the enzymes participate in. The latter details the chemical transformation of metabolites, step by step, into biomass and energy. The latter network is transformed into a system of equations and simulated using different methods. Prominent among these are constraint-based methods, especially Flux Balance Analysis, which utilizes linear programming tools to predict intracellular fluxes of single cells. Over the past 25 years, metabolic network modeling has had a range of applications in the fields of model-driven discovery, prediction of cellular phenotypes, analysis of biological network properties, multi-species interactions, engineering of microbes for product synthesis, and studying evolutionary processes. This thesis is concerned with the development and application of metabolic network modeling to cyanobacteria as well as E. coli. Chapter 1 is a brief survey of the past, present, and future of constraint-based modeling using flux balance analysis in systems biology. It includes discussion of (i) formulation, (ii) assumption, (iii) variety, (iv) availability, and (v) future directions in the field of constraint based modeling. Chapter 2, explores the enzyme-reaction networks of metabolic reconstructions belonging to various organisms; and finds that the distribution of the number of reactions an enzyme participates in, i.e. the enzyme-reaction distribution, is surprisingly similar. The role of this distribution in the robustness of the organism is also explored. Chapter 3, applies flux balance analysis on models of E. coli, Synechocystis sp. PCC6803, and C. reinhardtii to understand epistatic interactions between metabolic genes and pathways. We show that epistatic interactions are dependent on the environmental conditions, i.e. carbon source, carbon/oxygen ratio in E. coli, and light intensity in Synechocystis sp. PCC6803 and C. reinhardtii. Cyanobacteria are photosynthetic organisms and have great potential for metabolic engineering to produce commercially important chemicals such as biofuels, pharmaceuticals, and nutraceuticals. Chapter 4 presents our new genome scale reconstruction of the model cyanobacterium, Synechocystis sp. PCC6803, called iCJ816. This reconstruction was analyzed and compared to experimental studies, and used for predicting the capacity of the organism for (i) carbon dioxide remediation, and (ii) production of intracellular chemical species. Chapter 5 uses our new model iCJ816 for dynamic analysis under diurnal growth simulations. We discuss predictions of different optimization schemes, and present a scheme that qualitatively matches observations

Sphagnum physiology in the context of changing climate: emergent influences of genomics, modelling and host-microbiome interactions on understanding ecosystem function.

Peatlands harbour more than one-third of terrestrial carbon leading to the argument that the bryophytes, as major components of peatland ecosystems, store more organic carbon in soils than any other collective plant taxa. Plants of the genus Sphagnum are important components of peatland ecosystems and are potentially vulnerable to changing climatic conditions. However, the response of Sphagnum to rising temperatures, elevated CO2 and shifts in local hydrology have yet to be fully characterized. In this review, we examine Sphagnum biology and ecology and explore the role of this group of keystone species and its associated microbiome in carbon and nitrogen cycling using literature review and model simulations. Several issues are highlighted including the consequences of a variable environment on plant-microbiome interactions, uncertainty associated with CO2 diffusion resistances and the relationship between fixed N and that partitioned to the photosynthetic apparatus. We note that the Sphagnum fallax genome is currently being sequenced and outline potential applications of population-level genomics and corresponding plant photosynthesis and microbial metabolic modelling techniques. We highlight Sphagnum as a model organism to explore ecosystem response to a changing climate and to define the role that Sphagnum can play at the intersection of physiology, genetics and functional genomics

The utilization of carbon dioxide by the microalgal species Scenedesmus

Climate change, which is predominantly a result of anthropogenic global warming, is a major concern for humanity. The scientific consensus is that the rise of greenhouse gases, such as CO2, in the atmosphere has contributed to global warming. The increase in atmospheric CO2 is due to the burning of fossil fuels and deforestation. In South Africa, 77% of our primary energy needs are supplied by coal and our demand for energy is increasing. Cultivation of microalgae on flue gas from coal-fired plants and utilization of the algal biomass as an energy source has been proposed as a method for producing CO2-neutral fuels

The Physiological Effects of Phycobilisome Antenna Modification on the Cyanobacterium Synechocystis sp. PCC 6803

Phycobilisomes are the large, membrane extrinsic light harvesting antenna of cyanobacteria. They function to absorb light energy and deliver it efficiently to the photosystems, thereby increasing photosynthetic light absorption. Wild type phycobilisomes in the model organism Synechocystis sp. PCC 6803: Synechocystis 6803) consist of a tricylindrical core from which six rods radiate. The colored phycobiliproteins are held together by colorless linker polypeptides. Several phycobilisome truncation mutants have been generated in Synechocystis 6803. The first, CB, has truncated phycobilisome rods; the second, CK, has only the phycobilisome core; and the third, PAL, has no phycobilisomes at all. Together, these mutants construct a series of increasingly truncated phycobilisomes which are useful for studying the physiology of antenna truncation in cyanobacteria. In this dissertation, the physiological effects of antenna truncation are examined from three perspectives. First, the effect of partial and complete phycobilisome removal on the expression and activity of photosystem II is examined using a variety of assays that center around fluorescence and oxygen evolution. Second, the overall effects of antenna truncation on thylakoid membrane spacing and structure is explored using electron microscopy and small angle neutron scattering. Finally, the effects of antenna truncation on culture-wide biomass productivity are examined in a variety of setting, including a bench-scale photobioreactor. Together, these studies represent a comprehensive examination of the physiological effects of antenna truncation on Synechocystis 6803

Harnessing Solar Energy for the Production of Clean Fuel

The European Union and its member states are being urged by leading scientists to make a major multi million Euro commitment to solar driven production of environmentally clean electricity, hydrogen and other fuels, as the only sustainable long-term solution for global energy needs. The most promising routes to eventual full-scale commercial solar energy conversion directly into fuels were identified at a recent international meeting in Regensburg, sponsored by the European Science Foundation (ESF). An interdisciplinary task force was established at this meeting to make the case for substantial investments in these technologies to EU and national government decision makers. This report summarizes the outcome of this meeting. The fundamental issue is that total annual global energy consumption is set at least to double from its current level of 14 TW by 2050, while fossil fuels will start to run out and in any case would produce unacceptable levels of carbon dioxide, bringing global warming accompanied by disastrous effects in many areas, such as food production. Apart from solar energy, the shortfall can only be made up by renewable sources such as wind, along with the other nonfossil, non-renewable fuel source of energy, nuclear. But these will be unable to satisfy the expected increased energy needs, let alone replace fossil fuels entirely, even for electricity production. Another problem is that they will not readily yield stored fuels. Without an unexpected breakthrough in electricity storage, there will be a continued need for fuels for around 70% of total global energy requirements, particularly in transportation, manufacturing,and domestic heating. Electricity only accounts for 30% of global energy consumption at present. Solar energy, however, is plentiful since enough reaches the Earth’s surface every hour to meet the world’s annual energy needs. The problem lies in harnessing it, but nature has perfected in photosynthesis a highly efficient and flexible means of doing this across a wide variety of scales from isolated bacterial colonies to large forests. Substantial progress has been made recently, particularly in Europe, to understand and mimic these processes, sufficient for scientists to be confident that it can work to produce fuels on a commercial scale. The focus of research therefore should be on drawing inspiration from biological systems for the creation of both natural and artificial solar energy conversion systems that allow in the long run for a stable and sustainable energy supply. The focus should also be on reducing the ecological footprint of the human economy and thereby increasing the global ecological capacity using technology that is environmentally clean, for instance by conversion of carbon dioxide back into fuels in a cyclic process. The ESF task force is recommending that three parallel avenues of solar energy research for generating clean fuel cycles should be pursued in Europe: 1) Extending and adapting current photovoltaic technology to generate clean fuels directly from solar radiation. 2) Constructing artificial chemical and biomimetic devices mimicking photosynthesis to collect, direct, and apply solar radiation, for example to split water, convert atmospheric carbon dioxide and thus produce various forms of environmentally clean fuels. 3) Tuning natural systems to produce fuels such as hydrogen and methanol directly rather than carbohydrates that are converted into fuels in an indirect and inefficient process. These three research themes will overlap, and all will exploit fundamental research elucidating the precise molecular mechanisms involved in the splitting of water into hydrogen and oxygen in photosynthesis by both plants and bacteria. This process, which evolved 2.5 billion years ago, created the conditions for animal life by converting atmospheric carbon dioxide into carbohydrates, and also produced all the fossil fuels, which humans are turning back into carbon dioxide at an increasing rate, threatening catastrophic environmental effects. The same process now holds our salvation again. Although the principal products of photosynthesis in plants and bacteria are carbohydrates, certain algae and cyanobacteria can produce hydrogen directly from water using sunlight, providing a basis for genetic modification to increase yields, and for the creation of suitable artificial systems. Furthermore, photosynthesis is also capable of generating other chemicals currently made industrially, such as nitrates, and other high value compounds for chemical industry. The European research program will therefore also seek to develop systems for converting solar energy directly into such chemicals with much greater efficiency, offering the prospect not just of producing unlimited energy, but also fixing atmospheric carbon dioxide to bring concentrations back down to pre-industrial levels as part of the overall thrust for clean renewable energy. There are considerable challenges, with the first being to mimic the functioning of natural photosynthetic systems, particularly photosystem II, the enzyme complex in the leaves of plants that splits water into hydrogen and oxygen via a catalyst comprising four manganese atoms along with some calcium. Significant progress has been made recently on this front. Participants at the ESF’s brainstorming conference, describe the solar fuels project as the quest for building the “artificial leaf”. There is growing conviction in Europe and elsewhere that by 2050 a large proportion of our fuels will come from such “artificial leaves” and that there is no time to lose starting the crucial enabling research, in order to gain technology leadership in this important future key technology.White paper prepared by the ESF task force on converting solar energy into fuelEuropean Science FoundationPopulariserende publicatieFaculteit der Wiskunde en Natuurwetenschappe

Plant biosystems design research roadmap 1.0

Human life intimately depends on plants for food, biomaterials, health, energy, and a sustainable environment. Various plants have been genetically improved mostly through breeding, along with limited modification via genetic engineering, yet they are still not able to meet the ever-increasing needs, in terms of both quantity and quality, resulting from the rapid increase in world population and expected standards of living. A step change that may address these challenges would be to expand the potential of plants using biosystems design approaches. This represents a shift in plant science research from relatively simple trial-and-error approaches to innovative strategies based on predictive models of biological systems. Plant biosystems design seeks to accelerate plant genetic improvement using genome editing and genetic circuit engineering or create novel plant systems through de novo synthesis of plant genomes. From this perspective, we present a comprehensive roadmap of plant biosystems design covering theories, principles, and technical methods, along with potential applications in basic and applied plant biology research. We highlight current challenges, future opportunities, and research priorities, along with a framework for international collaboration, towards rapid advancement of this emerging interdisciplinary area of research. Finally, we discuss the importance of social responsibility in utilizing plant biosystems design and suggest strategies for improving public perception, trust, and acceptance

Molecular simulations unravel the molecular principles that mediate selective permeability of carboxysome shell protein

Bacterial microcompartments (BMCs) are nanoscale proteinaceous organelles that encapsulate enzymes from the cytoplasm using an icosahedral protein shell that resembles viral capsids. Of particular interest are the carboxysomes (CBs), which sequester the CO 2 -fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to enhance carbon assimilation. The carboxysome shell serves as a semi-permeable barrier for passage of metabolites in and out of the carboxysome to enhance CO 2 fixation. How the protein shell directs influx and efflux of molecules in an effective manner has remained elusive. Here we use molecular dynamics and umbrella sampling calculations to determine the free-energy profiles of the metabolic substrates, bicarbonate, CO 2 and ribulose bisphosphate and the product 3-phosphoglycerate associated with their transition through the major carboxysome shell protein CcmK2. We elucidate the electrostatic charge-based permeability and key amino acid residues of CcmK2 functioning in mediating molecular transit through the central pore. Conformational changes of the loops forming the central pore may also be required for transit of specific metabolites. The importance of these in-silico findings is validated experimentally by site-directed mutagenesis of the key CcmK2 residue Serine 39. This study provides insight into the mechanism that mediates molecular transport through the shells of carboxysomes, applicable to other BMCs. It also offers a predictive approach to investigate and manipulate the shell permeability, with the intend of engineering BMC-based metabolic modules for new functions in synthetic biology

The energy cost of primary metabolism vacuole expansion: central to shape toamto leaf development under ammonium nutrition

231 p.Ammonium (NH4+) is a nitrogen source of great interest in the context of sustainable agriculture. Its application in the field together with nitrification inhibitors has been extensively proven efficient to limit detrimental N losses compared to the use of nitrate (N03). NH4+ is a common intermediate involved in numerous metabolic routes. However, high NH4 concentrations may lead to a stress situation provoking a set of symptoms collectively known as "ammonium syndrome" mainly characterized by growth retardation. Those symptoms are caused by a combination of, among others, a profound metabolic reprogramming, disruption of photosynthesis, pH deregulation and ion imbalance. Numerous studies have described the way plant copes to ammonium nutrition. However, the organ developmental stage has been generally neglected.To fill in this gap, in the first chapter we first aimed studying how the metabolism is adapted in function of the leaf position in the vertical axis of the tomato plants (Solanum lycopersicum) grown with NH4+, N03- or NH4N03 supply. To do so, we dissected leaf biomass composition and metabolism through a complete analysis of metabolites, ions and enzyme activities. The results showed that C and N metabolic adjustment in function of the nitrogen source was more intense in older leaves compared to younger ones. Importantly, we propose a trade-off between NH4+ accumulation and assimilation to preserve young leaves from ammonium stress. Besides, NH4+-fed plants exhibited a rearrangement of carbon skeletons with a higher energy cost respect to plants supplied with N03-. We explain such reallocation by the action of the biochemical pH-stat, to compensate the differential proton production that depends on the nitrogen form provided.Ammonium nutrition may limit cell expansion, suggesting that the cellular processes involved would be altered. Among others, cell growth is largely dependent of the internal pressure exerted on the cell wall by the vacuole. However, the role of the vacuole in ammonium stress has been rarely addressed. In the second chapter, we evaluated the effect of ammonium stress on leaf development with a special focus on vacuole expansion and metabolism. To carry out this aim, we monitored the leaf development from its appearance until its complete expansion in plants grown under NH4+ or NO/ as unique nitrogen source. Cytological analysis evidenced that the reduced cell expansion under ammonium nutrition was associated with smaller vacuole size. Besides, we reported an acidification of the vacuole of NH4+-fed plants compared to nitrate nutrition. Moreover, a model was built to predict the thermodynamic equilibrium of different soluble species across the tonoplast. The model was set up through an extensive reviewing of vacuolar transporters and integrated subcellular volumes, vacuolar electrochemical gradients and the formation of ionic complex in the vacuole to fit the subcellular concentration of ions, organic acids and sugars measured in the leaf. Further, predictions obtained with the model were cross validated with data from non-aqueous fractionation. Firstly, the entrance of solutes was higher in vacuoles of N03--fed leaves but was not associated with higher vacuolar osmolarity likely because of the adjustment of the vacuolar volume. In this sense, we proposed that the lack of malate in cells of ammonium-fed leaves was central in the limitation of vacuolar expansion. Secondly, we conclude that the energy cost of solute transport into the vacuole is higher under NH4+ based nutrition because of the higher electrochemical gradient generated by the proton pumps across tonoplast.This work highlights the importance of considering leaf phenological state when studying nitrogen metabolism. In addition, our integrated approach place cytosolic pH control and vacuole expansion in the center of tomato leaf adaptation to ammonium stress and pave the way for future studies in the field of ammonium nutrition

Controlled Ecological Life Support System. First Principal Investigators Meeting

Control problems in autonomous life support systems, CELSS candidate species, maximum grain yield, plant growth, waste management, air pollution, and mineral separation are discussed

Geodynamic and metabolic cycles in the Hadean

International audienceHigh-degree melting of hot dry Hadean mantle at ocean ridges and plumes resulted in a crust about 30km thick, overlain in places by extensive and thick mafic volcanic plateaus. Continental crust, by contrast, was relatively thin and mostly submarine. At constructive and destructive plate boundaries, and above the many mantle plumes, acidic hydrothermal springs at ~400°C contributed Fe and other transition elements as well as P and H2 to the deep ocean made acidulous by dissolved CO2 and minor HCl derived from volcanoes. Away from ocean ridges, submarine hydrothermal fluids were cool (=100°C), alkaline (pH ~10), highly reduced and also H2-rich. Reaction of solvents in this fluid with those in ocean water was catalyzed in a hydrothermal mound, a natural self-restoring flow reactor and fractionation column made up of carbonates and freshly precipitated Fe-Ni sulfide and greenrust pores and bubbles, developed above the alkaline spring. Acetate and the amino acetate glycine were the main products, much of which was eluted to the ocean. Other organic byproducts were retained, concentrated and reacted within the compartments. These compartments comprising the natural hydrothermal reactor consisted partly of greigite (Fe5NiS8). It was from reactions between organic modules confined within these inorganic compartments that the first prokaryotic organism evolved. These acetogenic precursors to the Bacteria diversified and migrated down the mound and into the ocean floor to inaugurate the "deep biosphere". Once there the Bacteria, and the recently differentiated Archaea, were protected from cataclysmic heating events caused by large bolide impacts. Geodynamic forces led to the eventual obduction of the deep biosphere into the photic zone where, initially protected by a thin veneer of sediment, the use of solar energy was mastered and photosynthesis emerged. The further evolution to oxygenic photosynthesis was effected as catalytic [CaMn4+] bearing molecules that otherwise would have been interred in the mineral ranciéite in the shallow marine manganiferous sediments, were sequestered and invaginated within the cyanobacterial precursor where, energized by light, they could oxidize water with greater efficiency. Thus, chemical sediments were required both for the emergence of chemosynthesis and of oxygenic photosynthesis, the two innovations that did most to change the nature of our planet