Compared energetics of primordial and biological metabolisms

In model experiments of chemical evolution, the accumulation of redox energy has been achieved up to now only within gaseous phase. Such experiments lead to atmospheric precursors and involve an accumulation of large quantities of free enthalpy. A part of this energy can be released after dissolution of the precursors in aqueous media, either by simple multiple bond hydration, or by addition of heteroatomic reagents more nucleophilic than water, or by addition of carbonaceous nucleophiles. Energy balances of such processes are discussed.The non-enzymic photochemical accumulation of redox energy in aqueous phase appears later on feasible, but the main unsolved problem lies in the understanding of the primordial processes which made the conversion of redox energy into energy available from hydrolysis possible in aqueous media in the earliest stage of chemical evolution.In this respect, chemiosmotic or configurational interpretations of oxidative phosphorylations cannot be taken into consideration because they require complex structures which cannot be allowed for at this early stage.On the contrary, a discussion of the energetics and kinetics of electron transfers to and from substrates makes understandable the basic principles involved in the energy storage processes by means of the chemical hypothesis, as well as their likely occurrence, even in a non-enzymic form, from the very earliest stages of chemical evolution.     

Biochemical production capabilities of Escherichia coli

Microbial metabolism provides at mechanism for the conversion of substrates into useful biochemicals. Utilization of microbes in industrial processes requires a modification of their natural metabolism in order to increase the efficiency of the desired conversion. Redirection of metabolic fluxes forms the basis of the newly defined field of metabolic engineering. In this study we use a flux balance based approach to study the biosynthesis of the 20 amino acids and 4 nucleotides as biochemical products. These amino acids and nucleotides are primary products of biosynthesis as well as important industrial products and precursors for the production of other biochemicals. The biosynthetic reactions of the bacterium Escherichia coli have been formulated into a metabolic network, and growth has been defined as a balanced drain on the metabolite pools corresponding to the cellular composition. Theoretical limits on the conversion of glucose, glycerol, and acetate substrates to biomass as well as the biochemical products have been computed. The substrate that results in the maximal carbon conversion to a particular product is identified. Criteria have been developed to identify metabolic constraints in the optimal solutions. The constraints of stoichiometry, energy, and redox have been determined in the conversions of glucose, glycerol, and acetate substrates into the biochemicals. Flux distributions corresponding to the maximal production of the biochemicals are presented. The goals of metabolic engineering are the optimal redirection of fluxes from generating biomass toward producing the desired biochemical. Optimal biomass generation is shown to decrease in a piecewise linear manner with increasing product formation. In some cases, synergy is observed between biochemical production and growth, leading to an increased overall carbon conversion. Balanced growth and product formation are important in a bioprocess, particularly for nonsecreted products. (c) 1993 John Wiley & Sons, Inc.     

The role of sulphur in chemical evolution

Summary Sulphur may have played an important role, mainly as an energy converter, during the initial steps of Chemical Evolution.In atmospheric processes, sulphur, in the form of H₂S might have been a primary energy acceptor and a source of hot hydrogen atoms. The presence of H₂S in the primeval earth atmosphere with a molar ratio of about 10^–2 could have allowed the formation of several volatile S-containing compounds without inhibiting the synthesis of the reactive products which are formed in the absence of H₂S. An evaluation of the quantity of H₂S which could have been included in the primeval atmosphere suggests that such a molar ratio may have been reached.In the primitive soup, the thiols and sulphides formed in the gaseous phase may have evolved, giving rise to various prebiotic syntheses. Studies on the addition reaction of alkanethiols on malonic nitriles in aqueous solutions show two different condensation processes: the formation of thioethers and the formation of iminothioesters. Taking into account the values of the specific rate constants for the two reactions, it is shown that these reactions may have taken place in the primitive earth conditions. These two compounds may have played an important role in the prebiochemical evolution. In particular, iminothioesters can be considered as the immediate precursors of thioesters.     

Biosynthetic pathways of two polypeptide subunits of the light- harvesting chlorophyll a/b protein complex

We have used an in vitro reconstitution system, consisting of cell-free translation products and intact chloroplasts, to investigate the pathway from synthesis to assembly of two polypeptide subunits of the light-harvesting chlorophyll-protein complex. These polypeptides, designated 15 and 16, are integral components of the thylakoid membranes, but they are products of cytoplasmic protein synthesis. Double immunodiffusion experiments reveal that the two polypeptides share common antigenic determinants and therefore are structurally related. Nevertheless, they are synthesized in vitro from distinct mRNAs to yield separate precursors, p15 and p16, each of which is 4,000 to 5,000 daltons larger than its mature form. In contrast to the hydrophobic mature polypeptides, the precursors are soluble in aqueous solutions. Along with other cytoplasmically synthesized precursors, p15 and p16 are imported into purified intact chloroplasts by a post- translational mechanism. The imported precursors are processed to the mature membrane polypeptides which are recovered exclusively in the thylakoids. The newly imported polypeptides are assembled correctly in the thylakoid lipid bilayer and they bind chlorophylls. Thus, these soluble membrane polypeptide precursors must move from the cytoplasm through the two chloroplast envelope membranes, the stroma, and finally insert into the thylakoid membranes, where they assemble with chlorophyll to form the light-harvesting chlorophyll protein complex.     

Microbial engineering strategies to improve cell viability for biochemical production

Efficient production of biochemicals using engineered microbes as whole-cell biocatalysts requires robust cell viability. Robust viability leads to high productivity and improved bioprocesses by allowing repeated cell recycling. However, cell viability is negatively affected by a plethora of stresses, namely chemical toxicity and metabolic imbalances, primarily resulting from bio-synthesis pathways. Chemical toxicity is caused by substrates, intermediates, products, and/or by-products, and these compounds often interfere with important metabolic processes and damage cellular infrastructures such as cell membrane, leading to poor cell viability. Further, stresses on engineered cells are accentuated by metabolic imbalances, which are generated by heavy metabolic resource consumption due to enzyme overexpression, redistribution of metabolic fluxes, and impaired intracellular redox state by co-factor imbalance. To address these challenges, herein, we discuss a range of key microbial engineering strategies, substantiated by recent advances, to improve cell viability for commercially sustainable production of biochemicals from renewable resources.     

Establishing a Toolkit for Precursor-Directed Polyketide Biosynthesis: Exploring Substrate Promiscuities of Acid-CoA Ligases

Polyketides are chemically diverse and medicinally important biochemicals that are biosynthesized from acyl-CoA precursors by polyketide synthases. One of the limitations to combinatorial biosynthesis of polyketides has been the lack of a toolkit that describes the means of delivering novel acyl-CoA precursors necessary for polyketide biosynthesis. Using five acid-CoA ligases obtained from various plants and microorganisms, we biosynthesized an initial library of 79 acyl-CoA thioesters by screening each of the acid-CoA ligases against a library of 123 carboxylic acids. The library of acyl-CoA thioesters includes derivatives of cinnamyl-CoA, 3-phenylpropanoyl-CoA, benzoyl-CoA, phenylacetyl-CoA, malonyl-CoA, saturated and unsaturated aliphatic CoA thioesters, and bicyclic aromatic CoA thioesters. In our search for the biosynthetic routes of novel acyl-CoA precursors, we discovered two previously unreported malonyl-CoA derivatives (3-thiophenemalonyl-CoA and phenylmalonyl-CoA) that cannot be produced by canonical malonyl-CoA synthetases. This report highlights the utility and importance of determining substrate promiscuities beyond conventional substrate pools and describes novel enzymatic routes for the establishment of precursor-directed combinatorial polyketide biosynthesis.     

SO3H-functionalized ionic liquid: efficient catalyst for bagasse liquefaction

Liquefaction is a process for the production of biofuel or value-added biochemicals from non-food biomass. SO₃H-, COOH-functionalized and HSO₄-paired imidazolium ionic liquids were shown to be efficient catalysts for bagasse liquefaction in hot compressed water. Using SO₃H-functionalized ionic liquid, 96.1% of bagasse was liquefied and 50.6% was selectively converted to low-boiling biochemicals at 543 K. The degree of liquefaction and selectivity for low-boiling products increased and the average molecular weight of the tetrahydrofuran soluble products decreased with increasing acidic strength of ionic liquids. Analysis of products and comparative characterization of raw materials and residues suggested that both catalytic liquefaction and hydrolysis processes contribute to the high conversion of bagasse. A possible liquefaction mechanism based on the generation of 3-cyclohexyl-1-propanol, one of the main products, is proposed.     

Analysis of excess Gibbs energy of electrolyte solutions: a new model for aqueous solutions

This paper presents an analysis of the excess Gibbs free energy of aqueous electrolytes. The analysis of experimental data leads to the conclusion that the equilibrium state for dilute univalent electrolytes in water involves an intercalation of water and ionic liquid crystal domains. Excess free energy of the solution is determined by the Madelung energy of hydrated ion-pair liquid crystals, and the energy associated with a shift in the structural equilibrium of water. The data that point to such a model include: molecular orbital-molecular dynamics applied to electrolyte water systems; Raman spectra; infrared spectra; magnetic resonance spectra of ions; the apparent density of water; and the excess free energy of electrolytes in aqueous solutions. Molecular orbital-molecular dynamics calculations of relatively large water clusters containing a molecule of sodium iodide show that the solvent separated ion pair exists in a substantial potential well compared to other possible structures. Raman spectra of univalent electrolyte solutions as a function of concentration can be quantitatively modeled using only the spectra of pure water and electrolyte solution at the concentration of the solvent separated ion pair. The other observations are consistent with the structures proposed from the Raman spectral study. The new model provides a satisfactory account of the fact that the excess free energy of dilute (<0.2 mol/l) solutions is generally more negative than anticipated on the basis of Debye-Hückel theory, and that the equilibrium evidence points to the same functional behavior at very low concentrations as is seen at 0.05 mol/l. We present a testable hypothesis that the excess free energy, and other thermodynamic properties of the solutions do not follow the Debye-Hückel limiting law. The tests of this hypothesis must involve only equilibrium measurements at concentrations between 0.05 and 0.0005 mol/l. This hypothesis concerning the structure of aqueous electrolyte solutions is not in conflict in any way with the Debye-Hückel-Onsager theory of electrical conductivity.     

Culture and sociobiology

Sociobiological concepts are easily misapplied to human behavior because the latter is culturally as well as biologically organized. Because biological and cultural evolution are two linked but conceptually distinct processes, sociobiology is more readily applied to the evolution of cultural capacity than to contemporary cultural behavior. The extent to which the latter is consistent with sociobiological expectation must be determined empirically, although there are theoretical grounds for predicting a limited degree of concordance . [sociobiology, culture, evolution, reductionism, biosocial anthropology]     

Overtaking on migration: does longer distance migration always incur a penalty?

For many migratory bird species, the latitudinal range of the winter distribution spans thousands of kilometres, thus encompassing considerable variation in individual migration distances. Pressure to winter near breeding areas is thought to be a strong driver of the evolution of migration patterns, as individuals undertaking a shorter migration are generally considered to benefit from earlier arrival on the breeding grounds. However, the influence of migration distance on timing of arrival is difficult to quantify because of the large scales over which individuals must be tracked. Using a unique dataset of individually‐marked Icelandic black‐tailed godwits Limosa limosa islandica tracked throughout the migratory range by a network of hundreds of volunteer observers, we quantify the consequences of migrating different distances for the use of stop‐over sites and timing of arrival in Iceland. Modelling of potential flight distances and tracking of individuals from across the winter range shows that individuals wintering further from the breeding grounds must undertake a stop‐over during spring migration. However, despite travelling twice the distance and undertaking a stop‐over, individuals wintering furthest from the breeding grounds are able to overtake their conspecifics on spring migration and arrive earlier in Iceland. Wintering further from the breeding grounds can therefore be advantageous in migratory species, even when this requires the use of stop‐over sites which lengthen the migratory journey. As early arrival on breeding sites confers advantages for breeding success, the capacity of longer distance migrants to overtake conspecifics is likely to influence the fitness consequences of individual migration strategies. Variation in the quality of wintering and stopover sites throughout the range can therefore outweigh the benefits of wintering close to the breeding grounds, and may be a primary driver of the evolution of specific migration routes and patterns.     

Immobilized plant cell reactors

The use of plant cells for the production of biochemicals represents a new area of biotechnological exploration. The techniques envisioned for industrial processes are related to those developed for microorganisms and a strong emphasis should be placed on immobilized cell systems. This review examines the spectrum of products that are synthesized by higher plants and the immobilization techniques that are suited to entrap plant cells from suspension culture. Different reactor configurations are described. Both packed-bed reactors with alginate-entrapped cells and hollow-fibre cartridges with sequestered cells have utility for the continuous production of biochemicals.     

Infrared spectral characterization of peptidic material produced by ionizing radiation in aqueous cyanides

Summary Oligomers formed by ionizing radiation in aqueous cyanide solutions, under various experimental conditions, have been characterized by infrared spectroscopy. IR bands appear in the region known to be characteristic for amides and peptides. The results are discussed in relation to radiation-induced formation of peptidic material and the potential role of ionizing radiation as an energy source for some processes in prebiotic molecular evolution.     

Auger effects on bromo-deoxyuridine-monophosphate irradiated with monochromatic X-rays around bromine K-absorption edge

Summary In order to study the enhanced effect by Auger cascade, samples of bromo-deoxyuridine-monophosphate (Br-dUMP) in aqueous solutions were irradiated with monochromatic X-rays at 13.49 keV and 13.43 keV, just above and below the K-absorption edge of bromine, using synchrotron radiation as a source. Radiolytic products such as deoxyuridine-monophosphate (dUMP), uracil and bromo-uracil (Br-uracil) were isolated using high performance liquid chromatography. Their amounts were quantitatively analysed as a function of the absorbed dose in the solutions containing Br-dUMP for the energy of the X-rays.G values for these products were calculated on the basis of the absorbed energy. As the results, the ratios of G values of radiolytic products from Br-dUMP between X-rays of 13.49 keV and 13.43 keV were 2.2 for dUMP, 1.02 for Br-uracil and 1.23 for uracil, suggesting clearly the energy dependent enhancement. On the other hand, little significant difference between X-rays of 13.49 keV and 13.43 keV was observed for theG values of uracil released from dUMP irradiated in aqueous solutions. It seemed to confirm that the Auger electrons from K-shell of bromine atoms might play the main role for energy-dependent enhancement at induction of these radiolytic products.     

Modeling genome evolution with a diffusion approximation of a birth-and-death process

MOTIVATION: In our previous studies, we developed discrete-space birth, death and innovation models (BDIMs) of genome evolution. These models explain the origin of the characteristic Pareto distribution of paralogous gene family sizes in genomes, and model parameters that provide for the evolution of these distributions within a realistic time frame have been identified. However, extracting the temporal dynamics of genome evolution from discrete-space BDIM was not technically feasible. We were interested in obtaining dynamic portraits of the genome evolution process by developing a diffusion approximation of BDIM. RESULTS: The diffusion version of BDIM belongs to a class of continuous-state models whose dynamics is described by the Fokker-Plank equation and the stationary solution could be any specified Pareto function. The diffusion models have time-dependent solutions of a special kind, namely, generalized self-similar solutions, which describe the transition from one stationary distribution of the system to another; this provides for the possibility of examining the temporal dynamics of genome evolution. Analysis of the generalized self-similar solutions of the diffusion BDIM reveals a biphasic curve of genome growth in which the initial, relatively short, self-accelerating phase is followed by a prolonged phase of slow deceleration. This evolutionary dynamics was observed both when genome growth started from zero and proceeded via innovation (a potential model of primordial evolution), and when evolution proceeded from one stationary state to another. In biological terms, this regime of evolution can be tentatively interpreted as a punctuated-equilibrium-like phenomenon whereby evolutionary transitions are accompanied by rapid gene amplification and innovation, followed by slow relaxation to a new stationary state.     

Photosynthesis driven conversion of carbon dioxide to fatty alcohols and hydrocarbons in cyanobacteria

The production of high value biochemicals and high energy biofuels from sustainable resources through the use of microbial based, green conversion technologies could reduce the dependence on petrochemical resources. However, a sustainable source of carbon and a clean, cost effective method for its conversion to high quality biofuel products are obstacles that must be overcome. Here we describe the biosynthesis of fatty alcohols in a genetically engineered cyanobacterial system through heterologously expressing fatty acyl-CoA reductase and the effect of environmental stresses on the production of fatty alcohols in the mutant strains. Hydrocarbon production in three representative types of native cyanobacterial model strains and the mutant strain overexpressing acetyl-CoA carboxylase was evaluated. The results of this investigation demonstrate the potential for direct production of high value chemicals and high energy fuels in a single biological system that utilizes solar energy as the energy source and carbon dioxide as the carbon source.     

Lateral chain packing in lipids and membranes

The aliphatic chains of many biologically important lipids are heterogeneous and often related to the functions of the molecules. Certain phospholipids containing arachidonic acid may serve as precursors for prostaglandins, certain diglycerides may serve as second messengers for certain membrane-triggered reactions (43), and other phospholipids containing a very short chain in the two position may serve as vasoactive hormones (44). The packing of such molecules is of interest. The evidence is quite clear from both the conformation of saturated and unsaturated molecules and from mixing experiments in the solid state that long and short chains don't mix well, nor do unsaturated and saturated chains, even if they are of the same chain length. There is even some evidence to indicate that some degree of chain segregation occurs even in the liquid state. However, different chains are often associated through covalent bonds, e.g., in wax esters, diacylglycerols, triacylglycerols, and phospholipids. A variety of possibilities for chain segregation are present in the neat phases of wax esters, ceramides, diacylglycerols, and triacylglycerols. However, in the unique case of membrane lipids like phospholipids or sphingolipids, the two chains are forced to lie side by side by virtue of the interaction of the polar group with water, and thus interactions between different chains must occur. Most of the evidence suggests that, when a solid phase results in these systems, the nonspecific chain packing mode (hexagonal chain packing) is preferred. In fact, for all of the phospholipids studied thus far, clearcut evidence of specific chain-chain interaction in molecules having both unsaturated and saturated chains has never been observed. However, for mixed chain triacylglycerols, evidence of specific chain-chain interactions (beta' and even beta) has been found and some suggestions have been given as to how this might occur through chain segregation mechanisms in the neat state. The literature suggests that further work needs to be done on the interaction of different chains that are covalently linked to the same molecule. Such studies will lead to a better understanding of the structure of lipid bilayers, membranes, lipoproteins, and lipid deposits.     

On the role of periodism in the origin of proteins

Two different views have been proposed for origins of genes (or proteins). One is that primordial genes evolved from random sequences. This view underlies the concept of modern in vitro evolution experiments that functional molecules (even proteins) evolved from random sequence-libraries. On the contrary, the second view reminds that "random sequences" would be an unusual state in which to find RNA or DNA, because it is their inherent nature to yield periodic structures during the course of semi-conservative replication. In this second view, the periodicity of DNA (or RNA) is responsible for emergence of primordial genes. Although recent reports on the variety of periodicities present in proteins, genes and genomes are consistent with the second view, it has yet to be experimentally tested. We assessed the significance of periodicities of DNA in the origin of genes by constructing such periodic DNAs. The results showed that periodic DNA produced ordered proteins at very high rates, which is in contrast to the fact that proteins with random sequences lack secondary structures. We concluded that periodicity played a pivotal role in the origin of many genes. The observation should pave the way for new experimental evolution systems for proteins.     

Peptides and the origin of life.

Considering the state-of-the-art views of the geochemical conditions of the primitive earth, it seems most likely that peptides were produced ahead of all other oligomer precursors of biomolecules. Among all the reactions proposed so far for the formation of peptides under primordial earth conditions, the salt-induced peptide formation reaction in connection with adsorption processes on clay minerals would appear to be the simplest and most universal mechanism known to date. The properties of this reaction greatly favor the formation of biologically relevant peptides within a wide variation of environmental conditions such as temperature, pH, and the presence of inorganic compounds. The reaction-inherent preferences of certain peptide linkages make the argument of 'statistical impossibility' of the evolutionary formation of the 'right' peptides and proteins rather insignificant. Indeed, the fact that these sequences are reflected in the preferential sequences of membrane proteins of archaebacteria and prokaryonta distinctly indicates the relevance of this reaction for chemical peptide evolution. On the basis of these results and the recent findings of self-replicating peptides, some ideas have been developed as to the first steps leading to life on earth.     

Prebiological membranes: Synthesis and properties

Ultraviolet irradiation of alkanes on aqueous solutions of phosphate and magnesium yields complex structured products which bear many properties similar to simple membranes. The relevance of this product to prebiologic evolution is discussed from the point of view of phase spparation.     

Mechanism of electron transfer processes photoinduced by lumazine

UV-A (320-400 nm) and UV-B (280-320 nm) radiation causes damage to DNA and other biomolecules through reactions induced by different endogenous or exogenous photosensitizers. Lumazines are heterocyclic compounds present in biological systems as biosynthetic precursors and/or products of metabolic degradation. The parent and unsubstituted compound called lumazine (pteridine-2,4(1,3H)-dione; Lum) is able to act as photosensitizer through electron transfer-initiated oxidations. To get further insight into the mechanisms involved, we have studied in detail the oxidation of 2'-deoxyadenosine 5'-monophosphate (dAMP) photosensitized by Lum in aqueous solution. After UV-A or UV-B excitation of Lum and formation of its triplet excited state ((3)Lum*), three reaction pathways compete for the deactivation of the latter: intersystem crossing to singlet ground state, energy transfer to O(2), and electron transfer between dAMP and (3)Lum* yielding the corresponding pair of radical ions (Lum˙(-) and dAMP˙(+)). In the following step, the electron transfer from Lum˙(-) to O(2) regenerates Lum and forms the superoxide anion (O(2)˙(-)), which undergoes disproportionation into H(2)O(2) and O(2). Finally dAMP˙(+) participates in subsequent reactions to yield products.