Design Of Metabolic Pathways Research Articles

Controlling messenger RNA stability in bacteria: strategies for engineering gene expression.

Recent advances in the understanding of prokaryotic gene expression have led scientists to look beyond traditional promoter control for new methods of regulating gene expression. A promising, new technique centers on controlling the stability of messenger RNA. To exploit the potential of mRNA stability for gene expression control, it is important to understand the mechanisms of prokaryotic mRNA decay as well as the cellular factors that can be used to enhance bacterial gene expression through mRNA stabilization. Factors involved in controlling prokaryotic mRNA stability such as nucleases, secondary structures, translation influences, and transcription effects are discussed and analyzed within the context of three prevailing mRNA decay theories. Several strategies for manipulating mRNA stability in genetically-engineered cells are developed from these discussions and presented as a future direction in gene expression control. In the near future, it should be possible to use these strategies to control mRNA stability in such applications as pharmaceutical protein production and metabolic pathway design.

The Metabolic Productivity of the Cell Factory

It is widely accepted that some performance function has been optimized during the evolution of metabolic pathways. One can study the nature of such a function by analogy with the industrial manufacturing world, in which there have been efforts over recent decades to optimize production chains, and in which it is now accepted that fluxes are not the only important system variables that determine process efficiency, because inventory turnover must also be considered. Inspired by the parallels between living cells and manufacturing factories, we propose that fluxes and transit time may have simultaneously been major targets of natural selection in the optimization of the design, structure and kinetic parameters of metabolic pathways. Accordingly we define the ratio of flux to transit time as a performance index of productivity in metabolic systems: it measure the efficiency with which stocks are administered, and facilitates comparison of a pathway in different steady states or in different tissues or organisms. For a linear chain of two enzymes, at a fixed total equilibrium constant, we have analysed the variation of flux, transit time and productivity index as functions of the equilibrium constants of the two steps. The results show that only the productivity index has a maximum, which represents a good compromise in optimizing flux and transit time. We have extended control analysis to the productivity index and derived the summation theorem that applies to it. For linear chains of different length with maximum productivity index values, the distribution of control coefficients with regard to the three parameters has a characteristic profile independent of the length of the chain. Finally, this control profile changes when other variables are optimized, and we compare the theoretical results with the control profile of the first steps of glycolysis in rat liver.

The puzzle of the Krebs citric acid cycle: assembling the pieces of chemically feasible reactions, and opportunism in the design of metabolic pathways during evolution.

The evolutionary origin of the Krebs citric acid cycle has been for a long time a model case in the understanding of the origin and evolution of metabolic pathways: How can the emergence of such a complex pathway be explained? A number of speculative studies have been carried out that have reached the conclusion that the Krebs cycle evolved from pathways for amino acid biosynthesis, but many important questions remain open: Why and how did the full pathway emerge from there? Are other alternative routes for the same purpose possible? Are they better or worse? Have they had any opportunity to be developed in cellular metabolism evolution? We have analyzed the Krebs cycle as a problem of chemical design to oxidize acetate yielding reduction equivalents to the respiratory chain to make ATP. Our analysis demonstrates that although there are several different chemical solutions to this problem, the design of this metabolic pathway as it occurs in living cells is the best chemical solution: It has the least possible number of steps and it also has the greatest ATP yielding. Study of the evolutionary possibilities of each one-taking the available material to build new pathways-demonstrates that the emergence of the Krebs cycle has been a typical case of opportunism in molecular evolution. Our analysis proves, therefore, that the role of opportunism in evolution has converted a problem of several possible chemical solutions into a single-solution problem, with the actual Krebs cycle demonstrated to be the best possible chemical design. Our results also allow us to derive the rules under which metabolic pathways emerged during the origin of life.

The Puzzle of the Krebs Citric Acid Cycle: Assembling the Pieces of Chemically Feasible Reactions, and Opportunism in the Design of Metabolic Pathways During Evolution

The Puzzle of the Krebs Citric Acid Cycle: Assembling the Pieces of Chemically Feasible Reactions, and Opportunism in the Design of Metabolic Pathways During Evolution

Genetic and physiological approaches for the production of amino acids

The purposeful design of metabolic pathways and networks in order to increase the yield and productivity of microorganisms capable of amino acid production is still a major goal of modem biotechnology. Knowledge of fundamental aspects of physiology, biochemistry, molecular biology and bioprocess engineering is described which must be optimized further in order to reach this goal. These fundamentals refer to the following topics, (a) Improved knowledge of the relevant pathways in biotechnologically relevant amino-acid-producing microorganisms. This must include metabolic regulation and integration into global regulatory networks of the cell, (b) Detailed flux analysis of all relevant pathways. This refers to substrate uptake, to the central and peripheral carbon, redox and energy fluxes, as well as product excretion. A network of data is thus established, which provides a basis for a purposeful metabolic design, (c) Redirection of the carbon, redox and energy flux in a coordinated and fine-tuned way, thus, limiting precursor availability, by-product formation and imbalanced pool sizes, as well as avoiding problems with the redox and energy balance.

Pathway engineering for production of aromatics in Escherichia coli: Confirmation of stoichiometric analysis by independent modulation of AroG, TktA, and Pps activities

The synthesis of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) is the first commitment of resources toward aromatics production in Escherichia coli. DAHP is produced during a condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) catalyzed by DAHP synthases (coded by aroF, aroG, and aroH). Stoichiometric analysis has shown a severe PEP limitation in the theoretical yield of DAHP production from glucose due to the phosphotransferase system (PTS) for sugar uptake. This limitation can be relieved by (i) the recycling of pyruvate from PEP using PEP synthase (Pps) or (ii) use of non-PTS sugars such as xylose. Previous studies have shown the usefulness of overexpressing tktA (encoding transketolase), aroG, and pps (PEP synthase) for DAHP production in an aroB strain unable to utilize DAHP further. In the present study we confirm the predictions of the stoichiometric analysis by introducing pps, tktA, and aroG into vectors under independently controlled promoters. In glucose medium, although TktA has some positive effect on the final DAHP concentration, it has no effect on the yield (percent conversion). With Pps overexpression, the DAHP concentration produced from glucose is increased almost twofold and the yield is approaching the theoretical maximum, as predicted by the stoichiometric analysis. However, this Pps effect is observed only in the presence of both increased AroG and TktA. In xylose mimimal medium, the final DAHP concentration and the yield are completely determined by the AroG activity. TktA and Pps play no or insignificant roles, and the yield can reach the theoretical maximum without overexpression of these two enzymes. The results shown here are important for both rational design of metabolic pathways and industrial production of aromatics such as tryptophan, phenylalanine, indigo, quinic acid, and catechol.

Degradation of chlorinated aromatic compounds in UASB reactors

Data on anaerobic degradation of chloroaromatic compounds in Upflow Anaerobic Sludge Blanket Reactors (UASB-reactor) are presented and compared. Special attention is given to the metabolic pathways for degradation of chlorinated phenols by granular sludge. Results indicate that PCP can be degraded in UASB-reactors via stepwise dechlorination to phenol. Phenol will subsequently be converted to benzoate before ring cleavage. Dechlorination proceeds via different pathways dependent upon the inocula used. Results are further presented on the design of special metabolic pathways in granules which do not possess this activity using the dechlorinating organism, Desulfomonile tiedjei. Additionally, it is shown that it is possible to immobilize Dechlorosporium hafniense, a newly isolated dechlorinating anaerobe, into granular sludge, thereby introducing an ability not previously present in the granules.

Degradation of chlorinated aromatic compounds in UASB reactors

Data on anaerobic degradation of chloroaromatic compounds in Upflow Anaerobic Sludge Blanket Reactors (UASB-reactor) are presented and compared. Special attention is given to the metabolic pathways for degradation of chlorinated phenols by granular sludge. Results indicate that PCP can be degraded in UASB-reactors via stepwise dechlorination to phenol. Phenol will subsequently be converted to benzoate before ring cleavage. Dechlorination proceeds via different pathways dependent upon the inocula used. Results are further presented on the design of special metabolic pathways in granules which do not possess this activity using the dechlorinating organism, Desulfomonile tiedjei. Additionally, it is shown that it is possible to immobilize Dechlorosporium hafniense, a newly isolated dechlorinating anaerobe, into granular sludge, thereby introducing an ability not previosly present in the granules.

Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110.

Flux balance models of metabolism use stoichiometry of metabolic pathways, metabolic demands of growth, and optimality principles to predict metabolic flux distribution and cellular growth under specified environmental conditions. These models have provided a mechanistic interpretation of systemic metabolic physiology, and they are also useful as a quantitative tool for metabolic pathway design. Quantitative predictions of cell growth and metabolic by-product secretion that are experimentally testable can be obtained from these models. In the present report, we used independent measurements to determine the model parameters for the wild-type Escherichia coli strain W3110. We experimentally determined the maximum oxygen utilization rate (15 mmol of O2 per g [dry weight] per h), the maximum aerobic glucose utilization rate (10.5 mmol of Glc per g [dry weight] per h), the maximum anaerobic glucose utilization rate (18.5 mmol of Glc per g [dry weight] per h), the non-growth-associated maintenance requirements (7.6 mmol of ATP per g [dry weight] per h), and the growth-associated maintenance requirements (13 mmol of ATP per g of biomass). The flux balance model specified by these parameters was found to quantitatively predict glucose and oxygen uptake rates as well as acetate secretion rates observed in chemostat experiments. We have formulated a predictive algorithm in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures. In aerobic experiments we observed acetate secretion, accumulation in the culture medium, and reutilization from the culture medium. In fed-batch cultures acetate is cometabolized with glucose during the later part of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS)

Economy of design in metabolic pathways: Further remarks on the game of the pentose phosphate cycle

Sugar rearrangement in the pentose phosphate cycle and other related pathways were previously analysed by abstraction to a mathematical game of optimization based on certain hypotheses concerning enzymatic mechanisms of living cells (Meléndez-Hevia & Isidoro, 1985, J. theor. Biol. 117, 251-263). The solution of that problem shows that the metabolic pathway, as occurs in cells, is the simplest solution of the operative problem. However, in that work, only the number of carbons in every sugar was considered. In the present paper, all structural features of the sugars and reaction mechanisms are taken into consideration, and the problem is again considered by abstraction to a mathematical model which includes all structural features of the sugars as well as all structural requirements of the enzymes in the hypotheses of the mechanisms. As in the above-mentioned paper, the hypothesis of simplicity is also imposed in order to achieve the objective (to convert six ribulose 5-phosphate into five glucose 6-phosphate) in the least number of steps (or with the least number of free intermediates), and the least number of carbons in the intermediates. It is concluded that the optimal, or simplest, solution of this problem is the same procedure as that occurring in living cells. The Calvin cycle in photosynthesis and the "L-type" of the pentose phosphate cycle are also analysed arriving at similar conclusions in both cases. These results suggest some reflections about the logic in the design of metabolic pathways, and the possible role of the hypothesis of simplicity in cell evolution.

A theoretical approach to the evolution and structural design of enzymatic networks; Linear enzymatic chains, branched pathways and glycolysis of erythrocytes

A theoretical approach to the explanation of the structural design of metabolic pathways is presented. It is based on the hypothesis that due to natural selection during evolution the cellular metabolism of present-day organisms may be characterized by optimal properties. Two cardinal terms enter the theory: (i) the efficiency of a metabolic pathway and (ii) the evolutionary effort for the change of the kinetic parameters of enzymes by mutations of the corresponding genes. For both quantities simple mathematical expressions are proposed. While the efficiency is related to the reaction rates of the enzymes constituting the metabolic pathway, the evolutionary effort is considered to be a monotonically increasing function of the parameter values. By maximizing the efficiency under the constraint of a fixed evolutionary effort the theory allows the calculation of the optimal parameter distribution as the outcome of evolution processes. The methods developed are applied to the following systems: (a) linear reaction sequences with very low affinities of the enzymes towards substrates, (b) linear sequences consisting of saturable enzymatic reactions, (c) branched metabolic pathways consisting of segments of linear chains and (d) glycolysis of erythrocytes. The conclusion is derived that the optimal distribution of kinetic constants depends strongly on the equilibrium constants of the reactions as well as on the total osmolarity of the metabolic intermediates. Without osmotic constraints the evolutionary effort is mainly spent on the enzymes at the beginning of the chain. Using Michaelis-Menten equations the optimal state is characterized by a decrease of the maximal activities of the enzymes towards the end of the chain. These results are modified if osmotic constraints are taken into account. At the investigation of branched pathways the following results were obtained: firstly, if a certain end product may be synthesized along different pathways those which are thermodynamically more unfavourable (e.g. characterized by a small change of free energy) are eliminated in the course of evolution; secondly, if a branched pathway leads to several important end products those reaction segments which are thermodynamically unfavourable are characterized by a higher evolutionary effort. The application of the theory to a realistic model of glycolysis of erythrocytes leads to a correct desription of various functionally important properties of the system, such as the ratio between fluxes through different branches and theATP/ADPratio, whereas the theory cannot predict the strong separation of time constants observed in the real glycolytic system. It is concluded that the improvement of the predictive power of the theory necessitates the use of more complex functionals for the efficiency which take into account not only the fluxes but also other system properties such as the stability of the pathway or homoeostatic effects.