Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. I. Growth-rate-dependent metabolic efficiency at steady state

Kayser, Anke; Weber, Jan; Hecht, V.; Rinas, Ursula

doi:10.1099/mic.0.27481-0

Cited by 167 publications

(195 citation statements)

References 41 publications

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“…Now, the cell converts more sugar directly to cell growth. Maximal values of yield of biomass from ATP have been widely estimated before in aerobic E. coli grown in glucose and other carbon sources (30,31); our model is consistent with that data. Third, increasing sugar leads to upshifting the production of ribosomes relative to NRPs (6,10,32,33).…”

Section: The Cell Shifts Its Energy Flows Under Different Growth Condsupporting

confidence: 79%

Bacterial growth laws reflect the evolutionary importance of energy efficiency

Maitra

Dill

2014

Proc. Natl. Acad. Sci. U.S.A.

174

208

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We are interested in the balance of energy and protein synthesis in bacterial growth. How has evolution optimized this balance? We describe an analytical model that leverages extensive literature data on growth laws to infer the underlying fitness landscape and to draw inferences about what evolution has optimized in Escherichia coli. Is E. coli optimized for growth speed, energy efficiency, or some other property? Experimental data show that at its replication speed limit, E. coli produces about four mass equivalents of nonribosomal proteins for every mass equivalent of ribosomes. This ratio can be explained if the cell's fitness function is the the energy efficiency of cells under fast growth conditions, indicating a tradeoff between the high energy costs of ribosomes under fast growth and the high energy costs of turning over nonribosomal proteins under slow growth. This model gives insight into some of the complex nonlinear relationships between energy utilization and ribosomal and nonribosomal production as a function of cell growth conditions. Monod observed that increasing glucose increases Escherichia coli's growth rate, up to a maximum rate beyond which the cells cannot replicate any faster (1). On the one hand, such growth laws are experimentally observable. On the other hand, growth laws, per se, do not give insight into the evolutionary driving forces that lead to them.Evolutionary principles are expressed by fitness landscapes (13), which are mathematical surfaces that represent how the organism's fitness depends on some cellular property that can be altered by evolution over time. Peaks on fitness landscapes represent states of maximal fitness. To understand why a cell has a particular growth law, we need a mathematical model that relates its growth law (how the growth rate of the cellular population depends on food concentration) to its underlying fitness landscape (how the cell's growth parameters can be altered through evolution). Thus far, this is relatively uncharted territory for cellular modeling. Here, we develop a model to explore how bacteria balance their fluxes of energy and ribosomal (RPs) and nonribosomal proteins (NRPs). By comparing the model with data, we can explore possible fitness objectives for bacterial replication. Are bacteria evolutionarily optimized to maximize their duplication speed? Or, are bacteria evolutionarily optimized to maximize the energy efficiency of their duplication processes? Or, something else? By "evolutionarily optimized," we mean the tradeoffs that a cell must make. By evaluating extensive growth data on E. coli through the lens of the present model, which relates growth observables to fitness landscapes, we conclude that a principal evolutionary driving force for bacteria is the energy efficiency of the fastest-growing cells. Fig. 1 shows our kinetic model of bacteria growing in the exponential phase. This model defines relationships among four dynamical quantities: the rate of synthesis of ribosomal proteins, the synthesis and degradation rates of NRP...

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Section: The Cell Shifts Its Energy Flows Under Different Growth Condsupporting

confidence: 79%

Bacterial growth laws reflect the evolutionary importance of energy efficiency

Maitra

Dill

2014

Proc. Natl. Acad. Sci. U.S.A.

174

208

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“…(6) is the most popular one to describe the relationship between the specific substrate uptake rate and other cell culture parameters (Pirt, 1965). It has been widely used to study the maximum cell yield on substrate and maintenance coefficient (Abbott and Clamen, 1973;Christensen et al, 1995;Kayser et al, 2005). The maintenance coefficient indicates the amount of substrate needed per unit cell mass per hour to maintain cell in an active and proper physiological condition.…”

Section: Resultsmentioning

confidence: 99%

Kinetics of continuous cultivation of the oleaginous yeast Rhodosporidium toruloides

Shen

Gong

Yang

et al. 2013

Journal of Biotechnology

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“…4 and 5). Typically, wild-type E. coli strains, including E. coli JM101, form acetate above critical glucose uptake rates of 3.4 to 5.5 mmol (g CDW) Ϫ1 h Ϫ1 at growth rates and temperatures of between 0.35 and 0.4 h Ϫ1 and 28 and 37°C, respectively (16,31,44). The presence of NADH oxidase increased the critical glucose uptake rate from 4.44 to 6.67 mmol (g CDW) Ϫ1 h Ϫ1 at growth rates of between 0.40 and 0.36 h Ϫ1 (71), whereas styrene epoxidation caused pronounced acetate formation already at glucose uptake rates of above 2 mmol (g CDW) Ϫ1 h Ϫ1 and a low growth rate of 0.1 h Ϫ1 .…”

mentioning

confidence: 99%

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

Bühler

Park

Blank

et al. 2008

Appl Environ Microbiol

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Styrene can efficiently be oxidized to (S)-styrene oxide by recombinant Escherichia coli expressing the styrene monooxygenase genes styAB from Pseudomonas sp. strain VLB120. Targeting microbial physiology during whole-cell redox biocatalysis, we investigated the interdependency of styrene epoxidation, growth, and carbon metabolism on the basis of mass balances obtained from continuous two-liquid-phase cultures. Full induction of styAB expression led to growth inhibition, which could be attenuated by reducing expression levels. Operation at subtoxic substrate and product concentrations and variation of the epoxidation rate via the styrene feed concentration allowed a detailed analysis of carbon metabolism and bioconversion kinetics. Fine-tuned styAB expression and increasing specific epoxidation rates resulted in decreasing biomass yields, increasing specific rates for glucose uptake and the tricarboxylic acid (TCA) cycle, and finally saturation of the TCA cycle and acetate formation. Interestingly, the biocatalysis-related NAD(P)H consumption was 3.2 to 3.7 times higher than expected from the epoxidation stoichiometry. Possible reasons include uncoupling of styrene epoxidation and NADH oxidation and increased maintenance requirements during redox biocatalysis. At epoxidation rates of above 21 mol per min per g cells (dry weight), the absence of limitations by O 2 and styrene and stagnating NAD(P)H regeneration rates indicated that NADH availability limited styrene epoxidation. During glucose-limited growth, oxygenase catalysis might induce regulatory stress responses, which attenuate excessive glucose catabolism and thus limit NADH regeneration. Optimizing metabolic and/or regulatory networks for efficient redox biocatalysis instead of growth (yield) is likely to be the key for maintaining high oxygenase activities in recombinant E. coli.Oxygenases catalyze regio-and enantioselective oxyfunctionalization reactions and have a considerable potential in the area of asymmetric organic synthesis (2, 35). These enzymes typically depend on coenzymes such as NAD(P)H and are unstable outside cells, and they often consist of multiple components. Thus, for synthetic applications, their use in whole cells is favored over the use of isolated enzymes (7,18).The productivity of oxygenase-based whole-cell biocatalysts is influenced by various factors, such as maximal enzyme activity, enzyme level, substrate mass transfer, substrate and/or product inhibition/toxicity, and cofactor regeneration (7,13,54,68). Enzyme synthesis and cofactor regeneration are related to host metabolism, which in turn may be affected by the toxicity of organic substrates and products. Such toxicity can efficiently be attenuated by regulated substrate feeding (1, 11) and in situ product removal (7,38,61,66,74). Yet, microbial cells, especially in a nongrowing state, often lose biooxidation activity over time, which may be due to oxygenase inactivation and/or the metabolic burden imposed by redox-coenzyme withdrawal and reactive oxygen species formed via...

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Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. I. Growth-rate-dependent metabolic efficiency at steady state

Cited by 167 publications

References 41 publications

Bacterial growth laws reflect the evolutionary importance of energy efficiency

Bacterial growth laws reflect the evolutionary importance of energy efficiency

Kinetics of continuous cultivation of the oleaginous yeast Rhodosporidium toruloides

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

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