Quantifying and directing metabolite flux: Application to amino acid overproduction

Eggeling, Lothar; Sahm, Hermann; Graaf, Albert A. de

doi:10.1007/bfb0102331

Cited by 18 publications

(16 citation statements)

References 136 publications

(127 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, the recent development of metabolic engineering tools, allowing the rational design of microorganisms for metabolite production (27), prompted us to evaluate an alternative process for trehalose overproduction in the gram-positive bacterium Corynebacterium glutamicum (23). C. glutamicum was chosen for three major reasons: (i) it produces, and excretes, trehalose (15); (ii) it has a metabolic control architecture simpler than that of other microorganisms, maybe as a result of its comparatively small 3,500-kb genome size (10); and (iii) it is widely used in industrial biotechnological processes (10).…”

mentioning

confidence: 99%

Overproduction of Trehalose: Heterologous Expression of Escherichia coli Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Corynebacterium glutamicum

Padilla

Krämer

Stephanopoulos

et al. 2004

Appl Environ Microbiol

View full text Add to dashboard Cite

Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five-to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum.Trehalose (1-␣-glucopyranosyl-1-␣-glucopyranoside) is a nonreducing, particularly stable disaccharide formed by two glucose moieties (37). As a compatible osmolite (22) and protein stabilizer (39), trehalose shows a wide range of potential applications in biotechnology (increased stress tolerance of important crops, stability of recombinant proteins, etc.), as well as in the food industry (37).In the past, trehalose was produced by using Saccharomyces cerevisiae (32). The high cost of this system and the promising applications of the disaccharide led to the development of a new trehalose production process based on the enzymatic biotransformation of maltodextrins (25,26). The success of the enzymatic process has limited the interest in using microorganisms as alternative sources for trehalose synthesis. However, the recent development of metabolic engineering tools, allowing the rational design of microorganisms for metabolite production (27), prompted us to evaluate an alternative process for trehalose overproduction in the gram-positive bacterium Corynebacterium glutamicum (23). C. glutamicum was chosen for three major reasons: (i) it produces, and excretes, trehalose (15); (ii) it has a metabolic control architecture simpler than that of other microorganisms, maybe as a result of its comparatively small 3,500-kb genome size (10); and (iii) it is widely used in industrial biotechnological processes (10).Three pathways for trehalose synthesis have been characterized in C. glutamicum (45), similarly to that found in Mycobacterium species (7) (Fig. 1). The first is the TreS pathway, in which trehalose is formed by maltose isomerization (7, 31).The second is the TreY-TreZ pathway, le...

show abstract

mentioning

confidence: 99%

Overproduction of Trehalose: Heterologous Expression of Escherichia coli Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Corynebacterium glutamicum

Padilla

Krämer

Stephanopoulos

et al. 2004

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…It is also the most widely used microorganism for the industrial synthesis of amino acids (5). Although the physiological response of C. glutamicum to increases in osmotic pressure has been studied extensively, research has concentrated on individual pathways and not the organism's systemic response (7,9,10).…”

mentioning

confidence: 99%

Osmotic Stress Response: Quantification of Cell Maintenance and Metabolic Fluxes in a Lysine-Overproducing Strain of Corynebacterium glutamicum

Varela

Báez

Agosín

2004

Appl Environ Microbiol

View full text Add to dashboard Cite

Osmotic stress diminishes cell productivity and may cause cell inactivation in industrial fermentations. The quantification of metabolic changes under such conditions is fundamental for understanding and describing microbial behavior during bioprocesses. We quantified the gradual changes that take place when a lysineoverproducing strain of Corynebacterium glutamicum is grown in continuous culture with saline gradients at different dilution rates. The use of compatible solutes depended on environmental conditions; certain osmolites predominated at different dilution rates and extracellular osmolalities. A metabolic flux analysis showed that at high dilution rates C. glutamicum redistributed its metabolic fluxes, favoring energy formation over growth. At low dilution rates, cell metabolism accelerated as the osmolality was steadily increased. Flexibility in the oxaloacetate node proved to be key for the energetic redistribution that occurred when cells were grown at high dilution rates. Substrate and ATP maintenance coefficients increased 30-and 5-fold, respectively, when the osmolality increased, which demonstrates that energy pool management is fundamental for sustaining viability.In industrial fermentations, increases in osmotic pressure, mostly a direct result of product accumulation, disrupt bacterial growth and the production of desirable compounds and may inactivate cells in many bioprocesses (26).One strategy that microbes adopt to counteract higher osmotic pressure and to improve the likelihood of surviving such conditions is making use of organic intracellular solutes. Compatible solutes enable the cell to achieve osmotic balance without altering the cell's metabolic functions (35). The regulation of intracellular solute concentrations in response to the salinity of the medium provides the cell significant scope for adapting to changes in osmolality in the cell's environment (19).Aside from altering compatible solute concentrations, the higher energy requirements for cell maintenance under osmotic stress also disrupt energy pool management (18). "Maintenance" covers every cellular reaction involving the consumption of ATP that does not contribute to the net synthesis of biomass. Such reactions include the need to build up and maintain steep ion concentration gradients across the membrane and macromolecular turnover (27).Microbial growth and cellular productivity are driven by the flux balance generated between anabolic and catabolic reactions, as a higher osmotic pressure provokes numerous changes in the cell's metabolism. These fluxes vary considerably in response to changing environmental conditions (4, 34). Hence, a means for evaluating osmo-induced changes is necessary for the assessment and quantification of significant shifts within the metabolic network. Metabolic flux analysis is a powerful tool for quantifying such changes (22,(31)(32)(33).Corynebacterium glutamicum is particularly appropriate for studying responses to osmotic stress, as this microorganism adapts efficiently to changes in osmoti...

show abstract

“…Flux is the focal point of metabolic engineering. Different means of analyzing flux are (1) kinetic based models, (2) control theories, (3) tracer experiments, (4) magnetization transfer, (5) metabolite balancing, (6) enzyme analysis, and (7) genetic analysis [22]. Metabolic control analysis revealed that the overall flux through a metabolic pathway depends on several steps, not just a single rate-limiting reaction [31].…”

Section: Process Development In the Pilot Plant And Factorymentioning

confidence: 99%

From natural products discovery to commercialization: a success story

Demain

2006

J IND MICROBIOL BIOTECHNOL

116

View full text Add to dashboard Cite

In order for a natural product to become a commercial reality, laboratory improvement of its production process is a necessity since titers produced by wild strains could never compete with the power of synthetic chemistry. Strain improvement by mutagenesis has been a major success. It has mainly been carried out by "brute force" screening or selection, but modern genetic technologies have entered the scene in recent years. For every new strain developed genetically, there is further opportunity to raise titers by medium modifications. Of major interest has been the nutritional control by induction, as well as inhibition and repression by sources of carbon, nitrogen, phosphate and end products. Both strain improvement and nutritional modification contribute to the new process, which is then scaled up by biochemical engineers into pilot scale and later into factory size fermentors.

show abstract

Quantifying and directing metabolite flux: Application to amino acid overproduction

Abstract: targets of the L-lysine and L-isoleucine biosynthesis of Corynebacterium glutamicum, high amino acid overproduction can be obtained with this bacterium.

Cited by 18 publications

References 136 publications

Overproduction of Trehalose: Heterologous Expression of Escherichia coli Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Corynebacterium glutamicum

Overproduction of Trehalose: Heterologous Expression of Escherichia coli Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Corynebacterium glutamicum

Osmotic Stress Response: Quantification of Cell Maintenance and Metabolic Fluxes in a Lysine-Overproducing Strain of Corynebacterium glutamicum

From natural products discovery to commercialization: a success story

Contact Info

Product

Resources

About