Use of Feedback-Resistant Threonine Dehydratases of Corynebacterium glutamicum To Increase Carbon Flux towards l-Isoleucine

Morbach, Susanne; Sahm, Hermann; Eggeling, Lothar

doi:10.1128/aem.61.12.4315-4320.1995

Cited by 51 publications

(31 citation statements)

References 39 publications

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“…In the deletion mutant the value of the diffusion constant (k ) is 1.9 × 10 ¹3 min ¹1 , which shows that the diffusion component of L-lysine transport through the cytoplasmic membrane is negligible. Interestingly, it has become apparent that after deregulation of the assembling pathway of L-threonine (Reinscheid et al, 1994) and Lisoleucine (Morbach et al, 1995), these amino acids accumulate in the cytosol. The efflux of these two amino acids is also carrier dependent (Krä mer, 1994).…”

Section: Discussionmentioning

confidence: 99%

A new type of transporter with a new type of cellular function: l‐lysine export from Corynebacterium glutamicum

Vrljic

Sahm

Eggeling

1996

Molecular Microbiology

229

154

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We discovered that after deregulation of the L-lysine biosynthesis in Corynebacterium glutamicum, L-lysine accumulated in the cytosol and the efflux properties of this amino acid in mutants used for L-lysine production were altered. In this study we describe the cloning and molecular identification of lysE, which encodes the translocator specifically exporting L-lysine from the cell. The lysE gene product does not display homology to any known transporter. It is only 236 amino acids in size, with the potential to span the membrane six times. The LysE protein was oversynthesized to confirm its deduced M(r) of 25425 Da. A probable regulatory gene, lysG, is localized immediately adjacent to lysE and displays all the typical structural features of an autoregulatory transcriptional regulator of the LysR-type family. L-Lysine export is correlated with lysE expression. A null mutant is unable to excrete L-lysine, whereas with overexpressed lysE, L-lysine is exported at a rate of 3.76 nmol min-1 mg-1 dry weight, which is five times the rate that was obtained with the wild type. A deletion mutant was constructed to search for a natural function of this unique carrier. Surprisingly, growth of this mutant is abolished on a salt medium in the presence of the dipeptide Lys-Ala. The quantification of the intracellular L-lysine concentrations revealed that, in response to peptide addition, there was an accumulation of the exceptionally high concentration of more than 1100 mM L-lysine. These results distinguish LysE as an exporter, which: (i) structurally represents a new type of translocator; (ii) demonstrates that exporters are also present for primary metabolites such as amino acids; and (iii) serves in one physiological function to link import with export activity.

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Section: Discussionmentioning

confidence: 99%

A new type of transporter with a new type of cellular function: l‐lysine export from Corynebacterium glutamicum

Vrljic

Sahm

Eggeling

1996

Molecular Microbiology

229

154

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“…Consequently, we recently constructed an L-isoleucine producer of C. glutamicum by concentrating only on the assembling pathway and the conversion reactions of pyr plus oaa to L-isoleucine (Morbach et al, 1996a(Morbach et al, , 1996b. With this strain a product formation flux of up to 0.10 g of L-isoleucine/g dry cell weight per hour and a yield of 0.22 g of L-isoleucine/g glucose through the L-isoleucine biosynthesis pathway was obtained.…”

Section: Discussionmentioning

confidence: 99%

“…Unfortunately, however, in the central metabolism, the targets for obtaining high flux increase are not as well understood as in the final biosynthetic reactions. In the final biosynthetic reactions there are many examples of how the regulatory steps can be bypassed using feedback-resistant enzymes (Cremer et al, 1991;Morbach et al, 1996b;Reinscheid et al, 1994), gene overexpression (Morbach et al, 1996a;, or gene deletion to tailor the biosynthetic pathway. Instead, such general recommendations cannot be given for reactions of the central metabolism with its unusual structure and the many interconverting enzymes, as particularly evident within the carbon-3 (i.e., PEP and pyruvate) and carbon-4 (i.e., oxaloacetate and malate) pools at the entrance of the tricarboxylic acid cycle (TCC) or within the pentose phosphate pathway (PPP).…”

Section: Introductionmentioning

confidence: 99%

Response of the central metabolism ofCorynebacterium glutamicum to different flux burdens

Marx

Striegel

Graaf

et al. 1997

Biotechnol. Bioeng.

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To evaluate the importance of reactions within the central metabolism under different flux burdens the fluxes within the pentose phosphate pathway (PPP), as well as the other reactions of the central metabolism, were intensively analyzed and quantitated. For this purpose, Corynebacterium glutamicum was grown with [1‐13C]glucose to metabolic and isotopic steady state and the fractional enrichments in precursor metabolites (e.g., pentose 5‐phosphate) were quantified. Matrix calculus was used to express these data together with metabolite mass data. The detailed analysis of the dependence of 13C enrichments on exchange fluxes enabled the transketolase‐catalyzed exchange rate (2 pentose 5‐phosphate ↔ sedoheptulose 7‐phosphate + glyceraldehyde 3‐phosphate) to be quantified as 74.3% (molar metabolite flux) at a net flux of 10.3% and the exchange rate (pentose 5‐phosphate + erythrose 4‐phosphate ↔ fructose 6‐phosphate + glyceraldehyde 3‐phosphate) to be quantified as 5.6% at a net flux of 8.1%. The flux entering the tricarboxylic acid cycle was 93.3%. The same comprehensive flux analysis as performed for the nonexcreting condition was done with the identical strain that had been forced to excrete L‐glutamate. Because we had already quantified the fluxes for L‐lysine excretion with an isogenic strain, three directly comparable flux situations are thus available. Consequently, this comparison permits a direct cause‐and‐effect relationship to be specified. In response to the different flux burdens of the cell, the PPP flux decreased from a maximum of 67% to 26%, with the glycolytic flux increasing accordingly. The carbon flux through isocitrate dehydrogenase increased from 20% to 36%. The bidirectional carbon flux between pyruvate and oxaloacetate decreased from 36% to 9%. Since the cause of the three different flux states was the allelic exchange in the final L‐lysine assembling pathway or the glutamate export activity, respectively, the flexible response is the effect. This shows conclusively the enormous flexibility within the central metabolism of C. glutamicum to supply precursors upon their withdrawal for the synthesis of amino acids. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 168–180, 1997.

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“…In view of the discussion in the next section, it is notable that abolition of the feedback inhibition on the first enzyme of the branch to tryptophan made a relatively small contribution to the flux increase. Metabolic engineering for increased amino acid production in Corynebacteria species has been most successful when several enzymes have been over-expressed, in addition to introducing feedback resistance mutations (Colón et al, 1995b;Cremer et al, 1991;Katsumata and Ikeda, 1993;Morbach et al, 1995).…”

Section: Avoiding the Limitations On Flux Increasesmentioning

confidence: 99%

“…Furthermore, simulation studies Hofmeyr and Cornish-Bowden, 1991) suggest that the major effect of abolishing feedback inhibition will be to increase the levels of metabolic intermediates much more than the fluxes. Indeed, accumulation and excretion of intermediates is observed in feedback resistant mutants of amino acid synthetic pathways (Colón et al, 1995a;Katsumata and Ikeda, 1993;Morbach et al, 1995), and is thought to be a factor in causing plasmid instability.…”

Section: Does Feedback Inhibition Limit Pathway Flux?mentioning

confidence: 99%

Increasing the flux in metabolic pathways: A metabolic control analysis perspective

Fell

1998

Biotechnol. Bioeng.

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The problems of engineering increased flux in metabolic pathways are analyzed in terms of the understanding provided by metabolic control analysis. Overexpression of a single enzyme is unlikely to be effective unless it is known to have a high flux control coefficient, which can be used as an approximate predictive tool. This is likely to rule out enzymes subject to feedback inhibition, because it transfers control downstream from the inhibited enzyme to the enzymes utilizing the feedback metabolite. Although abolishing feedback inhibition can restore flux control to an enzyme, it is also likely to cause large increases in the concentrations of metabolic intermediates. Simultaneous and coordinated overexpression of most of the enzymes in a pathway can, in principle, produce substantial flux increases without changes in metabolite levels, though technically it may be difficult to achieve. It is, however, closer to the method used by cells to change flux levels, where coordinated changes in the level of activity of pathway enzymes are the norm. Another option is to increase the demand for the pathway product, perhaps by increasing its rate of excretion or removal.

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Use of Feedback-Resistant Threonine Dehydratases of Corynebacterium glutamicum To Increase Carbon Flux towards l-Isoleucine

Cited by 51 publications

References 39 publications

A new type of transporter with a new type of cellular function: l‐lysine export from Corynebacterium glutamicum

A new type of transporter with a new type of cellular function: l‐lysine export from Corynebacterium glutamicum

Response of the central metabolism ofCorynebacterium glutamicum to different flux burdens

Increasing the flux in metabolic pathways: A metabolic control analysis perspective

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