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John H. Mccusker - One of the best experts on this subject based on the ideXlab platform.
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fungal Homoserine Kinase thr1δ mutants are attenuated in virulence and die rapidly upon threonine starvation and serum incubation
Eukaryotic Cell, 2010Co-Authors: Joanne M. Kingsbury, John H. MccuskerAbstract:The fungally conserved subset of amino acid biosynthetic enzymes not present in humans offer exciting potential as an unexploited class of antifungal drug targets. Since threonine biosynthesis is essential in Cryptococcus neoformans, we further explored the potential of threonine biosynthetic enzymes as antifungal drug targets by determining the survival in mice of Saccharomyces cerevisiae Homoserine Kinase (thr1Δ) and threonine synthase (thr4Δ) mutants. In striking contrast to aspartate Kinase (hom3Δ) mutants, S. cerevisiae thr1Δ and thr4Δ mutants were severely depleted after only 4 h in vivo. Similarly, Candida albicans thr1Δ mutants, but not hom3Δ mutants, were significantly attenuated in virulence. Consistent with the in vivo phenotypes, S. cerevisiae thr1Δ and thr4Δ mutants as well as C. albicans thr1Δ mutants were extremely serum sensitive. In both species, serum sensitivity was suppressed by the addition of threonine, a feedback inhibitor of Hom3p. Because mutation of the HOM3 and HOM6 genes, required for the production of the toxic pathway intermediate Homoserine, also suppressed serum sensitivity, we hypothesize that serum sensitivity is a consequence of Homoserine accumulation. Serum survival is critical for dissemination, an important virulence determinant: thus, together with the essential nature of C. neoformans threonine synthesis, the cross-species serum sensitivity of thr1Δ mutants makes the fungus-specific Thr1p, and likely Thr4p, ideal antifungal drug targets.
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Homoserine Toxicity in Saccharomyces cerevisiae and Candida albicans Homoserine Kinase (thr1Δ) Mutants
Eukaryotic cell, 2010Co-Authors: Joanne M. Kingsbury, John H. MccuskerAbstract:In addition to threonine auxotrophy, mutation of the Saccharomyces cerevisiae threonine biosynthetic genes THR1 (encoding Homoserine Kinase) and THR4 (encoding threonine synthase) results in a plethora of other phenotypes. We investigated the basis for these other phenotypes and found that they are dependent on the toxic biosynthetic intermediate Homoserine. Moreover, Homoserine is also toxic for Candida albicans thr1Δ mutants. Since increasing levels of threonine, but not other amino acids, overcome the Homoserine toxicity of thr1Δ mutants, Homoserine may act as a toxic threonine analog. Homoserine-mediated lethality of thr1Δ mutants is blocked by cycloheximide, consistent with a role for protein synthesis in this lethality. We identified various proteasome and ubiquitin pathway components that either when mutated or present in high copy numbers suppressed the thr1Δ mutant Homoserine toxicity. Since the doa4Δ and proteasome mutants identified have reduced ubiquitin- and/or proteasome-mediated proteolysis, the degradation of a particular protein or subset of proteins likely contributes to Homoserine toxicity.
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Fungal Homoserine Kinase (thr1δ) mutants are attenuated in virulence and die rapidly upon threonine starvation and serum incubation.
Eukaryotic cell, 2010Co-Authors: Joanne M. Kingsbury, John H. MccuskerAbstract:The fungally conserved subset of amino acid biosynthetic enzymes not present in humans offer exciting potential as an unexploited class of antifungal drug targets. Since threonine biosynthesis is essential in Cryptococcus neoformans, we further explored the potential of threonine biosynthetic enzymes as antifungal drug targets by determining the survival in mice of Saccharomyces cerevisiae Homoserine Kinase (thr1Delta) and threonine synthase (thr4Delta) mutants. In striking contrast to aspartate Kinase (hom3Delta) mutants, S. cerevisiae thr1Delta and thr4Delta mutants were severely depleted after only 4 h in vivo. Similarly, Candida albicans thr1Delta mutants, but not hom3Delta mutants, were significantly attenuated in virulence. Consistent with the in vivo phenotypes, S. cerevisiae thr1Delta and thr4Delta mutants as well as C. albicans thr1Delta mutants were extremely serum sensitive. In both species, serum sensitivity was suppressed by the addition of threonine, a feedback inhibitor of Hom3p. Because mutation of the HOM3 and HOM6 genes, required for the production of the toxic pathway intermediate Homoserine, also suppressed serum sensitivity, we hypothesize that serum sensitivity is a consequence of Homoserine accumulation. Serum survival is critical for dissemination, an important virulence determinant: thus, together with the essential nature of C. neoformans threonine synthesis, the cross-species serum sensitivity of thr1Delta mutants makes the fungus-specific Thr1p, and likely Thr4p, ideal antifungal drug targets.
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Fungal Homoserine Kinase (thr1) Mutants Are Attenuated in Virulence and Die Rapidly upon Threonine Starvation and Serum Incubation†
2010Co-Authors: Joanne M. Kingsbury, John H. MccuskerAbstract:The fungally conserved subset of amino acid biosynthetic enzymes not present in humans offer exciting potential as an unexploited class of antifungal drug targets. Since threonine biosynthesis is essential in Cryptococcus neofor-mans, we further explored the potential of threonine biosynthetic enzymes as antifungal drug targets by determining the survival in mice of Saccharomyces cerevisiae Homoserine Kinase (thr1) and threonine synthase (thr4) mutants. In striking contrast to aspartate Kinase (hom3) mutants, S. cerevisiae thr1 and thr4 mutants were severely depleted after only 4 h in vivo. Similarly, Candida albicans thr1mutants, but not hom3mutants, were significantly attenuated in virulence. Consistent with the in vivo phenotypes, S. cerevisiae thr1 and thr4 mutants as well as C. albicans thr1 mutants were extremely serum sensitive. In both species, serum sensitivity was suppressed by the addition of threonine, a feedback inhibitor of Hom3p. Because mutation of the HOM3 and HOM6 genes, required for the production of the toxic pathway intermediate Homoserine, also suppressed serum sensitivity, we hypothesize that serum sensitivity is a consequence of Homoserine accumulation. Serum survival is critical for dissemination, an important virulence determinant: thus, together with the essential nature of C. neoformans threonine synthesis, the cross-species serum sensitivity of thr1 mutants makes the fungus-specific Thr1p, and likely Thr4p, ideal antifungal drug targets. Fungal infections are an increasingly significant cause o
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Threonine biosynthetic genes are essential in Cryptococcus neoformans.
Microbiology (Reading England), 2008Co-Authors: Joanne M. Kingsbury, John H. MccuskerAbstract:We identified and attempted to disrupt the Cryptococcus neoformans Homoserine and/or threonine biosynthetic genes encoding aspartate Kinase (HOM3), Homoserine Kinase (THR1) and threonine synthase (THR4); however, each gene proved recalcitrant to disruption. By replacing the endogenous promoters of HOM3 and THR1 with the copper-repressible CTR4-1 promoter, we showed that HOM3 and THR1 were essential for the growth of C. neoformans in rich media, when ammonium was the nitrogen source, or when threonine was supplied as an amino acid instead of a dipeptide. Moreover, the severity of the growth defect associated with HOM3 or THR1 repression increased with increasing incubation temperature. We believe this to be the first demonstration of threonine biosynthetic genes being essential in a fungus. The necessity of these genes for C. neoformans growth, particularly at physiologically relevant temperatures, makes threonine biosynthetic genes ideal anti-cryptococcal drug targets.
Joanne M. Kingsbury - One of the best experts on this subject based on the ideXlab platform.
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fungal Homoserine Kinase thr1δ mutants are attenuated in virulence and die rapidly upon threonine starvation and serum incubation
Eukaryotic Cell, 2010Co-Authors: Joanne M. Kingsbury, John H. MccuskerAbstract:The fungally conserved subset of amino acid biosynthetic enzymes not present in humans offer exciting potential as an unexploited class of antifungal drug targets. Since threonine biosynthesis is essential in Cryptococcus neoformans, we further explored the potential of threonine biosynthetic enzymes as antifungal drug targets by determining the survival in mice of Saccharomyces cerevisiae Homoserine Kinase (thr1Δ) and threonine synthase (thr4Δ) mutants. In striking contrast to aspartate Kinase (hom3Δ) mutants, S. cerevisiae thr1Δ and thr4Δ mutants were severely depleted after only 4 h in vivo. Similarly, Candida albicans thr1Δ mutants, but not hom3Δ mutants, were significantly attenuated in virulence. Consistent with the in vivo phenotypes, S. cerevisiae thr1Δ and thr4Δ mutants as well as C. albicans thr1Δ mutants were extremely serum sensitive. In both species, serum sensitivity was suppressed by the addition of threonine, a feedback inhibitor of Hom3p. Because mutation of the HOM3 and HOM6 genes, required for the production of the toxic pathway intermediate Homoserine, also suppressed serum sensitivity, we hypothesize that serum sensitivity is a consequence of Homoserine accumulation. Serum survival is critical for dissemination, an important virulence determinant: thus, together with the essential nature of C. neoformans threonine synthesis, the cross-species serum sensitivity of thr1Δ mutants makes the fungus-specific Thr1p, and likely Thr4p, ideal antifungal drug targets.
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Homoserine Toxicity in Saccharomyces cerevisiae and Candida albicans Homoserine Kinase (thr1Δ) Mutants
Eukaryotic cell, 2010Co-Authors: Joanne M. Kingsbury, John H. MccuskerAbstract:In addition to threonine auxotrophy, mutation of the Saccharomyces cerevisiae threonine biosynthetic genes THR1 (encoding Homoserine Kinase) and THR4 (encoding threonine synthase) results in a plethora of other phenotypes. We investigated the basis for these other phenotypes and found that they are dependent on the toxic biosynthetic intermediate Homoserine. Moreover, Homoserine is also toxic for Candida albicans thr1Δ mutants. Since increasing levels of threonine, but not other amino acids, overcome the Homoserine toxicity of thr1Δ mutants, Homoserine may act as a toxic threonine analog. Homoserine-mediated lethality of thr1Δ mutants is blocked by cycloheximide, consistent with a role for protein synthesis in this lethality. We identified various proteasome and ubiquitin pathway components that either when mutated or present in high copy numbers suppressed the thr1Δ mutant Homoserine toxicity. Since the doa4Δ and proteasome mutants identified have reduced ubiquitin- and/or proteasome-mediated proteolysis, the degradation of a particular protein or subset of proteins likely contributes to Homoserine toxicity.
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Fungal Homoserine Kinase (thr1δ) mutants are attenuated in virulence and die rapidly upon threonine starvation and serum incubation.
Eukaryotic cell, 2010Co-Authors: Joanne M. Kingsbury, John H. MccuskerAbstract:The fungally conserved subset of amino acid biosynthetic enzymes not present in humans offer exciting potential as an unexploited class of antifungal drug targets. Since threonine biosynthesis is essential in Cryptococcus neoformans, we further explored the potential of threonine biosynthetic enzymes as antifungal drug targets by determining the survival in mice of Saccharomyces cerevisiae Homoserine Kinase (thr1Delta) and threonine synthase (thr4Delta) mutants. In striking contrast to aspartate Kinase (hom3Delta) mutants, S. cerevisiae thr1Delta and thr4Delta mutants were severely depleted after only 4 h in vivo. Similarly, Candida albicans thr1Delta mutants, but not hom3Delta mutants, were significantly attenuated in virulence. Consistent with the in vivo phenotypes, S. cerevisiae thr1Delta and thr4Delta mutants as well as C. albicans thr1Delta mutants were extremely serum sensitive. In both species, serum sensitivity was suppressed by the addition of threonine, a feedback inhibitor of Hom3p. Because mutation of the HOM3 and HOM6 genes, required for the production of the toxic pathway intermediate Homoserine, also suppressed serum sensitivity, we hypothesize that serum sensitivity is a consequence of Homoserine accumulation. Serum survival is critical for dissemination, an important virulence determinant: thus, together with the essential nature of C. neoformans threonine synthesis, the cross-species serum sensitivity of thr1Delta mutants makes the fungus-specific Thr1p, and likely Thr4p, ideal antifungal drug targets.
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Fungal Homoserine Kinase (thr1) Mutants Are Attenuated in Virulence and Die Rapidly upon Threonine Starvation and Serum Incubation†
2010Co-Authors: Joanne M. Kingsbury, John H. MccuskerAbstract:The fungally conserved subset of amino acid biosynthetic enzymes not present in humans offer exciting potential as an unexploited class of antifungal drug targets. Since threonine biosynthesis is essential in Cryptococcus neofor-mans, we further explored the potential of threonine biosynthetic enzymes as antifungal drug targets by determining the survival in mice of Saccharomyces cerevisiae Homoserine Kinase (thr1) and threonine synthase (thr4) mutants. In striking contrast to aspartate Kinase (hom3) mutants, S. cerevisiae thr1 and thr4 mutants were severely depleted after only 4 h in vivo. Similarly, Candida albicans thr1mutants, but not hom3mutants, were significantly attenuated in virulence. Consistent with the in vivo phenotypes, S. cerevisiae thr1 and thr4 mutants as well as C. albicans thr1 mutants were extremely serum sensitive. In both species, serum sensitivity was suppressed by the addition of threonine, a feedback inhibitor of Hom3p. Because mutation of the HOM3 and HOM6 genes, required for the production of the toxic pathway intermediate Homoserine, also suppressed serum sensitivity, we hypothesize that serum sensitivity is a consequence of Homoserine accumulation. Serum survival is critical for dissemination, an important virulence determinant: thus, together with the essential nature of C. neoformans threonine synthesis, the cross-species serum sensitivity of thr1 mutants makes the fungus-specific Thr1p, and likely Thr4p, ideal antifungal drug targets. Fungal infections are an increasingly significant cause o
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Threonine biosynthetic genes are essential in Cryptococcus neoformans.
Microbiology (Reading England), 2008Co-Authors: Joanne M. Kingsbury, John H. MccuskerAbstract:We identified and attempted to disrupt the Cryptococcus neoformans Homoserine and/or threonine biosynthetic genes encoding aspartate Kinase (HOM3), Homoserine Kinase (THR1) and threonine synthase (THR4); however, each gene proved recalcitrant to disruption. By replacing the endogenous promoters of HOM3 and THR1 with the copper-repressible CTR4-1 promoter, we showed that HOM3 and THR1 were essential for the growth of C. neoformans in rich media, when ammonium was the nitrogen source, or when threonine was supplied as an amino acid instead of a dipeptide. Moreover, the severity of the growth defect associated with HOM3 or THR1 repression increased with increasing incubation temperature. We believe this to be the first demonstration of threonine biosynthetic genes being essential in a fungus. The necessity of these genes for C. neoformans growth, particularly at physiologically relevant temperatures, makes threonine biosynthetic genes ideal anti-cryptococcal drug targets.
Isabel L. Calderón - One of the best experts on this subject based on the ideXlab platform.
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Effect of gene amplification on threonine production by yeast.
Biotechnology and bioengineering, 1996Co-Authors: María-josé Farfán, Encarna Martin-rendon, Isabel L. CalderónAbstract:In this work, we have studied the effect of amplifying different alleles involved in the threonine biosynthesis on the amino acid production by Saccharomyces cerevisiae. The genes used were wild-type HOM3, HOM2, HOM6, THR1, and THR4, and two mutant alleles of HOM3 (namely HOM3-R2 and HOM3-R6), that code for feedback-insensitive aspartate Kinases. The results show that only the amplification of the HOM3 alleles leads to threonine and, in some instances, to Homoserine overproduction. In terms of the regulation of the pathway, the data indicate that the main control is exerted by inhibition of the aspartate Kinase and that, probably, a second and less important regulation takes place at the level of the Homoserine Kinase, the THR1 gene product. However, amplification of THR1 in two related Hom3-R2 strains does not increase the amount of threonine but, in one of them, it does induce accumulation of more Homoserine. This result probably reflects differences between these strains in some undetermined genetic factor/s related with threonine metabolism. In general, the data indicate that the common laboratory yeast strains are genetically rather heterogeneous and, thus, extrapolation of conclusions must be done carefully. (c) 1996 John Wiley & Sons, Inc.
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Identification of yeast cloned genes by genetic analysis
Microbiologia (Madrid Spain), 1992Co-Authors: Martín-rendón E, Isabel L. CalderónAbstract:Gene cloning in yeast is usually carried out by complementation of recessive mutations. However, the fact that a DNA fragment is able to complement a mutation in a certain gene does not necessarily mean that it contains that gene. The identification of a cloned gene can involve the use of Molecular and/or Classical Genetics techniques. In this paper we describe the strategy to be followed in order to establish the identity of a cloned gene, by using genetic crosses and tetrad analysis. As a practical example of the use of this strategy, we describe the cloning of the THR1 gene which codes for the Homoserine Kinase in S. cerevisiae. This gene has been isolated from a yeast genomic library by complementation of a thr1 mutation. The complementing DNA fragment has been subcloned and integrated into the yeast genome. By genetic crosses and tetrad analysis it has been demonstrated that integration has occurred at the THR1 locus. Since in this organism integration takes place mainly by homologous recombination, it can be inferred that we have, in fact, cloned the THR1 gene. Biochemical analysis of the transformant that carries multiple copies of the cloned gene confirms this result. It shows that this strain presents a Homoserine Kinase activity about 60 times higher than that of the wild type.
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Inhibition by different amino acids of the aspartate Kinase and the Homoserine Kinase of the yeast Saccharomyces cerevisiae.
FEBS letters, 1991Co-Authors: Cayo Ramos, Marco A. Delgado, Isabel L. CalderónAbstract:Abstract In this paper, we describe a simple method to measure the yeast Homoserine Kinase and asparate Kinase activities, independently but in the same extract. With this method, we have determined some kinetic parameters for the physiological substrates of both enzymes, and investigated the inhibition exerted by different amino acids on these activities. Off all natural amino acids tested, only threonine inhibits effectively both enzymatic activities, although to a different degree. We did not find the reported inhibition by L-Homoserine over the asparate Kinase. Altogether the data point to be asparate Kinase and to the threonine as the key factors in the regulation of this route.
Alan H. Fairlamb - One of the best experts on this subject based on the ideXlab platform.
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Homoserine and quorum sensing acyl Homoserine lactones as alternative sources of threonine a potential role for Homoserine Kinase in insect stage trypanosoma brucei
Molecular Microbiology, 2015Co-Authors: Han B. Ong, Wai S. Lee, Stephen Patterson, Susan Wyllie, Alan H. FairlambAbstract:Summary De novo synthesis of threonine from aspartate occurs via the β-aspartyl phosphate pathway in plants, bacte- ria and fungi. However, the Trypanosoma brucei genome encodes only the last two steps in this pathway: Homoserine Kinase (HSK) and threonine syn- thase. Here, we investigated the possible roles for this incomplete pathway through biochemical, genetic and nutritional studies. Purified recombinant TbHSK spe- cifically phosphorylates L-Homoserine and displays kinetic properties similar to other HSKs. HSK null mutants generated in bloodstream forms displayed no growth phenotype in vitro or loss of virulence in vivo. However, following transformation into procyclic forms, Homoserine, Homoserine lactone and certain acyl Homoserine lactones (AHLs) were found to sub- stitute for threonine in growth media for wild-type procyclics, but not HSK null mutants. The tsetse fly is considered to be an unlikely source of these nutrients as it feeds exclusively on mammalian blood. Bioinfor- matic studies predict that tsetse endosymbionts possess part (up to Homoserine in Wigglesworthia glossinidia) or all of the β-aspartyl phosphate pathway (Sodalis glossinidius). In addition S. glossinidius is known to produce 3-oxohexanoylHomoserine lactone which also supports trypanosome growth. We propose thatT. brucei has retained HSK and threonine synthase in order to salvage these nutrients when threonine availability is limiting.
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Homoserine and quorum‐sensing acyl Homoserine lactones as alternative sources of threonine: a potential role for Homoserine Kinase in insect‐stage Trypanosoma brucei
Molecular microbiology, 2014Co-Authors: Han B. Ong, Wai S. Lee, Stephen Patterson, Susan Wyllie, Alan H. FairlambAbstract:Summary De novo synthesis of threonine from aspartate occurs via the β-aspartyl phosphate pathway in plants, bacte- ria and fungi. However, the Trypanosoma brucei genome encodes only the last two steps in this pathway: Homoserine Kinase (HSK) and threonine syn- thase. Here, we investigated the possible roles for this incomplete pathway through biochemical, genetic and nutritional studies. Purified recombinant TbHSK spe- cifically phosphorylates L-Homoserine and displays kinetic properties similar to other HSKs. HSK null mutants generated in bloodstream forms displayed no growth phenotype in vitro or loss of virulence in vivo. However, following transformation into procyclic forms, Homoserine, Homoserine lactone and certain acyl Homoserine lactones (AHLs) were found to sub- stitute for threonine in growth media for wild-type procyclics, but not HSK null mutants. The tsetse fly is considered to be an unlikely source of these nutrients as it feeds exclusively on mammalian blood. Bioinfor- matic studies predict that tsetse endosymbionts possess part (up to Homoserine in Wigglesworthia glossinidia) or all of the β-aspartyl phosphate pathway (Sodalis glossinidius). In addition S. glossinidius is known to produce 3-oxohexanoylHomoserine lactone which also supports trypanosome growth. We propose thatT. brucei has retained HSK and threonine synthase in order to salvage these nutrients when threonine availability is limiting.
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Conference summary
2012Co-Authors: Han B. Ong, Wai S. Lee, Stephen Patterson, Susan Wyllie, Alan H. FairlambAbstract:Homoserine and quorum-sensing acyl Homoserine lactones as alternative sources of threonine: a potential role for Homoserine Kinase in insect-stage Trypanosoma bruce
Hermann Sahm - One of the best experts on this subject based on the ideXlab platform.
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Construction of l‐Lysine‐, l‐Threonine‐, or l‐Isoleucine‐Overproducing Strains of Corynebacterium glutamicum
Annals of the New York Academy of Sciences, 1996Co-Authors: Hermann Sahm, Lothar Eggeling, Bernd Eikmanns, Reinhard KrämerAbstract:The gram-negative bacterium Corynebacterium glutamicum is used for the industrial production of amino acids, for example, of L-glutamate and L-lysine. By cloning and expressing the various genes of the L-lysine pathway in C. glutamicum, we would demonstrate that an increase of the flux of L-aspartate semialdehyde to L-lysine could be obtained in strains with increased dihydrodipicolinate synthase activity. Recently we detected that in C. glutamicum two pathways exist for synthesis of D,L-diaminopimelate and L-lysine. Mutants defective in one pathway are still able to synthesize enough L-lysine for growth, but the L-lysine secretion is reduced to 50 to 70%. Using NMR spectroscopy, we could calculate how much of the L-lysine secreted into the medium is synthesized via either one or the other pathway. Amplification of the feedback inhibition insensitive Homoserine dehydrogenase and Homoserine Kinase in a high L-lysine-overproducing strain enabled channeling of the carbon flow from the intermediate aspartate semialdehyde towards Homoserine, resulting in a high accumulation of L-threonine. For a further flux from L-threonine to L-isoleucine, the allosteric control of threonine dehydratase was eliminated.
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Stable Expression of hom-1-thrB in Corynebacterium glutamicum and Its Effect on the Carbon Flux to Threonine and Related Amino Acids.
Applied and environmental microbiology, 1994Co-Authors: Dieter Reinscheid, Bernhard J. Eikmanns, W Kronemeyer, Lothar Eggeling, Hermann SahmAbstract:The hom-1-thrB operon encodes Homoserine dehydrogenase resistant to feedback inhibition by L-threonine and Homoserine Kinase. Stable expression of this operon has not yet been attained in different Corynebacterium glutamicum strains. We studied the use of chromosomal integration and of a low-copy-number vector for moderate expression of the hom-1-thrB operon to enable an analysis of the physiological consequences of its expression in C. glutamicum. Strains carrying one, two, or three copies of hom-1-thrB were obtained. They showed proportionally increased enzyme activity of feedback-resistant Homoserine dehydrogenase and of Homoserine Kinase. This phenotype was stably maintained in all recombinants for more than 70 generations. In a lysine-producing C. glutamicum strain which does not produce any threonine, expression of one copy of hom-1-thrB resulted in the secretion of 39 mM threonine. Additional copies resulted in a higher, although not proportional, accumulation of threonine (up to 69 mM). This indicates further limitations of threonine production. As the copy number of hom-1-thrB increased, increasing amounts of Homoserine (up to 23 mM) and isoleucine (up to 34 mM) were secreted. Determination of the cytosolic concentration of the respective amino acids revealed an increase of intracellular threonine from 9 to 100 mM and of intracellular Homoserine from 4 to 74 mM as the copy number of hom-1-thrB increased. These results suggest that threonine production with C. glutamicum is limited by the efflux system for this amino acid. Furthermore, the results show the successful use of moderate and stable hom-1-thrB expression for directing the carbon flux from aspartate to threonine.
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Amplification of three threonine biosynthesis genes inCorynebacterium glutamicum and its influence on carbon flux in different strains
Applied Microbiology and Biotechnology, 1991Co-Authors: Bernhard J. Eikmanns, Markus Metzger, Dieter Reinscheid, Manfred Kircher, Hermann SahmAbstract:The hom-thr B operon (Homoserine dehydrogenase/Homoserine Kinase) and the thr C gene (threonine synthase) of Corynebacterium glutamicum ATCC 13 032 and the hom _FBR (Homoserine dehydrogenase resistant to feedback inhibition by threonine) alone as well as hom _FBR- thr B operon of C. glutamicum DM 368-3 were cloned separately and in combination in the Escherichia coli/C. glutamicum shuttle vector pEK0 and introduced into different corynebacterial strains. All recombinant strains showed 8- to 20-fold higher specific activities of Homoserine dehydrogenase, Homoserine Kinase, and/or threonine synthase compared to the respective host. In wild-type C. glutamicum , amplification of the threonine genes did not result in secretion of threonine. In the lysine producer C. glutamicum DG 52-5 and in the lysine-plus-threonine producer C. glutamicum DM 368-3 overexpression of hom-thr B resulted in a notable shift of carbon flux from lysine to threonine whereas cloning of hom _FBR- thr B as well as of hom _FBR in C. glutamicum DM 368-3 led to a complete shift towards threonine or towards threonine and its precursor Homoserine, respectively. Overexpression of thr C alone or in combination with that of hom _FBR and thr B had no effect on threonine or lysine formation in all recombinant strains tested.
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Amplification of three threonine biosynthesis genes in Corynebacterium glutamicum and its influence on carbon flux in different strains.
Applied microbiology and biotechnology, 1991Co-Authors: Bernhard J. Eikmanns, Markus Metzger, Dieter Reinscheid, Manfred Kircher, Hermann SahmAbstract:The hom-thrB operon (Homoserine dehydrogenase/Homoserine Kinase) and the thrC gene (threonine synthase) of Corynebacterium glutamicum ATCC 13 032 and the homFBR (Homoserine dehydrogenase resistant to feedback inhibition by threonine) alone as well as homFBR-thrB operon of C. glutamicum DM 368-3 were cloned separately and in combination in the Escherichia coli/C. glutamicum shuttle vector pEK0 and introduced into different corynebacterial strains. All recombinant strains showed 8- to 20-fold higher specific activities of Homoserine dehydrogenase, Homoserine Kinase, and/or threonine synthase compared to the respective host. In wild-type C. glutamicum, amplification of the threonine genes did not result in secretion of threonine. In the lysine producer C. glutamicum DG 52-5 and in the lysine-plus-threonine producer C. glutamicum DM 368-3 overexpression of hom-thrB resulted in a notable shift of carbon flux from lysine to threonine whereas cloning of homFBR-thrB as well as of homFBR in C. glutamicum DM 368-3 led to a complete shift towards threonine or towards threonine and its precursor Homoserine, respectively. Overexpression of thrC alone or in combination with that of homFBR and thrB had no effect on threonine or lysine formation in all recombinant strains tested.