Amplification of three threonine biosynthesis genes inCorynebacterium glutamicum and its influence on carbon flux in different strains

Eikmanns, Bernhard J.; Metzger, Markus; Reinscheid, Dieter J.; Kircher, Manfred; Sahm, Hermann

doi:10.1007/bf00167910

Cited by 138 publications

(92 citation statements)

References 27 publications

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“…E. coli and all pre-cultures of C. glutamicum were grown aerobically in TY complex medium (Sambrook et al, 2001) at 37 and 30 uC, respectively, as 60 ml cultures in 500 ml baffled Erlenmeyer flasks on a rotary shaker at 120 r.p.m. For the main cultures of C. glutamicum, cells of an overnight pre-culture were washed with 0.9 % (w/v) NaCl (saline) and then inoculated into minimal medium (Eikmanns et al, 1991b) containing glucose or maltose at the concentrations indicated in Results. Cultivation was done aerobically at 30 uC as 250 ml cultures in a 'fedbatch-pro' parallel fermentation system from DASGIP, as described previously .…”

Section: Methodsmentioning

confidence: 99%

Roles of maltodextrin and glycogen phosphorylases in maltose utilization and glycogen metabolism in Corynebacterium glutamicum

2009

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Corynebacterium glutamicum transiently accumulates large amounts of glycogen, when cultivated on glucose and other sugars as a source of carbon and energy. Apart from the debranching enzyme GlgX, which is required for the formation of maltodextrins from glycogen, a-glucan phosphorylases were assumed to be involved in glycogen degradation, forming a-glucose 1-phosphate from glycogen and from maltodextrins. We show here that C. glutamicum in fact possesses two a-glucan phosphorylases, which act as a glycogen phosphorylase (GlgP) and as a maltodextrin phosphorylase (MalP). By chromosomal inactivation and subsequent analysis of the mutant, cg1479 was identified as the malP gene. The deletion mutant C. glutamicum DmalP completely lacked MalP activity and showed reduced intracellular glycogen degradation, confirming the proposed pathway for glycogen degradation in C. glutamicum via GlgP, GlgX and MalP. Surprisingly, the DmalP mutant showed impaired growth, reduced viability and altered cell morphology on maltose and accumulated much higher concentrations of glycogen and maltodextrins than the wild-type during growth on this substrate, suggesting an additional role of MalP in maltose metabolism of C. glutamicum. Further assessment of enzyme activities revealed the presence of 4-a-glucanotransferase (MalQ), glucokinase (Glk) and a-phosphoglucomutase (a-Pgm), and the absence of maltose hydrolase, maltose phosphorylase and b-Pgm, all three known to be involved in maltose utilization by Gram-positive bacteria. Based on these findings, we conclude that C. glutamicum metabolizes maltose via a pathway involving maltodextrin and glucose formation by MalQ, glucose phosphorylation by Glk and maltodextrin degradation via the reactions of MalP and a-Pgm, a pathway hitherto known to be present in Gram-negative rather than in Gram-positive bacteria. INTRODUCTIONCorynebacterium glutamicum is a Gram-positive soil bacterium employed in the production of various amino acids (Eggeling & Bott, 2005), and serves as a model organism for the mycolic acid-containing suborder Corynebacterianeae, which includes pathogenic species such as Corynebacterium diphtheriae, Mycobacterium tuberculosis and Mycobacterium leprae. The organism grows on a variety of mono-and disaccharides, alcohols and organic acids as single or combined sources of carbon and energy (Liebl, 2005). and transiently forms intracellular glycogen when cultivated on sugar substrates (Tzvetkov et al., 2003;. We recently showed that C. glutamicum forms maltodextrins in the course of glycogen degradation , and thus we speculate that glycogen is degraded in C. glutamicum by a pathway which proceeds similarly or identically to the one present in Escherichia coli. This pathway involves the interplay of six enzymes (Fig. 1). Glycogen phosphorylase (GlgP) cleaves a-1,4-glycosidic bonds at the non-reducing ends of glycogen and forms glucose 1-phosphate and phosphorylase-limited dextrins (pl-dextrins) (Dauvillee et al., 2005;Alonso-Casajus et al., 2006). Cleavage of the a-1,6-glycosidic bon...

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Section: Methodsmentioning

confidence: 99%

Roles of maltodextrin and glycogen phosphorylases in maltose utilization and glycogen metabolism in Corynebacterium glutamicum

2009

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“…E. coli and all pre-cultures of C. glutamicum were grown aerobically in TY complex medium (Sambrook & Russel, 2001) at 37 and 30 uC, respectively, as 60 ml cultures in 500 ml baffled Erlenmeyer flasks on a rotary shaker at 120 r.p.m. For the main cultures of C. glutamicum, cells of an overnight pre-culture were washed with 0.9 % (w/v) NaCl, and then inoculated into CgXII minimal medium (Eikmanns et al, 1991) containing glucose at the concentrations indicated in Results. C. glutamicum was grown aerobically at 30 uC as 50 ml cultures in 500 ml baffled Erlenmeyer flasks on a rotary shaker at 120 r.p.m., or as 250 ml cultures in a parallel fermentation system (Fedbatch-pro system; DASGIP) as described previously (Seibold et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

The glgX gene product of Corynebacterium glutamicum is required for glycogen degradation and for fast adaptation to hyperosmotic stress

Seibold

Eikmanns

2007

Microbiology

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Corynebacterium glutamicum cells growing in medium containing sugars accumulate glycogen in the early exponential-growth phase, and start to degrade this polymer at entry into the stationary phase. In a first attempt to investigate glycogen degradation, the C. glutamicum glgX gene, which encodes a protein with 46 % identity to the isoamylase-type debranching enzyme of Escherichia coli, was analysed, expressed and inactivated. The purified C. glutamicum gene product showed debranching activity towards glycogen, amylopectin and starch. Chromosomal inactivation of glgX in C. glutamicum wild-type led to slower growth and to a higher intracellular glycogen pool throughout growth, when compared to those in the parental strain. This result suggests that glycogen synthesis and degradation occur simultaneously in C. glutamicum. When exposed to hyperosmotic shock, C. glutamicum rapidly degrades glycogen, and at the same time, synthesizes the osmoprotectant trehalose. The glgX mutant, however, synthesized only minor amounts of trehalose throughout cultivation, and its growth ceased after hyperosmotic shock. Taken together, the results indicate that the glgX gene product is essential for glycogen degradation in C. glutamicum, that glycogen is constantly recycled in C. glutamicum, and that it serves as a carbon store for trehalose synthesis via the TreYZ pathway after hyperosmotic shock.

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“…The plasmids used in this study are pUC18 and pUC19 (56), the C. glutamicum-E. coli shuttle vector pEKO (22), cosmid ppc42 (cosmid pHC79 containing a 26-kb chromosomal DNA fragment from C. glutamicum AS019) (20), and pUC-ppcII (pUC8 containing a 4.5-kb DNA fragment from ppc42) (20). The minimal medium used for C. glutamicum has been described previously (23); LB medium (37) was the complex medium for C. glutamicum and for E. coli. When appropriate, ampicillin (50 ,ug/ml) or kanamycin (15 or 50 ,ug/ml) was added to the medium.…”

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Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase

Eikmanns

1992

J Bacteriol

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To investigate a possible chromosomal clustering of glycolytic enzyme genes in Corynebacterium glutamicum, a 6.4-kb DNA 27,198). The amino acid sequences of the three enzymes show up to 62% (GAPDH), 48% (PGK), and 44% (TPI) identity in comparison with respective enzymes from other organisms. The gap, pgk, tpi, and ppc genes were cloned into the C. glutamicum-Escherichia coli shuttle vector pEKO and introduced into C. glutamicum. Relative to the wild type, the recombinant strains showed up to 20-fold-higher specific activities of the respective enzymes. On the basis of codon usage analysis of gap, pgk, tpi, and previously sequenced genes from C. glutamicum, a codon preference profile for this organism which differs significantly from those of E. coli and Bacillus subtilis is presented.

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Amplification of three threonine biosynthesis genes inCorynebacterium glutamicum and its influence on carbon flux in different strains

Cited by 138 publications

References 27 publications

Roles of maltodextrin and glycogen phosphorylases in maltose utilization and glycogen metabolism in Corynebacterium glutamicum

Roles of maltodextrin and glycogen phosphorylases in maltose utilization and glycogen metabolism in Corynebacterium glutamicum

The glgX gene product of Corynebacterium glutamicum is required for glycogen degradation and for fast adaptation to hyperosmotic stress

Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase

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