Amino Acids – Technical Production and Use

Leuchtenberger, Wolfgang

doi:10.1002/9783527620999.ch14af

Cited by 37 publications

(36 citation statements)

References 113 publications

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“…The type strain, C. glutamicum ATCC 13032, was initially selected because of its ability to secrete large amounts of L-glutamic acid, and it is widely used in the production of L-lysine, L-glutamic acid and other amino acids used as flavour enhancers in oriental foods, as supplements of animal feeds and in human medicine (Leuchtenberger, 1996;Hermann, 2003). Because of its industrial interest, the molecular genetics of C. glutamicum has received considerable attention (reviewed by Martín, 1989;Martín & Gil, 1999;Kirchner & Tauch, 2003).…”

Section: Introductionmentioning

confidence: 99%

Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH

et al. 2006

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Corynebacterium glutamicum, a soil Gram-positive bacterium used for industrial amino acid production, was found to grow optimally at pH 7?0-9?0 when incubated in 5 litre fermenters under pH-controlled conditions. The highest biomass was accumulated at pH 9?0. Growth still occurred at pH 9?5 but at a reduced rate. The expression of the pH-regulated F 0 F 1 ATPase operon (containing the eight genes atpBEFHAGDC) was induced at alkaline pH. A 7?5 kb transcript, corresponding to the eight-gene operon, was optimally expressed at pH 9?0. The same occurred with a 1?2 kb transcript corresponding to the atpB gene. RT-PCR studies confirmed the alkaline pH induction of the F 0 F 1 operon and the existence of the atpI gene. The atpI gene, located upstream of the F 0 F 1 operon, was expressed at a lower level than the polycistronic 7?5 kb mRNA, from a separate promoter (P-atp1). Expression of the major promoter of the F 0 F 1 operon, designated P-atp2, and the P-atp1 promoter was quantified by coupling them to the pET2 promoter-probe vector. Both P-atp1 and P-atp2 were functional in C. glutamicum and Escherichia coli. Primer extension analysis identified one transcription start point inside each of the two promoter regions. The P-atp1 promoter fitted the consensus sequence of promoters recognized by the vegetative s factor of C. glutamicum, whereas the "35 and "10 boxes of P-atp2 fitted the consensus sequence for s H -recognized Mycobacterium tuberculosis promoters C C / G GG A / G AC 17-22 nt C / G GTT C / G , known to be involved in expression of heat-shock and other stress-response genes. These results suggest that the F 0 F 1 operon is highly expressed at alkaline pH, probably using a s H RNA polymerase.

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Section: Introductionmentioning

confidence: 99%

Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH

et al. 2006

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“…The world market for amino acids amounts to about 3 billion US dollars, whereby lysine with a worldwide production of about 250 000 tonnes per annum is one of the most important products [1]. Optimization of cultivation strategies and producer strains have led to increased yields and rates of amino-acid production by Corynebacteria.…”

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Application of MALDI‐TOF MS to lysine‐producing Corynebacterium glutamicum

Wittmann

Heinzle

2001

European Journal of Biochemistry

113

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In the present work, a novel comprehensive approach of 13 C-tracer studies with labeling measurements by MALDI-TOF MS, and metabolite balancing was developed to elucidate key fluxes in the central metabolism of lysine producing Corynebacterium glutamicum during batch culture. MALDI-TOF MS methods established allow the direct quantification of labeling patterns of low molecular mass Corynebacterium products from 1 mL of diluted culture supernatant. A mathematical model of the central Corynebacterium metabolism was developed, that describes the carbon transfer through the network via matrix calculations in a generally applicable way and calculates steady state mass isotopomer distributions of the involved metabolites. The model was applied for both experimental planning of tracer experiments and parameter estimation. Metabolic fluxes were calculated from stoichiometric data and from selected mass intensity ratios of lysine, alanine, and trehalose measured by MALDI-TOF MS in tracer experiments either with 1-13 C glucose or with mixtures of 13 C 6 / 12 C 6 glucose. During the phase of maximum lysine production C. glutamicum ATCC 21253 exhibited high relative fluxes into the pentose phosphate pathway of 71%, a highly reversible glucose-6-phosphate isomerase, significant backfluxes from the tricarboxylic acid cycle to the pyruvate node consuming the lysine precursor oxaloacetate, 36% net flux of anaplerotic carboxylation and 63% contribution of the dehydrogenase branch in the lysine biosynthetic pathway. Due to the straightforward and simple measurements of selected labeling patterns by MALDI-TOF MS sensitively reflecting the flux parameters of interest, the presented approach has an excellent potential to extend metabolic flux analysis from single experiments with enormous experimental effort to a broadly applied technique.Keywords: MALDI-TOF mass spectrometry; metabolic flux analysis; Corynebacterium glutamicum; lysine; 13 C tracer.Production of amino acids by Corynebacterium glutamicum belongs to the major processes in industrial biotechnology. The world market for amino acids amounts to about 3 billion US dollars, whereby lysine with a worldwide production of about 250 000 tonnes per annum is one of the most important products [1]. Optimization of cultivation strategies and producer strains have led to increased yields and rates of amino-acid production by Corynebacteria. To a large extent, the strain improvement was based on random mutagenesis and subsequent selection [2,3]. In the last decade the powerful tools of genetic engineering allowed the targeted amplification and disruption of selected genes, which led to a number of mutants with increased capabilities of lysine secretion [4]. However, practically achieved yields are still far from theoretical optima, leaving a significant potential for further improvement. To achieve such improvements in a targeted and efficient way, detailed knowledge of intracellular carbon flux distributions and their regulation is needed. Data on genome, transcriptome and proteome ...

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“…Today, industrial amino acid production processes using genetically improved strains of this micro-organism are used to satisfy the growing world market for amino acids, in particular L-glutamate and L-lysine (Leuchtenberger, 1996). In the classification system of bacteria, the genus Corynebacterium, together with mycobacteria, nocardia, rhodococci and some other phylogenetically related taxa, belongs the group of mycolic-acid-containing actinomycetes (see Liebl, 2001).…”

Section: Introductionmentioning

confidence: 99%

Genetic dissection of trehalose biosynthesis in Corynebacterium glutamicum: inactivation of trehalose production leads to impaired growth and an altered cell wall lipid composition

et al. 2003

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The analysis of the available Corynebacterium genome sequence data led to the proposal of the presence of all three known pathways for trehalose biosynthesis in bacteria, i.e. trehalose synthesis from UDP-glucose and glucose 6-phosphate (OtsA-OtsB pathway), from malto-oligosaccharides or α-1,4-glucans (TreY-TreZ pathway), or from maltose (TreS pathway). Inactivation of only one of the three pathways by chromosomal deletion did not have a severe impact on C. glutamicum growth, while the simultaneous inactivation of the OtsA-OtsB and TreY-TreZ pathway or of all three pathways resulted in the inability of the corresponding mutants to synthesize trehalose and to grow efficiently on various sugar substrates in minimal media. This growth defect was largely reversed by the addition of trehalose to the culture broth. In addition, a possible pathway for glycogen synthesis from ADP-glucose involving glycogen synthase (GlgA) was discovered. C. glutamicum was found to accumulate significant amounts of glycogen when grown under conditions of sugar excess. Insertional inactivation of the chromosomal glgA gene led to the failure of C. glutamicum cells to accumulate glycogen and to the abolition of trehalose production in a ΔotsAB background, demonstrating that trehalose production via the TreY-TreZ pathway is dependent on a functional glycogen biosynthetic route. The trehalose-non-producing mutant with inactivated OtsA-OtsB and TreY-TreZ pathways displayed an altered cell wall lipid composition when grown in minimal broth in the absence of trehalose. Under these conditions, the mutant lacked both major trehalose-containing glycolipids, i.e. trehalose monocorynomycolate and trehalose dicorynomycolate, in its cell wall lipid fraction. The results suggest that a dramatically altered cell wall lipid bilayer of trehalose-less C. glutamicum mutants may be responsible for the observed growth deficiency of such strains in minimal medium. The results of the genetic and physiological dissection of trehalose biosynthesis in C. glutamicum reported here may be of general relevance for the whole phylogenetic group of mycolic-acid-containing coryneform bacteria.

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Amino Acids – Technical Production and Use

Cited by 37 publications

References 113 publications

Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH

Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH

Application of MALDI‐TOF MS to lysine‐producing Corynebacterium glutamicum

Genetic dissection of trehalose biosynthesis in Corynebacterium glutamicum: inactivation of trehalose production leads to impaired growth and an altered cell wall lipid composition

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