A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA

Rest, M E van der; Lange, Christian; Molenaar, Douwe

doi:10.1007/s002530051557

Cited by 402 publications

(238 citation statements)

References 16 publications

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“…DNA fragments were isolated from agarose gels using the GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences). E. coli cells were transformed by standard methods (Sambrook & Russell, 2001), whereas C. glutamicum cells were transformed by electroporation (van der Rest et al, 1999).…”

Section: Methodsmentioning

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: Methodsmentioning

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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“…Plasmid DNA was isolated with the QIAprep spin miniprep kit (Qiagen, Hilden, Germany). Transformation of E. coli was performed using the rubidium chloride method (Hanahan 1985), whereas C. glutamicum was transformed by electroporation as described by van der Rest et al (1999).…”

Section: Preparation Of Dna and Transformationmentioning

confidence: 99%

Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves l-lysine formation

Kabus

Georgi

Wendisch

et al. 2007

Appl Microbiol Biotechnol

121

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A critical factor in the biotechnological production of L-lysine with Corynebacterium glutamicum is the sufficient supply of NADPH. The membrane-integral nicotinamide nucleotide transhydrogenase PntAB of Escherichia coli can use the electrochemical proton gradient across the cytoplasmic membrane to drive the reduction of NADP + via the oxidation of NADH. As C. glutamicum does not possess such an enzyme, we expressed the E. coli pntAB genes in the genetically defined C. glutamicum lysineproducing strain DM1730, resulting in membrane-associated transhydrogenase activity of 0.7 U/mg protein. When cultivated in minimal medium with 10% (w/v) carbon source, the presence of transhydrogenase slightly reduced glucose consumption, whereas the consumption of fructose, glucose plus fructose, and, in particular, sucrose was stimulated. Biomass was increased by pntAB expression between 10 and 30% on all carbon sources tested. Most importantly, the lysine concentration was increased in the presence of transhydrogenase by ∼10% on glucose, ∼70% on fructose, ∼50% on glucose plus fructose, and even by ∼300% on sucrose. Thus, the presence of a proton-coupled transhydrogenase was shown to be an efficient way to improve lysine production by C. glutamicum. In contrast, pntAB expression had a negative effect on growth and glutamate production of C. glutamicum wild type.

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“…E. coli was transformed by the RbCl 2 method (14). C. glutamicum was transformed via electroporation (56). All transformants were analyzed by plasmid analysis and/or PCR with appropriate primers.…”

Section: Methodsmentioning

confidence: 99%

Cometabolism of a Nongrowth Substrate:l-Serine Utilization byCorynebacterium glutamicum

Netzer

Peters‐Wendisch

Eggeling

et al. 2004

Appl Environ Microbiol

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C-labeled L-serine and analysis of cellularly derived metabolites by nuclear magnetic resonance spectroscopy revealed that the carbon skeleton of L-serine is mainly converted to pyruvate-derived metabolites such as L-alanine. The sdaA gene was identified in the genome of C. glutamicum, and overexpression of sdaA resulted in (i) functional L-serine dehydratase (L-SerDH) activity, and therefore conversion of L-serine to pyruvate, and (ii) growth of the recombinant strain on L-serine as the single substrate. In contrast, deletion of sdaA decreased the L-serine cometabolism rate with glucose by 47% but still resulted in degradation of L-serine to pyruvate. Cystathionine ␤-lyase was additionally found to convert L-serine to pyruvate, and the respective metC gene was induced 2.4-fold under high internal L-serine concentrations. Upon sdaA overexpression, the growth rate on glucose is reduced 36% from that of the wild type, illustrating that even with glucose as a single substrate, intracellular L-serine conversion to pyruvate might occur, although probably the weak affinity of L-SerDH (apparent K m , 11 mM) prevents substantial L-serine degradation.Cometabolism is often observed for xenobiotic compounds which do not enable growth as a single carbon-and energy source (21). This is due, for example, to long degradation pathways and unnatural structures. On the other hand, microorganisms with a restricted metabolism, such as Lactococcus lactis, are dependent on cometabolism of essential natural compounds, e.g., amino acids (35). The amino acid L-serine is characterized by the fact that several organisms have the ability to introduce it into the central metabolism via pyruvate (35,2,16). Another distinguishing feature is its high cellular demand, exceeding the simple provision of L-serine for protein synthesis, since L-serine is additionally required for glycine, cysteine, tryptophan, and phospholipid synthesis as well as for 1-carbonunit generation (52). Glycine, in turn, is a precursor for purines and heme. In Corynebacterium glutamicum about 7.5% of the total carbon flux toward L-serine is utilized for these purposes (28). Prior estimates for Escherichia coli determined that as much as 15% of the carbon assimilated from glucose involves L-serine (42). Due to the high demand and its key position in the precursor supply, L-serine has to be regarded as an intermediate of the central metabolism (52).In spite of the presence of L-serine dehydratase (L-SerDH) (47) and the key position of L-serine, growth of E. coli on L-serine as a carbon source is very poor, allowing doubling times of about 60 h only (63). When the organism is additionally exposed to low concentrations of glycine, isoleucine, and threonine, growth is enhanced (36). Interestingly, during growth on tryptone broth, where a number of amino acids are present, L-serine is utilized immediately and earlier than any other amino acid (43). Taken together, L-serine is clearly a poor growth substrate for E. coli and is preferably cometabolized. Klebsiella aerogenes and ...

show abstract

A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA

Cited by 402 publications

References 16 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

Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves l-lysine formation

Cometabolism of a Nongrowth Substrate:l-Serine Utilization byCorynebacterium glutamicum

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