This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gubler, M Articles by Sinskey, A J Search for Related Content PubMed PubMed Citation Articles by Gubler, M Articles by Sinskey, A J Agricola Articles by Gubler, M Articles by Sinskey, A J

 Previous Article  |  Next Article 

Appl Environ Microbiol. 1994 July; 60(7): 2494-2500

Cloning of the pyruvate kinase gene (pyk) of Corynebacterium glutamicum and site-specific inactivation of pyk in a lysine-producing Corynebacterium lactofermentum strain.

Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.

ABSTRACT

The pyruvate kinase gene pyk from Corynebacterium glutamicum was cloned by applying a combination of PCR, site-specific mutagenesis, and complementation. A 126-bp DNA fragment central to the C. glutamicum pyk gene was amplified from genomic DNA by PCR with degenerate oligonucleotides as primers. The cloned DNA fragment was used to inactivate the pyk gene in C. glutamicum by marker rescue mutagenesis via homologous recombination. The C. glutamicum pyk mutant obtained was unable to grow on minimal medium containing ribose as the sole carbon source. Complementation of this phenotype by a gene library resulted in the isolation of a 2.8-kb PstI-BamHI genomic DNA fragment harboring the C. glutamicum pyk gene. Multiple copies of plasmid-borne pyk caused a 20-fold increase of pyruvate kinase activity in C. glutamicum cell extracts. By using large internal fragments of the cloned C. glutamicum gene, pyk mutant derivatives of the lysine production strain Corynebacterium lactofermentum 21799 were generated by marker rescue mutagenesis. As determined in shake flask fermentations, lysine production in pyk mutants was 40% lower than that in the pyk+ parent strain, indicating that pyruvate kinase is essential for high-level lysine production. This finding questions an earlier hypothesis postulating that redirection of carbon flow at the phosphoenol pyruvate branch point of glycolysis through elimination of pyruvate kinase activity results in an increase of lysine production in C. glutamicum and its close relatives.

This article has been cited by other articles:

• Han, S. O., Inui, M., Yukawa, H. (2007). Expression of Corynebacterium glutamicum glycolytic genes varies with carbon source and growth phase. Microbiology 153: 2190-2202 [Abstract] [Full Text]  
• Loos, A., Glanemann, C., Willis, L. B., O'Brien, X. M., Lessard, P. A., Gerstmeir, R., Guillouet, S., Sinskey, A. J. (2001). Development and Validation of Corynebacterium DNA Microarrays. Appl. Environ. Microbiol. 67: 2310-2318 [Abstract] [Full Text]