Cloning of the pyruvate kinase gene (pyk) of Corynebacterium glutamicum and site-specific inactivation of pyk in a lysine-producing Corynebacterium lactofermentum strain
| Autor: | Mike S. M. Jetten, Sang Ho Lee, M. E. Gubler, Anthony J. Sinskey |
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| Rok vydání: | 1994 |
| Předmět: |
DNA
Bacterial Molecular Sequence Data Pyruvate Kinase Mutant Corynebacterium Mutagenesis (molecular biology technique) Molecular cloning Polymerase Chain Reaction Applied Microbiology and Biotechnology Corynebacterium glutamicum Amino Acid Sequence Cloning Molecular Site-directed mutagenesis DNA Primers Base Sequence Sequence Homology Amino Acid Ecology biology Lysine Chromosome Mapping Gene Expression Regulation Bacterial biology.organism_classification Molecular biology Complementation Biochemistry Genes Bacterial Mutagenesis Fermentation bacteria Pyruvate kinase Research Article Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 60:2494-2500 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.60.7.2494-2500.1994 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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