CLONING OF THE PYRUVATE-KINASE GENE (PYK) OF CORYNEBACTERIUM-GLUTAMICUM AND SITE-SPECIFIC INACTIVATION OF PYK IN A LYSINE-PRODUCING CORYNEBACTERIUM-LACTOFERMENTUM STRAIN

The pyruvate kinase gene pyk from Corynebacterium glutamicum was clone d by applying a combination of PCR, site-specific mutagenesis, and com plementation. A 126-bp DNA fragment central to the C. glutamicum pyk g ene was amplified from genomic DNA by PCR with degenerate oligonucleot ides as primers. The cloned DNA fragment was used to inactivate the py k gene in C. glutamicum by marker rescue mutagenesis via homologous re combination. The C. glutamicum pyk mutant obtained was unable to grow on minimal medium containing ribose as the sole carbon source. Complem entation of this phenotype by a gene library resulted in the isolation of a 2.8-kb PstI-BamHI genomic DNA fragment harboring the C. glutamic um pyk gene. Multiple copies of plasmid-borne pyk caused a 20-fold inc rease of pyruvate kinase activity in C. glutamicum cell extracts. By u sing large internal fragments of the cloned C. glutamicum gene, pyk mu tant derivatives of the lysine production strain Corynebacterium lacto fermentum 21799 were generated by marker rescue mutagenesis. As determ ined in shake flask fermentations, lysine production in pyk mutants wa s 40% lower than that in the pyk(+) parent strain, indicating that pyr uvate kinase is essential for high-level lysine production. This findi ng questions an earlier hypothesis postulating that redirection of car bon flow at the phosphoenol pyruvate branch point of glycolysis throug h elimination of pyruvate kinase activity results in an increase of ly sine production in C. glutamicum and its dose relatives.