| abstract |
The present invention is a method for the isolation and characterization of C. glutamicum genes involved in amino acid biosynthesis, specifically, encoding hom, thrB, and thrC, and sequences regulating their expression. Techniques for modifying or replacing these sequences and means for facilitating further isolations and characterizations, including promotor probe vectors which are useful in screening for high efficiency and regulatable promoters and repressors, are also disclosed. A C. glutamicum genomic library was constructed by cleaving chromosomal DNA with restriction enzymes, inserting the DNA fragments into an appropriate vector, and transforming the resulting recombinant molecules (rDNA) into C. glutamicum. Amino acid biosynthetic genes hom, thrB, and thrC, encoding homoserine dehydrogenase, homoserine kinase, and threonine synthetase, respectively, were isolated by complementation of C. glutamicum auxotrophs. The hom-thrB genes were subcloned on a 3.6 kb Sal1 generated chromosomal fragment while thrC activity was isolated from a second recombinant plasmid within the genomic library and subcloned on a 2.7 kb Sph1 generated fragment. The hom-thrB and thrC loci, and regulatory sequences, were identified by enzyme assays, complementation of defined E. coli auxotrophs, S1 nuclease and deletion mapping. |