ANALYSIS OF DIFFERENT DNA FRAGMENTS OF CORYNEBACTERIUM-GLUTAMICUM COMPLEMENTING DAPE OF ESCHERICHIA-COLI

In Corynebacterium glutamicum L-lysine is synthesized simultaneously v ia the succinylase and dehydrogenase variant of the diaminopimelate pa thway. Starting from a strain with a disrupted dehydrogenase gene, thr ee different-sized DNA fragments were isolated which complemented defe ctive Escherichia coli mutants in the succinylase pathway. Enzyme stud ies revealed that in one case the dehydrogenase gene had apparently be en reconstituted in the heterologous host. The two other fragments res ulted in desuccinylase activity; one of them additionally in succinyla se activity. However, the physical analysis showed that structural cha nges had taken place in all fragments. Using a probe derived from one of the fragments we isolated a 3.4 kb BamHI DNA fragment without selec tive pressure (by colony hybridization). This was structurally intact and proved functionally to result in tenfold desuccinylase overexpress ion. The nucleotide sequence of a 1966 bp fragment revealed the presen ce of one truncated open reading frame of unknown function and that of dapE encoding N-succinyl diaminopimelate desuccinylase (EC 3.5.1.18). The deduced amino acid sequence of the dapE gene product shares 23% i dentical residues with that from E. coli. The C. glutamicum gene now a vailable is the first gene from the succinylase branch of lysine synth esis of this biotechnologically important organism.