EFFECT OF AMPLIFICATION OR TARGETED DISRUPTION OF THE BETA-LACTAMASE GENE OF NOCARDIA LACTAMDURANS ON CEPHAMYCIN BIOSYNTHESIS

The bla gene of the cephamycin cluster of Nocardia lactamdurans has be en subcloned in the shuttle plasmids pULVK2 and pULVK2A and amplified in N. lactamdurans LC411. The transformants showed two- to threefold h igher beta-lactamase activity. Formation of beta-lactamase preceded th e onset of cephamycin biosynthesis. The beta-lactamase of N. lactamdur ans inactivated penicillins and, to a lesser extent, cephalosporin C b ut did not hydrolyse cephamycin C. This beta-lactamase was highly sens itive to clavulanic acid (50% inhibition was observed at 0.48 mu g/ml clavulanic acid). The N. lactamdurans bla gene was disrupted in vivo b y insertion of the kanamycin-resistance gene. Three bin-disrupted muta nts, BD4, BD8 and BD12, were selected that lacked beta-lactamase activ ity. Overexpression of the bla gene resulted in N. lactamdurans transf ormants that were resistant to penicillin whereas mutants in which the bin gene was disrupted were supersensitive to this antibiotic. The th ree N. lactamdurans mutants with the bla gene disrupted showed a signi ficant increase of cephamycin biosynthesis in solid medium, whereas tr ansformants with the amplified bla gene produced reduced levels of cep hamycin. The cephamycin-overproducing Merck strain N. lactamdurans MA4 213 showed no detectable levels of beta-lactamase activity. The beta-l actamase plays a negative role in cephamycin biosynthesis in solid med ium, but not in liquid medium.