Obtaining a penicillin-biosynthesis-gene-cluster-free strain. A: Double homologous recombination strategy to delete the final biosynthesis gene cluster. A denotes the pcbAB gene, B the pcbC and penDE genes and M the marker gene amdS. B: Southern blot analysis to determine relative gene-copy number. Arrows indicate putative 'single copy' penicillin biosynthetic gene cluster candidates. Each number represents a single mutant. N, the non-producing isolate npe10; W, the lab strain Wisconsin54-1255, P, the parent strain DS47274, C, strain DS47276 with ± 8 copies and wt, the high-producing strain DS17690. C: Relative penicillinV production by putative single copy isolates (DS47274; DS48081; DS48083; DS48088) in shake flasks. D: Southern blot analysis to characterize the cluster free strain. Genomic DNA digested with Hin dIII was probed with a 425-bp fragment targeting the 3'pcbAB flanking region, cluster containing strain exhibited a band at 5.3-kb while cluster free strain exhibited a band of 10.2-kb resulting from the deletion of the last copy of the cluster. E: Relative penicillinG production by putative zero-amplicon mutants (DS50650–DS50671) in shake flasks.