Functional Analysis Of The Regions Of The Gene Cluster Governing The Antibiotic FR-008 Biosynthesis

FR-008 is a heptaene macrolide antibiotic with antifungal and mosquito larvicidal activity. It has a similar 4-aminoacetophenone-containing aglycone as candicidin D from Streptomyces griseus IMRU 3570.The chief aim of the present study is to locate the gene(s) for the sugar moiety and potential regulator(s) in the region upstream of the pabAB gene, to truncate PKS gene cluster by gene replacement so as to generate potentially new products, and to clone a large DNA fragment using Streptomyces low copy number plasmid SCP2*.I focused on an 18kb region upstream of the pabAB gene in the FR-008 biosynthetic pathway. Using gene replacement techniques, the 5.2kb fragment in this region was substituted from the chromosome by an apramycin resistance gene (aac3(IV)). Analysis of the resulting mutant, HJ1, revealed that it couldn't synthesize the antibiotic FR-008. Reference to the corresponding region in the candicidin D biosynthetic pathway, which had been sequenced and was found to be mostly identical with the FR-008 gene cluster, suggested that the deleted 5.2kb region included an ORF whose deduced amino acid sequence showed homology to the transcriptional regulator found in nystatin biosynthetic gene cluster of S. noursei ATCC 11455.pHZ2039, pHZ2012 and pHZ2016 were constructed for the replacements of c. 40kb, 7kb and 20kb regions in the PKS gene cluster, respectively. The former two constructs were introduced into Streptomyces sp. FR-008 by conjugation. The resulting strains, HJ8 and HJ9, after gene replacement were analyzed by bioassay and HPLC and were found to produce some seemingly novel compounds, which need to be further characterized.Streptomyces low copy number plasmid SCP2* was employed for the cloning and recovery of a large DNA fragment. Two fragments of the cluster, 1.5kb and 4.0kb in size respectively, were inserted into pIJ903 derived from SCP2* as two arms for gene replacement, giving rise to pHZ2029. By using the gene replacement construct pHZ2029, approximate 80kb fragment from chromosome was substituted by an apramycin resistance gene (aac(3)IV) ofpHZ2029 through double crossing over by homologous recombination event. However, the attempts to recover the 80kb fragment of the PKS gene cluster to the heterologous hosts, S. lividans 3200, S. coelicolor M145 and S. coelicolor J1501, by conjugation, were not successful.