Application Of NsdA, A Negative Regulator Of Streptomyces Differentiation In Antibiotic Breeding

nsdA is a globally negative regulator gene first found by our laboratory. It negatively regulates antibiotics production and sporulation in Streptomyces coelicolor. S.coelicolor nsdA null mutants produce more actinorhodin, CDA, methylenomycin and spores than wild type strain. The goal of this study is to test whether nsdA homologous gene is wildly existent in other Streptomyces species, especially producers of some important antibiotics, and whether it can be exploited in strain improvement.In this study, Southern blot using an nsdA fragment as probe was carried out to test the existence of nsdA gene in S. avermitilis NRRL8165, S.hygroscopicus 5008, S.albus JA3453, S.aureofaciens 211, S.spp.FR-008 derivative DX600, S.hygroscopicus 10-22 and S.lividans ZX64. The hybridization result indicated that all of these strains have at least one copy of nsdA homologue.In S. lividans genome, there is a silent gene cluster contributing to the biosynthesis of actinorhodin. In this study, PCR amplification and sequencing showed that the 5. lividans nsdA homologue was identical with the S.coelicolor nsdA. By means of homologous recombination, the ZX64 nsdA gene was disrupted to yield a null mutant strain WQ2. WQ2 was able to produce actinorhodin whereas the parent strain ZX64 was not. And the actinorhodin production of WQ2 could be eliminated by re-introducing a wild-type copy of nsdA gene into it. These results suggested that nsdA gene represses the actinorhodin gene cluster, and the disruption of it activate the silent actinorhodin biosynthesis in S. lividans.Based on the published S. avermitilis genome sequence, primers were designed to PCR amplify the 5.8kb fragment containing the nsdA homologue nsdAav and flanking sequences from S. avermitilis NRRL8165. The cloned PCR product was sequenced. Then an nsdA disruption plasmid pHL214 were constructed and conjugated into S.avermitilis NRRL8165. Two nsdAav disruption isolates, WQ5-1 and WQ5-2, were obtained and the nsdAav disruption was confirmed by nested PCR. HPLC analysis of fermentation culture indicated that the avermectins production of the nsdAav disrupted strains was elevated by 3-5 folds.Using primers designed based on the S.coelicolor genome sequence, a 4.7 kb fragment containing the nsdA homologue nsdAjg and flanking sequence was also PCR amplified and cloned from S.hygroscopicus 5008, a producer of the wildly used agricultural antibiotic jinggangmycin. Sequencing of the cloned fragment showed thatnsdAjg has an 86% nucleotides similarity with the S.coelicolor nsdA. Two disruption plasmids pHL202 and pHL213 were constructed. Efforts to transfer these plasmids into SMygroscopicus 5008 via conjugation failed until now. It is remained to elucidate the regulatory role oinsdAjg on jinggangmycin production.