Functional Analysis Of Rela And Nsda Gene Disruption In Moenomycin Producing Strains

The moenomycins are phosphoglycolipid antibiotics mainly produced by Streptomyces ghanaensis and Streptomyces bambergiensis. Moenomycin A is the prototypical member of the phosphoglycolipid family of antibiotics. It has been widely used as an animal growth promoter.relA was first observed in Escherichia coli as the repression of stable RNA synthesis in response to amino acid depletion. Subsequent studies showed tha relA homologous gene was wildly existed and conserved in many Streptomyces, pleiotropic regulating sporulation and antibiotic production. The relA gene has been shown to encode a ribosome bound (p)ppGpp synthetase. nsdA gene was globally negative regulator gene, which was first found in Streptomyces coelicolor A3(2), negatively regulating sporulation and antibiotic production. Subsequent studies showed that nsdA gene was wildly existed and conserved in Streptomyces.On account of the complexity of regulation in Streptomyces and universality and pleiotropy of relA and nsdA gene, functions of regulatory genes relA and nsdA in the morphological differentiation and antibiotic production of S. ghanaensis and S. bambergiensis were studied by gene disruption.Primers were designed according to the data obtained from the S. coelicolor A(3)2genome sequences. nsdA gene and relA genes from S. ghanaensis ATCC14672and S. bambergiensis ATCC13879were cloned, respectively. Sequence analysis showed that the relA gene in S. bambergiensis has a83.55%similarity in DNA sequence compared with that of other Streptomyces, while relA gene in S. ghanaensis has a85.37%similarity; nsdA gene in S. bambergiensis shared a86.39%sequence similarity in the DNA sequence compared with that of other Streptomyces, while nsdA gene in S. ghanaensis shared a87.52%sequence similarity.To further study the function of relA and nsdA gene, the plasmid pKC1139was used to construct the homologous recombination vectors pKQRK, pKBRK, pKQNK and pKBNK. These gene disruption recombinant plasmids were introduced into S. bambergiensis and S. ghanaensis by intergeneric conjugal transfer. Under the high temperature and antibiotic selection pressure, the double cross-over recombination mutants were obtained and gene disruptions were confirmed by PCR and DNA sequencing. In the level of flask, HPLC assays on relA gene mutant S. bambergiensis1203revealed that disruption of relA remarkably increased the yield of moenomycin A by18.59times, while the yield of moenomycin A in relA gene disruption S. ghanaensis0613was lower than that of the original strain S. ghanaensis ATCC13879. HPLC assays on nsdA gene mutant S. bambergiensis0107revealed that disruption of nsdA remarkably increased yields of moenomycin A by22.41times in the level of flask. Changes of phenotype and antibiotic production were reversed to the ancestral level by the introduction of nsdA back into S. bambergiensis0107, excluding potential polar effects on adjacent genes as an explanation. The results suggested that nsdA played a negative role in morphological differentiation and antibiotic production in S. bambergiensis ATCC13879; relA gene pleiotropic regulated morphological differentiation and antibiotic production in S. bambergiensis ATCC13879and S. ghanaensis ATCC14672.