ABSTRACT
Roseoflavin (RoF), and its metabolic precursor 8-dimethylaminoriboflavin (AF), produced by Gram-positive bacteria Streptomyces davawensis and Streptomyces cinnabarinus, reveal a strong antibiotic effect against Staphyloccus aureus, Bacillus subtilis, Bacillus cereus, Micrococcus luteus and other Gram-positive bacteria and could be promising for developing the new effective antibacterial drugs. In this work, we present the optimization of basic molecular tools for S. davawensis including total DNA preparation, PCR amplification of target gene, cell transformation. In addition, the compositions of cultivation media were studied and the optimal cultivation conditions for increased of RoF production have been developed.
Subject(s)
Anti-Bacterial Agents/metabolism , DNA, Bacterial/genetics , Genes, Bacterial , Riboflavin/analogs & derivatives , Streptomyces/genetics , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Bacillus cereus/drug effects , Bacillus cereus/growth & development , Bacillus subtilis/drug effects , Bacillus subtilis/growth & development , Culture Media/chemistry , Culture Media/pharmacology , DNA, Bacterial/metabolism , Microbial Sensitivity Tests , Micrococcus luteus , Plasmids/chemistry , Plasmids/metabolism , Polymerase Chain Reaction/methods , Riboflavin/biosynthesis , Riboflavin/isolation & purification , Riboflavin/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development , Streptomyces/drug effects , Streptomyces/metabolism , Transformation, GeneticABSTRACT
The riboflavin overproducing mutants of the flavinogenic yeast Candida famata isolated by conventional selection methods are used for the industrial production of vitamin B2. Recently, a transformation system was developed for C. famata using the leu2 mutant as a recipient strain and Saccharomyces cerevislae LEU2 gene as a selective marker. In this paper the cloning of C. famata genes for riboflavin synthesis on the basis of developed transformation system for this yeast species is described. Riboflavin autotrophic mutants were isolated from a previously selected C. famata leu2 strain. C. famata genomic DNA library was constructed and used for cloning of the corresponding structural genes for riboflavin synthesis by complementation of the growth defects on a medium without leucine and riboflavin. As a result, the DNA fragments harboring genes RIB1, RIB2, RIB5, RIB6 and RIB7 encoding GTP cyclohydrolase, reductase, dimethylribityllumazine synthase, dihydroxybutanone phosphate synthase and riboflavin synthase, were isolated and subsequently subcloned to the smallest possible fragments. The plasmids with these genes successfully complemented riboflavin auxotrophies of the corresponding mutants of another flavinogenic yeast Pichia guilliermondii. This suggested that C. famata structural genes for riboflavin synthesis and not some of the supressor genes were cloned.
