Study On Founctional Gene In An Alkaphilic Actinomycetes And Realtive Gene Involved In Medermycin Biosynthesis

Extreme microbes become a new resource of functional genes for that they probably contain important enzymes with unique physiological features. As well, investigations of some functional genes from terrace microbes still are attractive, especially in the genetic engineering of some genes for antibiotic biosynthesis and regulation.The present research covers two aspects:(1) Study on alkaline protease activity in an alkaphilic actinomycetesBased on the optimization of agar assay method using Coomassie brilliant blue G250as staining agent, we firstly reported an alkaphilic actinomycete strain HSL10isolated from a salt-alkaline lake sediments in Xinjiang could secrete alkaline proteases. Analysis of crude enzymes from this strain showed that it possesses the greatest activity under alkaline conditions, supported that it could produce alkaline proteases. Further, we cloned an alkaline protease-encoding gene, expressed it in E.coli, and observed the obvious proteolytic activity in agar media containing a substrate.(2) Study on a regulator involved in medermycin biosynthesisMedermycin is an antitumor antibiotic with unique mechanism against many types of tumor cells. It could be produced by Streptomyces sp. AM7161.A proposed small regulatory-protein-encoding gene was proved to be involved in medermycin biosynthesis, through knocking out of this gene and expressing the resulting mutant medermycin gene cluster in a heterologous host. However, the med-ORF10-deletion mutant derived from the wild type strain AM7161has yet to be obtained.Here, we firstly introduced a suicide plasmid into AM-7161and obtained successfully a med-ORF10-deficient mutant, conformed by PCR. Agar assay revealed that the wild type strain and mutant strain gave complete different pigmentation, implying differencein metabolite accumulation, especially, the dark brown pigment indicatingproduction of medermycin in AM7161became light brown in the mutant. HPLC analysis also supported different metabolic profiles between the mutant and wild type strains.Additionally, we obtained a mutant by UV mutagenesis which showed stronger antibacterial activity and another antibiotic kalafungin was produced at a higher level in this mutant.