Impact. Metk Nosiheptide Biosynthesis And Streptomyces Efficient Transfer Block System

Part OneStreptomyces produce two-thirds of antibiotics, therefore Streptomyces play an important role in pharmaceutical industry. Genetic engineering of Streptomyces is an active field in these years. Nosiheptide (Nsh), a metabolite of Streptomyces actuosus, belongs to a family of highly modified cyclic peptide antibiotics. Based on the former work, this paper promote the production of Nsh by genetic engineering and medium optimizing.First of all, constructed an E.coeli-Streptomyces shuttle plasmid containing metK from Saccharomyces cerevisiase and transformed it into Streptomyces actuosus using protoplast to get strain Z-SAM. The level of intracellular SAM in Z-SAM was found to be 9 times higher than that in original strain Z-10.Second, to explore the synthesis pathway of Nsh, fermentation medium was optimized, a medium contains 2% maltose,2% glucose,4% soluble starch,4% soy meal, 0.2% ammonium sulfate,0.2% sodium sulfate,0.04% K2HPO4,0.01% MnSO4,0.05% MgSO4,0.25% NaCl,0.003% FeSO4,0.005% ZnSO4 is used and a mixture of 2% soy meal, 0.01% serine 1.25% maltose,1.25% glucose,0.15% NaSO4 and 0.15% (NH4)2SO4 were fed at 72,120 and 168 hours respectively. The maximum yield of nosiheptide in Z-SAM was increased by 80% to 1746μg/ml.Last but not least, the quantity of intracellular cysteine in Z-SAM was found to be higher than that in Z-10, According to these results, we supposed there is another nosiheptide syhthesis pathway which is dominated by SAM in S.actuosus. The over-expressed SAM in Z-SAM, coupled with exogenous serine, formed excess cysteine, and the raised quantity of cysteine further stimulated the biosynthesis of nosiheptide Part TwoThe basic research on Streptomyces spp enters the post-genomics era since the punishment of Streptomyces coelicolor A3(2). Functions of many novel genes are to be identified. Researches on such genes will be helpful to clarify gene regulation between primary and secondary metabolites and provide more experimental evidence for the antibiotic synthesis mechanism. Construction and screen of a mutant library is an important strategy, and transposon is a useful tool to construct mutant libraries.Transposon is a very useful tool on gene function analysis. However, currently utilized transposons in streptomyces have many shortages. In this thesis, we constructed a minitransposon plasmid pDZY101 using a natural existing transposon IS204 in Nocardia asteroids(mexicana)YP21. It contained oriT, the origion of transfer in conjugation between E.coli and Streptomycete, therefore it could be easily transferred into streptomycetes. The whole system overcame some limitations of current transposons used in streptomycetes, easy to operate with high efficiency, randomized, stability and other advantages. We did some preliminary study on the mechanism of the transposase by construction a series of plasmid based on pDZY101. Results show that a circle junction which composed of two abutted inverted repeats is essential for transposition of IS204 derived transposable plasmids in S.coelicolor. Insertional randomness was verified by plenty of sequencing results of the mutants and Southern Blot. Results show that transposon insertions were not only yield a high efficiency and genetic stability, but recovered at widely scattered locations around the chromosome as well. The mini-transposon system provided a new approach in the study of functional genomics in Streptomycete.Furthermore, a plasmid pF12 which is used for homologous recombination with streptomyces phageφC31 was constructed based on pDZY101. A phage transposition system was studied.