Cloning And Analysis Of Moenomycin Biosynthesis Gene Cluster In Streptomyces Bambergiensis

Moenomycin is a potential antibiotic which was first found in fermentation product of Stretomyces bambergiensis,It has been widely used as an animal growth promoter.In the decades use of moenomycin,it has been never reported that microflora in animal had significantly resistant to moenomycin.In this paper,S.bambergiensis ATCC 13879 was used as analysis material and its genomic DNA was extracted.In order to obtain about 40 kb fragment,genomic DNA was randomly sheared.Then the inserted fragments which were repaired by end-repaire enmyzes were separated by PFGE and then successfully ligated with pCC1FOS vector.After that they were packaged into phages with packaging extracts,transfected into host bacteria.In this study,3,0000 clones were obtained,and the titer of the genomic library was 6.0×104 cfu/mL.In the end,2880 clones were stored in 96-well plates at-80?,the number of clones is far greater than minimum value needed in the library.Then chose 10 clones,tested the extracted plasmid DNA and the recombination rate was close to 100%.The constructed S.bambergiesis genomic library met the requirements.The moenomycin biosynthetic gene M5 fragments in S.ghanaensis was taken as probes.S.bambergiesis genomic library was screened by narrow-down PCR amplification.After the first round screening of thirty 96-well plates,clones in 5 positive plates containing objective gene fragments were obtained.Then clones in 7 positive lines of the 5 plates were got by the same way,at last 8 positive clones containing M5 gene fragments were obtained by narrow-down PCR amplification.In order to focus on one positive containing complete moenomycin biosynthetic gene cluster,moenomycin biosynthesis gene R5 and A5 in S.ghanaensis ATCC 14672 were used to design primers.After PCR amplification,24F12 were the objective clone containing moenomycin biosynthesis gene cluster.After sequencing,moenomycin biosynthesis gene cluster sequence in S.bambergiesis was obtained.According to the sequence analysis,the lengh of moenomycin biosynthesis gene cluster sequence in S.bambergiesis was identified as 27763 bp,in which contains 20 complete Open Reading Frames(ORF).The identity of the gene sequences between S.bambergiesis and S.ghanaensis ATCC 14672 was 90%.The similarity of most amino acids between them was 85%?93%.