链霉菌工业菌种HS007的基因组标记

通过小片段基因组文库的构建获得工业生产菌HS007的若干基因组片段,并以大肠杆菌-链霉菌穿梭质粒pHJL400为载体,构建了5个插入了特异性标记序列及抗性筛选标记的重组质粒pHJL02AFOH,pHJL07AFOH,pHJL08AFOH,pHJL10AFOH和pHJL12AFOH.利用这些质粒转化工业生产菌株HS007,获得具有特异性标记序列和相应抗性的标记菌株02-72,07-44,08-02,10-81和12-58,其中02-72和12-58的生产能力不受插入片段的影响.利用重组质粒pSP02AFOH上抗性标记两端两个FRT序列的分子内重组去除抗性标记,并以大肠杆菌一链霉菌穿梭质粒pGH112替换该质粒的载体部分,得到重组质粒pGH02FH.以pGH02FH转化标记菌株02-72,获得具有特异性标记序列而没有相应抗性的菌株02-72-36.发酵结果表明,标记片段的插入不影响菌株02-72-36的生产能力.本方法建立了链霉菌工业菌种基因组标记的技术平台.

Genome labeling of the Streptomyces strain HS007

By construction of small fragments genome bank, several different fragments from chromosomal DNA of streptomyces production strain HS007 were cloned into cloning vector pSP-SIM. According to the sequencing results, five of them, which were 3- to 7- kb in size with single restriction sites in the middle, were cut down and re-cloned into bacteria-streptomyces shuttle vector pHJL400. Subsequently, the marker cassette, composed of special labeling sequence, oriT, 2 FLP recognition target sites and apramycin resistance gene, was inserted into the single restriction sites to create recombinant plasmids pHJL02AFOH, pHJL07AFOH, pHJL08AFOH, pHJL10AFOH and pHJL 12AFOH. These recombinant plasmids were then transformed into target production strain HS007 by conjugal transfer method, and corresponding marked mutants named 02-72, 07-44, 08-02, 10-81 and 12-58 were screened. Two of these mutants, 02-72 and 12-58, did not show changes in fermentation ability, whereas the others lost partially or completely fermentation ability. The apramycin resistance gene and oriT, flanked by two FLP recognition target sites, were then removed by FLP-mediated deletion from recombinant plasmid pSP02AFOH to give pSP02F, whereas the special labeling sequence was still reserved in pSP02F because of being located out of the two FLP recognition target sites. Finally, replacement plasmid pGH02FH was constructed by replacing the vector part of pSP02F with bacteria-streptomyces shuttle vector pGHll2 and transformed into mutant 02-72. By selecting apramycin sensitive colonies, marked mutant 02-72-36, whose chromosome was inserted by special labeling sequence without apramycin resistance gene, was screened. Fermentation confirmed that its production ability was not reduced. Such genome labeling technique might be used in other strains of streptomyces to protect the property marks.