In vivo random mutagenesis of streptomycetes using mariner-based transposon Himar1

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• 作者：Bohdan Bilyk (1)
  Stephen Weber (2)
  Maksym Myronovskyi (1)
  Oksana Bilyk (1)
  Lutz Petzke (3)
  Andriy Luzhetskyy (1)
  
• 关键词：Himar1 ; Actinobacteria ; Transposon mutagenesis ; Recombinases ; Rescue plasmids
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2013
• 出版时间：January 2013
• 年：2013
• 卷：97
• 期：1
• 页码：351-359
• 全文大小：312KB
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• 作者单位：Bohdan Bilyk (1)
  Stephen Weber (2)
  Maksym Myronovskyi (1)
  Oksana Bilyk (1)
  Lutz Petzke (3)
  Andriy Luzhetskyy (1)
  
  1. Helmholtz-Institute for Pharmaceutical Research Saarland, Saarland University, Campus C2.3, Saarbrücken, 66123, Germany
  2. Pharmazeutische Biologie und Biotechnologie, Albert-Ludwigs-University of Freiburg, Stefan-Meier-Str. 19, Freiburg, 79104, Germany
  3. BASF SE, Carl-Bosch Strasse 38, Ludwigshafen am Rhein, 67056, Germany
  
• ISSN：1432-0614

文摘

We report here the in vivo expression of the synthetic transposase gene himar1(a) in Streptomyces coelicolor M145 and Streptomyces albus. Using the synthetic himar1(a) gene adapted for Streptomyces codon usage, we showed random insertion of the transposon into the streptomycetes genome. The insertion frequency for the Himar1-derived minitransposons is nearly 100?% of transformed Streptomyces cells, and insertions are stably inherited in the absence of an antibiotic selection. The minitransposons contain different antibiotic resistance selection markers (apramycin, hygromycin, and spectinomycin), site-specific recombinase target sites (rox and/or loxP), I-SceI meganuclease target sites, and an R6Kγ origin of replication for transposon rescue. We identified transposon insertion loci by random sequencing of more than 100 rescue plasmids. The majority of insertions were mapped to putative open-reading frames on the S. coelicolor M145 and S. albus chromosomes. These insertions included several new regulatory genes affecting S. coelicolor M145 growth and actinorhodin biosynthesis.