Use of the meganuclease I-SceI of Saccharomyces cerevisiae to select for gene deletions in actinomycetes.
The search for new natural products is leading to the isolation of novel actinomycete species, many of which will ultimately require genetic analysis. Some of these isolates will likely exhibit low intrinsic frequencies of homologous recombination and fail to sporulate under laboratory conditions, exacerbating the construction of targeted gene deletions and replacements in genetically uncharacterised strains. To facilitate the genetic manipulation of such species, we have developed an efficient method to generate gene or gene cluster deletions in actinomycetes by homologous recombination that does not introduce any other changes to the targeted organism's genome. We have synthesised a codon optimised I-SceI gene for expression in actinomycetes that results in the production of the yeast I-SceI homing endonuclease which produces double strand breaks at a unique introduced 18 base pair recognition sequence. Only those genomes that undergo homologous recombination survive, providing a powerful selection for recombinants, approximately half of which possess the desired mutant genotype. To demonstrate the efficacy and efficiency of the system, we deleted part of the gene cluster for the red-pigmented undecylprodiginine complex of compounds in Streptomyces coelicolor M1141. We believe that the system we have developed will be broadly applicable across a wide range of actinomycetes.
Department of Molecular Microbiology, John Innes Centre, Norwich, NR4 7UH, U.K.
Lorena T
LT
Fernández-Martínez
Department of Molecular Microbiology, John Innes Centre, Norwich, NR4 7UH, U.K.
Mervyn J
MJ
Bibb
D001483:Base Sequence; D003062:Codon; D053903:DNA Breaks, Double-Stranded; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D059765:Homologous Recombination; D008969:Molecular Sequence Data; D005810:Multigene Family; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D011353:Prodigiosin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D003062:Codon; D004269:DNA, Bacterial; D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; C528938:undecylprodiginine; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific; D011353:Prodigiosin