title: An efficient procedure for marker-free mutagenesis of S. coelicolor by site-specific recombination for secondary metabolite overproduction.
abstract: Streptomyces bacteria are known for producing important natural compounds by secondary metabolism, especially antibiotics with novel biological activities. Functional studies of antibiotic-biosynthesizing gene clusters are generally through homologous genomic recombination by gene-targeting vectors. Here, we present a rapid and efficient method for construction of gene-targeting vectors. This approach is based on Streptomyces phage φBT1 integrase-mediated multisite in vitro site-specific recombination. Four 'entry clones' were assembled into a circular plasmid to generate the destination gene-targeting vector by a one-step reaction. The four 'entry clones' contained two clones of the upstream and downstream flanks of the target gene, a selectable marker and an E. coli-Streptomyces shuttle vector. After targeted modification of the genome, the selectable markers were removed by φC31 integrase-mediated in vivo site-specific recombination between pre-placed attB and attP sites. Using this method, part of the calcium-dependent antibiotic (CDA) and actinorhodin (Act) biosynthetic gene clusters were deleted, and the rrdA encoding RrdA, a negative regulator of Red production, was also deleted. The final prodiginine production of the engineered strain was over five times that of the wild-type strain. This straightforward φBT1 and φC31 integrase-based strategy provides an alternative approach for rapid gene-targeting vector construction and marker removal in streptomycetes.
authors: Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China.
authors: Bo
authors: B
authors: Zhang
authors: Lin
authors: L
authors: Zhang
authors: Ruixue
authors: R
authors: Dai
authors: Meiying
authors: M
authors: Yu
authors: Guoping
authors: G
authors: Zhao
authors: Xiaoming
authors: X
authors: Ding
mesh_terms: D000880:Anthraquinones / Q000378:metabolism; D017353:Gene Deletion; D023061:Gene Order; D018390:Gene Targeting; D005798:Genes, Bacterial; D005822:Genetic Vectors; D059765:Homologous Recombination; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D011353:Prodigiosin / Q000031:analogs & derivatives / Q000096:biosynthesis; D013302:Streptomyces / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D000095622:Benzoisochromanequinones
chemical_list: D000880:Anthraquinones; D011353:Prodigiosin; C010307:prodiginine; C013390:actinorhodin; D000095622:Benzoisochromanequinones