An Efficient Procedure for Marker-Free Mutagenesis of S. coelicolor by Site-Specific Recombination for Secondary Metabolite Overproduction
Publication in refereed journal

香港中文大學研究人員
替代計量分析
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其它資訊
摘要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. © 2013 Zhang et al.
著者Zhang B., Zhang L., Dai R., Yu M., Zhao G., Ding X.
期刊名稱PLoS ONE
出版年份2013
月份2
日期7
卷號8
期次2
出版社Public Library of Science
出版地United States
國際標準期刊號1932-6203
語言英式英語

上次更新時間 2020-25-10 於 02:28