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

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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 phi BT1 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 phi C31 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 wildtype strain. This straightforward phi BT1 and phi C31 integrase-based strategy provides an alternative approach for rapid genetargeting vector construction and marker removal in streptomycetes.
著者Zhang B, Zhang L, Dai RX, Yu MY, Zhao GP, Ding XM
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 SCIENCE
國際標準期刊號1932-6203
語言英式英語
Web of Science 學科類別Multidisciplinary Sciences; MULTIDISCIPLINARY SCIENCES; Science & Technology - Other Topics

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