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

Zhang, Bo; Zhang, Lin; Dai, Ruixue; Yu, Meiying; Zhao, Guoping; Ding, Xiaoming
February 2013
PLoS ONE;Feb2013, Vol. 8 Issue 2, p1
Academic Journal
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.


Related Articles

  • The use of molecular biology to reprogram Streptomyces to make polyketide antibiotics more efficiently, and create novel secondary metabolites. Petkovič, Hrvoje; Hrauneli, Daslav; Raspor, Peter; Hunter, Iain // Pflugers Archiv European Journal of Physiology;Apr2000, Vol. 439 Issue 7, pr087 

    Recent advances in the molecular genetics of Streptomyces have increased our understanding of polyketide antibiotic biosynthesis, to the point where recombinant DNA approaches to generate novel structures are possible. Our understanding of how antibiotic pathways are regulated and integrated...

  • recombinant DNA.  // Taber's Cyclopedic Medical Dictionary;2005, p1860 

    A definition of the term "recombinant DNA" is presented. It refers to the employment of gene splicing technique to manipulate or insert segments of DNA from an organism into DNA of another. With the help of this technique, the properties and actions of particular genes can be isolated and examined.

  • The cultural mouse. Evans, Martin J. // Nature Medicine;Oct2001, Vol. 7 Issue 10, p1081 

    Focuses on the Lasker Basic Medical Research Award given to researchers for their work in mouse gene targeting. Description of the development system used in the study; Testing of the similarity of ICM of mouse embryos; Techniques developed for mutagenesis.

  • Size Of Gene Specific Inverted Repeat - Dependent Gene Deletion In Saccharomyces cerevisiae. Lim, Chanyuen; Luhe, Annette Lin; JingYing, Crystal Tear; Balagurunathan, Balaji; Wu, Jinchuan; Zhao, Hua // PLoS ONE;Aug2013, Vol. 8 Issue 8, p1 

    We describe here an approach for rapidly producing scar-free and precise gene deletions in S. cerevisiae with high efficiency. Preparation of the disruption gene cassette in this approach was simply performed by overlap extension-PCR of an invert repeat of a partial or complete sequence of the...

  • High-Precision Genome Surgery in Human Stem Cells. Zhou, Ruijie; Dröge, Peter // Current Genomics;Nov2006, Vol. 7 Issue 7, p427 

    The great potential of human embryonic and adult stem cells in regenerative medicine, gene therapy, drug discovery, and basic research is widely recognized. Many future applications depend on our ability to manipulate stem cell genomes with exogenous DNA in a safe and controllable way. Foreign...

  • Technology: The holy grail for plant biologists. Skipper, Magdalena // Nature Reviews Genetics;Jun2009, Vol. 10 Issue 6, p350 

    The article highlights two studies on successful gene targeting in maize and tobacco, providing an improvement on the conventional mutagenesis or trangenesis approaches in plant biology research. Both studies have relied on zinc-finger nucleases (ZFNs), an engineered enzyme that create...

  • Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Tetsushi Sakuma; Ayami Nishikawa; Satoshi Kume; Kazuaki Chayama; Takashi Yamamoto // Scientific Reports;6/27/2014, p1 

    CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications, not only for single gene targeting, but also for multiple targeted mutagenesis. To make the most of the multiplexity of CRISPR/Cas9, we established a system for constructing all-in-one expression vectors...

  • A Novel Selection Marker for Efficient DNA Cloning and Recombineering in E. coli. Jang, Chuan-Wei; Magnuson, Terry // PLoS ONE;Feb2013, Vol. 8 Issue 2, p1 

    Production of recombinant DNA in bacterial cells is an essential technique in molecular biology. Plasmids are usually maintained in an E. coli host by antibiotic selection. However, there are only a few antibiotic-resistance markers available in common use. Here we report the adoption of a novel...

  • Recombinant DNA--On Our Own. Sinsheimer, Robert L. // BioScience;Oct1976, Vol. 26 Issue 10, p599 

    The article presents information on the role of biotechnology in human lives and the recombinant DNA issues. The author observes that the great discoveries in molecular and cellular biology, in particular the elucidation of the structure and functions of the nucleic acids have provided the world...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics