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    A Targetron System for Gene Targeting in Thermophiles and Its Application in Clostridium thermocellum

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    journal.pone.0069032.pdf (1.638Mb)
    Date
    2013-07-09
    Author
    Mohr, Georg
    Hong, Wei
    Zhang, Jie
    Cui, Gu-zhen
    Yang, Yunfeng
    Cui, Qiu
    Liu, Ya-jun
    Lambowitz, Alan M.
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    Abstract
    Background: Targetrons are gene targeting vectors derived from mobile group II introns. They consist of an autocatalytic intron RNA (a “ribozyme”) and an intron-encoded reverse transcriptase, which use their combined activities to achieve highly efficient site-specific DNA integration with readily programmable DNA target specificity. -- Methodology/Principal Findings: Here, we used a mobile group II intron from the thermophilic cyanobacterium Thermosynechococcus elongatus to construct a thermotargetron for gene targeting in thermophiles. After determining its DNA targeting rules by intron mobility assays in Escherichia coli at elevated temperatures, we used this thermotargetron in Clostridium thermocellum, a thermophile employed in biofuels production, to disrupt six different chromosomal genes (cipA, hfat, hyd, ldh, pta, and pyrF). High integration efficiencies (67–100% without selection) were achieved, enabling detection of disruptants by colony PCR screening of a small number of transformants. Because the thermotargetron functions at high temperatures that promote DNA melting, it can recognize DNA target sequences almost entirely by base pairing of the intron RNA with less contribution from the intron-encoded protein than for mesophilic targetrons. This feature increases the number of potential targetron-insertion sites, while only moderately decreasing DNA target specificity. Phenotypic analysis showed that thermotargetron disruption of the genes encoding lactate dehydrogenase (ldh; Clo1313_1160) and phosphotransacetylase (pta; Clo1313_1185) increased ethanol production in C. thermocellum by decreasing carbon flux toward lactate and acetate. -- Conclusions/SignificanceThermotargetron provides a new, rapid method for gene targeting and genetic engineering of C. thermocellum, an industrially important microbe, and should be readily adaptable for gene targeting in other thermophiles.
    Department
    Microbiology
     
    Chemistry
     
    Biochemistry
     
    Institute for Cellular and Molecular Biology
     
    Description
    Georg Mohr, Alan M. Lambowitz, Section of Molecular Genetics and Microbiology, Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, School of Biological Sciences, University of Texas at Austin, Austin, Texas, United States of America
    Wei Hong, Jie Zhang, Gu-zhen Cui, Qiu Cui, Ya-jun Liu, Shandong Provincial Key Laboratory of Energy Genetics, and Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, People’s Republic of China
    Yunfeng Yang, State Key Joint Laboratory of Environment, Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing, People’s Republic of China
    Wei Hong, Jie Zhang, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing, People’s Republic of China
    Subject
    DNA sequeces
    ethanol
    extremophiles
    gene disrution
    gene targeting
    introns
    plasmid construction
    polymerase chain reaction
    URI
    http://hdl.handle.net/2152/27925
    Citation
    Mohr G, Hong W, Zhang J, Cui G-z, Yang Y, et al. (2013) A Targetron System for Gene Targeting in Thermophiles and Its Application in Clostridium thermocellum. PLoS ONE 8(7): e69032. doi:10.1371/journal.pone.0069032
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