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A Targetron System for Gene Targeting in Thermophiles and Its Application in Clostridium thermocellum
Mohr, Georg1; Hong, Wei2,3,5; Zhang, Jie2,3,5; Cui, Gu-zhen2,3; Yang, Yunfeng4; Cui, Qiu2,3; Liu, Ya-jun2,3; Lambowitz, Alan M.1
2013-07-09
Source PublicationPLOS ONE
Volume8Issue:7
Abstract Abstract: OPEN ACCESS
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/Significance: Thermotargetron 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.
 
; 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.
SubtypeArticle
Subject Area代谢物组学
WOS HeadingsScience & Technology
DOI10.1371/journal.pone.0069032
WOS KeywordGROUP-II INTRON ; HOST-RANGE VECTOR ; STAPHYLOCOCCUS-AUREUS ; REVERSE-TRANSCRIPTASE ; LACTOCOCCUS-LACTIS ; KNOCKOUT SYSTEM ; DNA ; CELLULOSE ; RESTRICTION ; SEQUENCES
Indexed BySCI
Language英语
WOS Research AreaScience & Technology - Other Topics
WOS SubjectMultidisciplinary Sciences
WOS IDWOS:000321736900129
Citation statistics
Cited Times:35[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://ir.qibebt.ac.cn/handle/337004/1682
Collection代谢物组学研究组
Affiliation1.Univ Texas Austin, Sect Mol Genet & Microbiol, Dept Chem & Biochem, Inst Cellular & Mol Biol,Sch Biol Sci, Austin, TX 78712 USA
2.Chinese Acad Sci, Shandong Prov Key Lab Energy Genet, Qingdao Inst Bioenergy & Bioproc Technol, Qingdao, Peoples R China
3.Chinese Acad Sci, Key Lab Biofuels, Qingdao Inst Bioenergy & Bioproc Technol, Qingdao, Peoples R China
4.Tsinghua Univ, State Key Joint Lab Environm Simulat & Pollut Con, Sch Environm, Beijing 100084, Peoples R China
5.Chinese Acad Sci, Univ Chinese Acad Sci, Beijing, Peoples R China
Recommended Citation
GB/T 7714
Mohr, Georg,Hong, Wei,Zhang, Jie,et al. A Targetron System for Gene Targeting in Thermophiles and Its Application in Clostridium thermocellum[J]. PLOS ONE,2013,8(7).
APA Mohr, Georg.,Hong, Wei.,Zhang, Jie.,Cui, Gu-zhen.,Yang, Yunfeng.,...&Lambowitz, Alan M..(2013).A Targetron System for Gene Targeting in Thermophiles and Its Application in Clostridium thermocellum.PLOS ONE,8(7).
MLA Mohr, Georg,et al."A Targetron System for Gene Targeting in Thermophiles and Its Application in Clostridium thermocellum".PLOS ONE 8.7(2013).
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