A targetron system for gene targeting in thermophiles and its application in Clostridium thermocellum

• Published in: PloS one 2013-07, Vol.8 (7), p.e69032
• Main Authors: Mohr, Georg, Hong, Wei, Zhang, Jie, Cui, Gu-zhen, Yang, Yunfeng, Cui, Qiu, Liu, Ya-jun, Lambowitz, Alan M
• Format: Article
• Language: English
• Subjects: Acetic acid; Agriculture; Base Pairing; Base Sequence; Binding Sites; Biochemistry; Biodiesel fuels; Biofuels; Biology; Biomass energy; Catalytic RNA; Cellulose; Chromosomes, Bacterial; Climate change; Clostridium thermocellum; Clostridium thermocellum - genetics; Clostridium thermocellum - metabolism; Cyanobacteria - genetics; Deoxyribonucleic acid; DNA; DNA polymerases; E coli; Earth Sciences; Elongation; Energy; Engineering; Enzymes; Escherichia coli; Escherichia coli - genetics; Ethanol; Gene Order; Gene sequencing; Gene Targeting; Genes; Genetic engineering; Genetic Vectors; Genetically modified organisms; Genomes; High temperature; Insertion; Introns; L-Lactate dehydrogenase; L-Lactate Dehydrogenase - genetics; L-Lactate Dehydrogenase - metabolism; Laboratories; Lac Operon; Lactate dehydrogenase; Lactic acid; Lactococcus lactis; Metabolism; Metabolites; Metabolome; Metabolomics - methods; Molecular biology; Mutagenesis, Insertional; Nucleic Acid Conformation; Nucleotide sequence; Phosphate Acetyltransferase - genetics; Phosphate Acetyltransferase - metabolism; Plasmids; Plasmids - genetics; Ribonucleic acid; RNA; RNA, Bacterial - chemistry; RNA, Bacterial - genetics; RNA, Catalytic; RNA-directed DNA polymerase; Target recognition; Temperature; Thermophiles
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0069032

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. 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. 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.