Abstract
BackgroundTargetrons 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 FindingsHere, 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.
Highlights
Renewable fuels like bioethanol are urgently needed due to ever increasing global energy demands, limited quantities of fossil fuels, and climate change [1,2]
That TeI4h* is not retargetable, likely due to difficulties with its exon-binding site 2 (EBS2), one of the sequence elements that base pairs to the DNA target site
We describe the construction of a thermotargetron derived from a mobile group II intron found in the thermophilic cyanobacterium T. elongatus
Summary
Renewable fuels like bioethanol are urgently needed due to ever increasing global energy demands, limited quantities of fossil fuels, and climate change [1,2]. Clostridium thermocellum is a thermophilic anaerobic bacterium well known for its robust cellulose-degrading system [7]. It is considered one of the most promising candidates for consolidated bioprocessing (CBP) of cellulolytic ethanol [8]. The recently determined genome sequences of C. thermocellum strains enable metabolic engineering by targeting specific genes and pathways to improve ethanol production. Novel gene targeting methods are required for the efficient metabolic engineering of C. thermocellum, as well as for other industrially important thermophiles. 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
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