[2013.7.10]We and American collaborators developed a novel thermotargetron system for gene targeting in thermophiles based on a mobile group II intron, which has been successfully applied in the targeted gene engineering inClostridium thermocellum, one of the most promising candidates for production of lignocellulosic ethanol. This study has been published in PloS ONE on 9th July.
Targetron is a widely used gene targeting tool derived from the mobile group II intron. However, the targetrons in hand are developed on the basis of mesophilic group II introns, hense cannot be applied in thermophiles. On the other hand, many thermophilic bacteria can be potentially used in various industrialization due to their unique biochemical and metabolic characterizations.C. thermocellumis one of the most promising candidates for cellulosic ethanol production via consolidated bioprocess (CBP), because the oxygen-free growth condition at high temperature promotes the hydrolysis of lignocellulose substrates, reduces the cost of refrigeration and product retrievement, and decreases the frequency of contamination during fermentation. However, the wild type thermophilic candidates contain shortcomings of low stress resistance, restricted substrate, or low production of ethanol. Meanwhile, few genetic tools have been developed for thermophiles so far. Thus, novel targeted genetic technology for thermophiles is emergently needed to meet the requirement of industrialization.
Targetron consists of an autocatalytic intron RNA 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. In this study, Here we contructed a thermotargetron system for gene targeting in thermophiles based on a mobile group II intron from the thermophilic cyanobacteriumThermosynechococcus elongates. The thermotargetron system was used inC. thermocellumto disrupt six different chromosomal genes (cipA, hfat, hyd, ldh, pta, and pyrF), and high integration efficiencies were achieved (67-100%). 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. Besides, the fermentation analysis demonstrated that the disruption of lactate dehydragenase (Ldh) and phosphotransacetylase (Pta) could enhance the ethanol production ofC. thermocellum, which also indicated that this novel technique could be used in the metabolic engineering of this important thermophilic microorganism to support the industrial utilization of lignocellulosic biomass and production of cellulosic ethanol in future. The thermotargetron system developed in this study should also be readily adaptable for gene targeting in other thermophiles.
The work in this paper was accomplished through a research collaboration between us and the research group of Prof. Alan M. Lambowitz from University of Texas at Austin (USA). Dr. Georg Mohr from Prof. Lambowitz’s lab and the Ph.D student Wei Hong from our group are the co-first authors. Prof. Lambowitz and the assitant professor Ya-jun Liu from our group are the corresponding authors. The Ph.D student Jie Zhang and the assistant professor Dr. Gu-zhen Cui also participated in this sutdy. The work in Prof. Lambowitz’s lab was supported by the NIH (grant GM37949) and the Welch Foundation (grant F-1607); while the study in our group was supported by 973 Program (grant 2011CB707404) and the 863 Program (grant 2011BAD22B02) from Ministry of Science and Technology of China to Prof. Qui Cui.
NCBI PubMed
Georg Mohr, Wei Hong, Jie Zhang, Gu-zhen Cui, Yunfeng Yang, Qiu Cui, Ya-jun Liu*, Alan M. Lambowitz* (2013) A targetron system for gene targeting in thermophiles and its Application inClostridium thermocellum.PLoS ONE8(7), e69032. [Full text (Publisher website)]
