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首页> 外文期刊>Applied and Environmental Microbiology >Development of pyrF-Based Genetic System for Targeted Gene Deletion in Clostridium thermocellum and Creation of a pta Mutant
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Development of pyrF-Based Genetic System for Targeted Gene Deletion in Clostridium thermocellum and Creation of a pta Mutant

机译：基于pyrF的热纤梭菌靶向基因删除遗传系统的开发和pta突变体的创建

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摘要

We report development of a genetic system for making targeted gene knockouts in Clostridium thermocellum, a thermophilic anaerobic bacterium that rapidly solubilizes cellulose. A toxic uracil analog, 5-fluoroorotic acid (5-FOA), was used to select for deletion of the pyrF gene. The ΔpyrF strain is a uracil auxotroph that could be restored to a prototroph via ectopic expression of pyrF from a plasmid, providing a positive genetic selection. Furthermore, 5-FOA was used to select against plasmid-expressed pyrF, creating a negative selection for plasmid loss. This technology was used to delete a gene involved in organic acid production, namely pta, which encodes the enzyme phosphotransacetylase. The C. thermocellum Δpta strain did not produce acetate. These results are the first examples of targeted homologous recombination and metabolic engineering in C. thermocellum, a microbe that holds an exciting and promising future in the biofuel industry and development of sustainable energy resources.
机译：我们报告了一种遗传系统的发展，该系统可用于快速溶解纤维素的嗜热厌氧细菌中的靶向基因敲除。使用有毒的尿嘧啶类似物5-氟乳清酸（5-FOA）来选择 pyrF 基因的缺失。 Δ pyrF 菌株是尿嘧啶营养缺陷型，可通过质粒中 pyrF 的异位表达恢复为原养型，提供了积极的遗传选择。此外，使用5-FOA来选择质粒表达的 pyrF ，从而对质粒丢失产生阴性选择。这项技术被用于删除涉及有机酸生产的基因，即 pta ，该基因编码磷酸转乙酰酶。 C。 Thermocellum Δ pta 菌株不产生乙酸盐。这些结果是 C中靶向同源重组和代谢工程的第一个实例。 Thermocellum ，在生物燃料行业和可持续能源发展中拥有令人兴奋且充满希望的未来的微生物。

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