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首页> 外文期刊>Applied 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 Δ pyrF 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 Δ pta 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：我们报道了在热纤梭菌（一种快速溶解纤维素的嗜热厌氧细菌）中进行靶向基因敲除的遗传系统的发展。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。 ΔpyrF菌株是尿嘧啶营养缺陷型，可通过从质粒中对pyrF进行异位表达而恢复为原生营养型，从而提供了积极的遗传选择。此外，使用5-FOA来针对表达质粒的pyrF进行选择，从而对质粒丢失产生阴性选择。这项技术被用来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。热纤梭菌Δpta菌株不产生乙酸盐。这些结果是在热纤梭菌中进行有针对性的同源重组和代谢工程的第一个实例，该微生物在生物燃料行业和可持续能源的发展中拥有令人兴奋且充满希望的未来。

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