A Genetic System for the Thermophilic Acetogenic Bacterium Thermoanaerobacter kivui
A Genetic System for the Thermophilic Acetogenic Bacterium Thermoanaerobacter kivui
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DOI：
10.1128/aem.02210-17
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发表时间：
2018-02-01
影响因子：
4.4
通讯作者：
Mueller, Volker
中科院分区：
文献类型：
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作者：
Basen, Mirko;Geiger, Irina;Mueller, Volker
Kivui高温厌氧菌是为数不多的嗜热产乙酸菌之一。它在66摄氏度的糖上生长得最好，但在H-2+CO2或CO的情况下也有营养作用，产生的主要产物是醋酸酯。虽然基因组衍生的乙酰化模型已经被开发出来，但关于重要的酶在碳和能量代谢中的功能的生理或生化实验只进行了很少。为了解决这个问题，我们开发了一种定向无标记基因缺失和基因整合到基氏锥虫基因组中的方法。该菌株在指数生长期自然携带质粒DNA，转化频率高达3.9×10(-6)。利用非复制型质粒和5-氟代甲酸盐法筛选，缺失了转磷酸核糖基转移酶(PYRE)基因，获得了一株Delta Pre尿液营养缺陷型菌株TKV002。在没有尿嘧啶的情况下，将PYRE重新引入到质粒上，或将PYRE插入到基因组内的不同位置，恢复了生长。随后，我们研究了猕猴桃果糖代谢。编码1-磷酸果糖激酶(1-PFK)的基因FruK(TKV_C23150)被缺失，通过两个单一的同源重组事件以PYRE作为选择标记。由此产生的Delta FruK菌株TKV003不能在果糖上生长；然而，在葡萄糖(或甘露糖)上的生长不受影响。将PYRE作为一种选择标记与菌株吸收DNA的天然能力相结合，将为进一步研究这种嗜热产乙酸菌的二氧化碳减排、节能及其调控奠定基础。重要的产乙酸菌由于具有从H-2+CO2或合成气中从头合成乙酸酯、丁酸酯或乙醇等碳化合物的潜力，目前是生物技术应用研究的重点。根据现有的基因组序列和生化实验，乙酸菌在能量代谢方面有所不同。因此，迫切需要了解通过Wood-LJungdahl途径的碳和电子流动及其与能量守恒的联系，这需要进行遗传操作，如删除或过度表达编码可能的关键酶的基因。不幸的是，只有少数产乙酸菌的遗传系统被报道过。在这里，我们证明了对嗜热性产乙酸菌的基因改造的概念证明。该遗传系统将用于研究涉及生物合成和能量代谢的基因，并可能进一步应用于代谢工程T.kivui生产燃料和化学品。
Thermoanaerobacter kivui is one of the very few thermophilic acetogenic microorganisms. It grows optimally at 66 degrees C on sugars but also lithotrophically with H-2 + CO2 or with CO, producing acetate as the major product. While a genome-derived model of acetogenesis has been developed, only a few physiological or biochemical experiments regarding the function of important enzymes in carbon and energy metabolism have been carried out. To address this issue, we developed a method for targeted markerless gene deletions and for integration of genes into the genome of T. kivui. The strain naturally took up plasmid DNA in the exponential growth phase, with a transformation frequency of up to 3.9 x 10(-6). A nonreplicating plasmid and selection with 5-fluoroorotate was used to delete the gene encoding the orotate phosphoribosyltransferase (pyrE), resulting in a Delta pyrE uracil-auxotrophic strain, TKV002. Reintroduction of pyrE on a plasmid or insertion of pyrE into different loci within the genome restored growth without uracil. We subsequently studied fructose metabolism in T. kivui. The gene fruK (TKV_c23150) encoding 1-phosphofructosekinase (1-PFK) was deleted, using pyrE as a selective marker via two single homologous recombination events. The resulting Delta fruK strain, TKV003, did not grow on fructose; however, growth on glucose (or on mannose) was unaffected. The combination of pyrE as a selective marker and the natural competence of the strain for DNA uptake will be the basis for future studies on CO2 reduction and energy conservation and their regulation in this thermophilic acetogenic bacterium.IMPORTANCE Acetogenic bacteria are currently the focus of research toward biotechnological applications due to their potential for de novo synthesis of carbon compounds such as acetate, butyrate, or ethanol from H-2 + CO2 or from synthesis gas. Based on available genome sequences and on biochemical experiments, acetogens differ in their energy metabolism. Thus, there is an urgent need to understand the carbon and electron flows through the Wood-Ljungdahl pathway and their links to energy conservation, which requires genetic manipulations such as deletion or overexpression of genes encoding putative key enzymes. Unfortunately, genetic systems have been reported for only a few acetogenic bacteria. Here, we demonstrate proof of concept for the genetic modification of the thermophilic acetogenic species Thermoanaerobacter kivui. The genetic system will be used to study genes involved in biosynthesis and energy metabolism, and may further be applied to metabolically engineer T. kivui to produce fuels and chemicals.