Shao Xiongjun, Zhou Jilai, Olson Daniel G, Lynd Lee R
14 Engineering Drive, Thayer School of Engineering, Dartmouth College, Hanover, NH 03755 USA ; DOE BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831 USA.
Biotechnol Biofuels. 2016 May 4;9:100. doi: 10.1186/s13068-016-0514-1. eCollection 2016.
Thermoanaerobacter ethanolicus produces a considerable amount of ethanol from a range of carbohydrates and is an attractive candidate for applications in bioconversion processes. A genetic system with reusable selective markers would be useful for deleting acid production pathways as well as other genetic modifications.
The thymidine kinase (tdk) gene was deleted from T. ethanolicus JW200 to allow it to be used as a selectable marker, resulting in strain X20. Deletion of the tdk gene reduced growth rate by 20 %; however, this could be reversed by reintroducing the tdk gene (strain X20C). The tdk and high-temperature kanamycin (htk) markers were tested by using them to delete lactate dehydrogenase (ldh). During positive selection of ldh knockouts in strain X20 on kanamycin agar plates, six out of seven picked colonies were verified transformants. Deletion of ldh reduced lactic acid production by 90 %. The tdk and 5-fluoro-2'-deoxyuridine (FUDR) combination worked reliably as demonstrated by successful tdk removal in all 21 colonies tested.
A gene deletion and integration system with reusable markers has been developed for Thermoanaerobacter ethanolicus JW200 with positive selection on kanamycin and negative selection on FUDR. Gene deletion was demonstrated by ldh gene deletion and gene integration was demonstrated by re-integration of the tdk gene. Transformation via a natural competence protocol could use DNA PCR products amplified directly from Gibson Assembly mixture for efficient genetic modification.
嗜热栖热放线菌能利用多种碳水化合物大量生产乙醇，是生物转化过程中颇具吸引力的候选菌株。具有可重复使用选择标记的遗传系统对于删除产酸途径以及进行其他基因改造很有用。
从嗜热栖热放线菌JW200中删除胸苷激酶（tdk）基因，使其可作为选择标记，得到菌株X20。tdk基因的缺失使生长速率降低了20%；然而，通过重新引入tdk基因（菌株X20C）可使其恢复。通过利用tdk和高温卡那霉素（htk）标记删除乳酸脱氢酶（ldh）来进行测试。在菌株X20的ldh基因敲除在卡那霉素琼脂平板上进行阳性选择期间，挑选的7个菌落中有6个被证实为转化体。ldh基因的缺失使乳酸产量降低了90%。tdk和5-氟-2'-脱氧尿苷（FUDR）组合的效果可靠，如在所有测试的21个菌落中tdk成功去除所证明的那样。
已为嗜热栖热放线菌JW200开发了一种具有可重复使用标记的基因缺失和整合系统，在卡那霉素上进行阳性选择，在FUDR上进行阴性选择。通过ldh基因缺失证明了基因缺失，通过tdk基因的重新整合证明了基因整合。通过自然感受态方案进行的转化可以使用直接从吉布森组装混合物中扩增的DNA PCR产物进行高效的基因改造。
