Development of a Markerless Knockout Method for Actinobacillus succinogenes
Development of a Markerless Knockout Method for Actinobacillus succinogenes
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DOI：
10.1128/aem.00492-14
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发表时间：
2014-05-01
影响因子：
4.4
通讯作者：
Vieille, Claire
中科院分区：
文献类型：
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作者：
Joshi, Rajasi V.;Schindler, Bryan D.;Vieille, Claire
产琥珀酸放线杆菌是最好的天然产琥珀酸生物之一，但它仍然需要工程改造，以进一步提高琥珀酸产量和生产力。在本研究中，我们开发了一种无标记敲除A。使用自然转化或电穿孔将琥珀酸原酶转化为琥珀酸原酶。以大肠杆菌异柠檬酸脱氢酶基因为阳性选择标记，利用A.琥珀酸菌的谷氨酸营养缺陷型选择异柠檬酸生长。使用酿酒酵母翻转酶重组酶（Flp）去除选择标记，允许其重复使用。最后，用吖啶橙子固化表达flp的质粒。我们证明，使用这种方法可以将至少两个连续的缺失引入到同一菌株中，在选择盒的每一侧上总共不超过1kb的DNA被需要以在转化期间保护免受核酸外切酶活性的影响，并且在每一侧上不超过200bp的同源DNA被需要以进行有效重组。我们还表明，电穿孔可以作为一种替代的转化方法，以获得敲除突变体和富集的限定培养基可以用于直接选择敲除突变体的琼脂平板上具有高效率。单敲除突变体的延胡索酸还原酶和丙酮酸甲酸裂解酶编码基因，获得使用这种敲除策略。通过在丙酮酸甲酸裂解酶敲除突变株中删除柠檬酸裂解酶、β-半乳糖苷酶和乌头酸酶编码基因，也获得了双敲除突变株。
Actinobacillus succinogenes is one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method for A. succinogenes using natural transformation or electroporation. The Escherichia coli isocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use of A. succinogenes's auxotrophy for glutamate to select for growth on isocitrate. The Saccharomyces cerevisiae flippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressing flp was cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, beta-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain.