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New Vector System for Random, Single-Step Integration of Multiple Copies of DNA into the Rhodococcus Genome

机译：新的矢量系统，用于随机，一步整合多份DNA到红球菌基因组中。

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We designed a new vector system for creating a random mutant library with multiple integrations of DNA fragments into the Rhodococcus genome in a single step. For this, we cotransformed two vectors into Rhodococcus by electroporation: pTip- istAB - sacB regulates the expression of the transposase (IstA) and its helper protein (IstB) under the influence of a thiostrepton-inducible promoter, and pRTSK- sacB provides the transposable-marker DNA. Both are multicopy vectors that are stable in the host cells; transposition of the transposable-marker DNA occurs only after the induction of IstA/IstB expression. With the addition of thiostrepton, all cultured cells harboring the two vectors, irrespective of the volume, can be mutated by random insertion of the transposable-marker DNA into their genome. Among the generated mutants examined, 30% showed multiple (two to five) insertion copies. The multiple integrated DNA copies were stable in the genome for more than 80 generations of serial growth without the addition of any selective antibiotics. This system can also be used for integrating various copy numbers of stably maintained protein expression cassettes in the host cell genome to modulate the expression level of biologically active recombinant proteins. We successfully applied this system to integrate multiple copies of expression cassettes for proline iminopeptidase and vitamin D_(3) hydroxylase into the Rhodococcus genome and verified that the clones containing double or multiple copies of the integrated cassettes produced higher levels and showed higher enzymatic activities of the target protein than clones with only a single copy of integration.
机译：我们设计了一个新的载体系统，用于创建一个随机突变体文库，可在一个步骤中将DNA片段多次整合到红球菌基因组中。为此，我们通过电穿孔将两个载体共转化为红球菌：pTipistAB-sacB在硫代链霉菌诱导型启动子的影响下调节转座酶（IstA）及其辅助蛋白（IstB）的表达，而pRTSK- sacB提供可转座的标记DNA。两者都是在宿主细胞中稳定的多拷贝载体。仅在诱导IstA / IstB表达后才发生转座标记DNA的转座。通过添加硫代链霉菌素，可以通过将转座标记DNA随机插入其基因组来突变所有带有两个载体的培养细胞，而不论其体积如何。在检查的生成的突变体中，有30％显示出多（两到五个）插入拷贝。多个整合的DNA拷贝在基因组中稳定了80多个世代，无需添加任何选择性抗生素。该系统还可以用于整合宿主细胞基因组中各种拷贝数的稳定维持的蛋白表达盒，以调节生物活性重组蛋白的表达水平。我们成功地应用了该系统，将脯氨酸亚肽酶和维生素D_（3）羟化酶的表达盒的多个副本整合到红球菌基因组中，并验证了包含双重或多个副本的整合盒的克隆产生了更高的水平，并显示了更高的酶活性。靶蛋白比仅具有单个拷贝的克隆高。

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