Orotidine 5′-phosphate decarboxylase-based reusable in situ genetic editing system: Development and application in taxol-producing Pestalotiopsis microspora
Orotidine 5′-phosphate decarboxylase-based reusable in situ genetic editing system: Development and application in taxol-producing Pestalotiopsis microspora
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DOI：
10.1002/elsc.201400220
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发表时间：
2015-07-01
影响因子：
2.7
通讯作者：
Zhu, Xudong
中科院分区：
文献类型：
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作者：
Chen, Longfei;Wei, Dongsheng;Zhu, Xudong
拟盘多毛孢小孢子菌NK17产生抗肿瘤药物紫杉醇和拟盘多毛内酯B，一种类似胆固醇酯转移蛋白抑制剂。然而，由于缺乏分子工具，人们对它们的生物合成知之甚少。我们在此报道了从该真菌中鉴定出的欧罗替丁5-磷酸脱羧酶编码基因pm-ura3的功能。我们利用它作为一个标记物，构建了一个可重复使用的系统，用于基因无疤痕的删除、恢复和标记。作为阳性标记物，pm-ura3可用于启动尿嘧啶营养不良宿主的靶向兴趣位点。然后，在5-氟原酸的负选择下，它可以被任何人工DNA序列取代，例如突变的ORF。通过这种方法，我们可以就地编辑感兴趣的基因并回收标记。不需要异质标记，避免了外源DNA序列如HisG、FRP和LoxP的积累。我们利用该系统对pm-mus53的功能进行了研究，发现该基因对农杆菌介导的转化介导的非同源末端连接过程影响不大。我们还研究了一种推测的杉二烯合成酶在小孢子假单胞菌紫杉醇生物合成途径中的表达，发现该合成酶在固体和液体培养基中的表达存在差异。
Pestalotiopsis microspora NK17 produces the antitumor drug paclitaxel and pestalotiollide B, an analog of cholesterol ester transfer protein inhibitors. However, little is known about their biosynthesis due to the lack of molecular tools. We report here the functional identification of the orotidine 5-phosphate decarboxylase encoding gene, pm-ura3, from this fungus. We utilized it as a marker in the construction of a reusable system for gene scarless deletion, restoration, and tagging. As a positive marker, pm-ura3 can be used to targeted locus of interest in a start uracil auxotrophic host. It then can be replaced by any artificial DNA sequence, e.g. a mutated ORF, under the negative selection of 5-fluoroorotic acid. By this method, we can edit genes of interest in situ and recycle the marker. No heterogeneous markers are needed, and the accumulation of foreign DNA sequences such as HisG, FRP, and LoxP is avoided. We applied the system to study the function of pm-mus53 and found that the gene had little effect on the nonhomologous end-joining process mediated by agrobacterium tumefaciens-mediated transformation. We also investigated the expression of a putative taxadiene synthase in taxol biosynthetic pathways of P. microspora and found it was differentially expressed in solid and liquid medium.