Su Chun, Zhao Xin-Qing, Wang Hai-Na, Qiu Rong-Guo, Tang Li
Research Center for Molecular Medicine, Faculty of Chemical, Environmental and Biological Science and Technology, Dalian University of Technology, Dalian 116024, China.
Research Center for Molecular Medicine, Faculty of Chemical, Environmental and Biological Science and Technology, Dalian University of Technology, Dalian 116024, China; School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China.
Gene. 2015 Jan 10;554(2):233-40. doi: 10.1016/j.gene.2014.10.022. Epub 2014 Oct 11.
Type I polyketides are natural products with diverse functions that are important for medical and agricultural applications. Manipulation of large biosynthetic gene clusters containing type I polyketide synthases (PKS) for heterologous expression is difficult due to the existence of conservative sequences of PKS in multiple modules. Red/ET mediated recombination has permitted rapid manipulation of large fragments; however, it requires insertion of antibiotic selection marker in the cassette, raising the problem of interference of expression by leaving "scar" sequence. Here, we report a method for precise seamless stitching of large polyketide biosynthetic gene cluster using a 48.4kb fragment containing type I PKS involved in fostriecin biosynthesis as an example. rpsL counter-selection was used to assist seamless stitching of large fragments, where we have overcome both the size limitations and the restriction on endonuclease sites during the Red/ET recombination. The compatibility and stability of the co-existing vectors (p184 and pMT) which respectively accommodate 16kb and 32.4kb inserted fragments were demonstrated. The procedure described here is efficient for manipulation of large DNA fragments for heterologous expression.
I型聚酮化合物是具有多种功能的天然产物，对医学和农业应用具有重要意义。由于多个模块中存在I型聚酮合酶（PKS）的保守序列，操纵包含I型聚酮合酶的大型生物合成基因簇进行异源表达很困难。Red/ET介导的重组允许对大片段进行快速操作；然而，它需要在盒式结构中插入抗生素选择标记，这就产生了留下“疤痕”序列干扰表达的问题。在此，我们以一个包含参与磷霉素生物合成的I型PKS的48.4kb片段为例，报告一种精确无缝拼接大型聚酮生物合成基因簇的方法。利用rpsL反选择来辅助大片段的无缝拼接，在此过程中我们克服了Red/ET重组过程中的大小限制和对核酸内切酶位点的限制。证明了分别容纳16kb和32.4kb插入片段的共存载体（p184和pMT）的兼容性和稳定性。本文所述方法对于操纵用于异源表达的大型DNA片段是有效的。
