Seamless deletion of a large DNA fragment in the taxol-producing fungus Pestalotiopsis microspora |
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Authors: | Longfei Chen Yingying Li Qian Zhang Oren Akhberdi Dongsheng Wei Jiao Pan Xudong Zhu |
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Institution: | 1. State Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China;2. Beijing Key Laboratory of Gene Engineering Drug and Biotechnology, Institute of Biochemistry and Biotechnology, College of Life Sciences, Beijing Normal University, Beijing 100875, China |
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Abstract: | To reduce the unnecessary gene clusters in the taxol-producing fungus Pestalotiopsis microspora, we report the development of an effective DNA deletion method that relies on a deletion cassette constructed with the Gateway-technique and overlap extension PCR, using the orotidine 5′-phosphate decarboxylase as recyclable marker for selection. By this approach, two adjacent DNA sequences can be sequentially deleted in a single transformation mediated by Agrobacterium tumefaciens, resulting in the deletion of a large DNA fragment. Additionally, the selection marker is spontaneously eliminated in this process. We used this method to successfully remove the mus53 locus of P. microspora. |
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Keywords: | ATMT 5-FOA Counter selection Orotidine 5′-phosphate decarboxylase Seamless genome reduction |
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