Seamless deletion of a large DNA fragment in the taxol-producing fungus Pestalotiopsis microspora

Longfei Chen; Yingying Li; Qian Zhang; Oren Akhberdi; Dongsheng Wei; Jiao Pan; Xudong Zhu

doi:10.1016/j.myc.2016.08.003

Short communication

Seamless deletion of a large DNA fragment in the taxol-producing fungus Pestalotiopsis microspora

Longfei Chen, Yingying Li, Qian Zhang, Oren Akhberdi, Dongsheng Wei, Jiao Pan, Xudong Zhu

Author information

Longfei Chen
State Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University
Yingying Li
State Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University
Qian Zhang
State Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University
Oren Akhberdi
State Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University
Dongsheng Wei
State Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University
Jiao Pan
State Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University
Xudong Zhu Corresponding author
State Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University
Beijing Key Laboratory of Gene Engineering Drug and Biotechnology, Institute of Biochemistry and Biotechnology, College of Life Sciences, Beijing Normal University

Keywords: ATMT, 5-FOA, Counter selection, Orotidine 5′-phosphate decarboxylase, Seamless genome reduction

JOURNAL OPEN ACCESS

2016 Volume 58 Issue 1 Pages 35-39

DOI https://doi.org/10.1016/j.myc.2016.08.003

Details

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.

Corresponding author

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