RedEx: a method for seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters |
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| Authors: | Chaoyi Song Ji Luan Ruijuan Li Chanjuan Jiang Yu Hou Qingwen Cui Tianqi Cui Long Tan Zaichao Ma Ya-Jie Tang A Francis Stewart Jun Fu Youming Zhang Hailong Wang |
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| Affiliation: | State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Helmholtz International Lab for Anti-infectives, Shandong University–Helmholtz Institute of Biotechnology, Shandong University, Qingdao, Shandong, 266237, China;Genomics, Biotechnology Center, Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Tatzberg 47–51, 01307 Dresden, Germany |
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| Abstract: | Biosynthesis reprograming is an important way to diversify chemical structures. The large repetitive DNA sequences existing in polyketide synthase genes make seamless DNA manipulation of the polyketide biosynthetic gene clusters extremely challenging. In this study, to replace the ethyl group attached to the C-21 of the macrolide insecticide spinosad with a butenyl group by refactoring the 79-kb gene cluster, we developed a RedEx method by combining Redαβ mediated linear-circular homologous recombination, ccdB counterselection and exonuclease mediated in vitro annealing to insert an exogenous extension module in the polyketide synthase gene without any extra sequence. RedEx was also applied for seamless deletion of the rhamnose 3′-O-methyltransferase gene in the spinosad gene cluster to produce rhamnosyl-3′-desmethyl derivatives. The advantages of RedEx in seamless mutagenesis will facilitate rational design of complex DNA sequences for diverse purposes. |
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