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RedEx: a method for seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters.


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 Red?? 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.

SUBMITTER: Song C 

PROVIDER: S-EPMC7736807 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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RedEx: a method for seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters.

Song Chaoyi C   Luan Ji J   Li Ruijuan R   Jiang Chanjuan C   Hou Yu Y   Cui Qingwen Q   Cui Tianqi T   Tan Long L   Ma Zaichao Z   Tang Ya-Jie YJ   Stewart A Francis AF   Fu Jun J   Zhang Youming Y   Wang Hailong H  

Nucleic acids research 20201201 22


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 reco  ...[more]

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