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

Publisher: Oxford University Press Country of Publication: England NLM ID: 0411011 Publication Model: Print Cited Medium: Internet ISSN: 1362-4962 (Electronic) Linking ISSN: 03051048 NLM ISO Abbreviation: Nucleic Acids Res Subsets: MEDLINE

Publication: 1992- : Oxford : Oxford University Press
Original Publication: London, Information Retrieval ltd.

Gene Deletion*; Mutagenesis, Insertional/*genetics ; Polyketide Synthases/*genetics ; Protein Domains/*genetics; Base Sequence/genetics ; Cloning, Molecular ; DNA/genetics ; Homologous Recombination/genetics ; Multigene Family/genetics

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.
(© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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79956-01-7 (Polyketide Synthases)
9007-49-2 (DNA)

Date Created: 20201029 Date Completed: 20210113 Latest Revision: 20210113

20221213

PMC7736807

10.1093/nar/gkaa956

33119745