Site-Directed Mutagenesis of Large Biosynthetic Gene Clusters via Oligonucleotide Recombineering and CRISPR/Cas9 Targeting.

ABSTRACT: Genetic engineering of natural product biosynthetic gene clusters represents an attractive approach to access new and complex bioactive small molecules. However, due to the large number and size of some genes involved in specialized metabolism, notably those encoding modular polyketide synthase and nonribosomal peptide synthetase megaproteins, it remains difficult to introduce precise genetic mutations to probe domain activity or alter chemical product formation. Here, we report the development and validation of a robust method combining oligonucleotide recombineering and CRISPR/Cas9 targeting for rapid site-directed mutagenesis of cloned pathways, which can be directly transferred to a heterologous host for expression. We rapidly generated 12 point mutations and identified several importa

SUBMITTER: Zhang JJ 

PROVIDER: S-EPMC8142379 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature