ArticleACS synthetic biology2020
Site-Directed Mutagenesis of Large Biosynthetic Gene Clusters
Article in ACS synthetic biology, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.
What it found
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
6 citing papers in PubMed, 10 citations in OpenAlex.
- MOSAIC: A Highly Efficient, One-Step Recombineering Approach to Plasmid Editing and Diversification.ACS synthetic biology · 2025Article
- Parallelized gene cluster editing illuminates mechanisms of epoxyketone proteasome inhibitor biosynthesis.Nucleic acids research · 2023Article
- Striving for sustainable biosynthesis: discovery, diversification, and production of antimicrobial drugs in Escherichia coli.Biochemical Society transactions · 2022Review
- Engineering the stambomycin modular polyketide synthase yields 37-membered mini-stambomycins.Nature communications · 2022Article
- Engineering Modular Polyketide Biosynthesis in Streptomyces Using CRISPR/Cas: A Practical Guide.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Gene editing enables rapid engineering of complex antibiotic assembly lines.Nature communications · 2021Article
Corrections and comments
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
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 important determinants of successful mutagenesis, including the protospacer/PAM sequence and presence of regions of local homology. Our approach may be broadly applicable for researchers interested in probing natural product biosynthesis or performing pathway engineering.
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Registered trials
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