Gene editing enables rapid engineering of complex antibiotic assembly lines

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Re-engineering megasynthase enzymes like nonribosomal peptide synthetases (NRPS) offers a pathway to complex antibiotics.
• Current methods for engineering these large enzymes are challenging and inefficient.

Purpose of the Study:

• To demonstrate the utility of CRISPR-Cas9 gene editing for rapidly engineering complex NRPS assembly lines.
• To create novel lipopeptide variants by altering substrate selectivity within the enduracidin NRPS.

Main Methods:

• Utilized CRISPR-Cas9 gene editing to exchange subdomains within the 2.0 MDa enduracidin NRPS.
• Compared CRISPR-Cas9 approach with conventional homologous recombination methods.
• Incorporated subdomains from diverse bacterial NRPS enzymes.

Main Results:

• Successfully engineered the enduracidin NRPS, generating ten new lipopeptide variants in good yields.
• CRISPR-Cas9 significantly outperformed homologous recombination, which yielded only trace amounts of new variants.
• Demonstrated successful integration of heterologous NRPS subdomains.

Conclusions:

• CRISPR-Cas9 is a powerful and efficient tool for engineering complex NRPS assembly lines.
• This approach enables rapid generation of diverse bioactive natural products, including novel antibiotics.
• Facilitates the creation of new lipopeptide variants by modifying substrate selectivity and incorporating diverse domains.