High-throughput reprogramming of an NRPS condensation domain

Area of Science:

• Biotechnology
• Synthetic Biology
• Enzyme Engineering

Background:

• Engineered biosynthetic assembly lines offer sustainable routes to bioactive natural product analogs.
• Yeast display effectively alters substrate specificity in nonribosomal peptide synthetase (NRPS) adenylation domains.
• Engineering strategies for other NRPS components, like condensation (C) domains, are less developed.

Purpose of the Study:

• To develop a high-throughput method for engineering NRPS condensation (C) domains.
• To enable the modification of C-domain substrate specificity for novel product synthesis.
• To advance the precision engineering of NRPS-based molecular assembly lines.

Main Methods:

• A yeast display system was developed to present a 120-kDa functional NRPS module.
• The displayed NRPS module was engineered to interact with an upstream module in solution.
• A library of C-domains was screened using this system to identify improved variants.

Main Results:

• The engineered NRPS system successfully produced amide products tethered to the yeast surface.
• A surfactin synthetase C-domain was reprogrammed to accept a fatty acid donor.
• Catalytic efficiency for the noncanonical fatty acid substrate increased over 40-fold.

Conclusions:

• This high-throughput C-domain engineering method facilitates the precision modification of NRPS assembly lines.
• The developed strategy enables the creation of novel bioactive natural product analogs.
• This work expands the toolkit for engineering complex enzymatic systems for sustainable chemical production.