High-throughput reprogramming of an NRPS condensation domain.

Engineered biosynthetic assembly lines could revolutionize the sustainable production of bioactive natural product analogs. Although yeast display is a proven, powerful tool for altering the substrate specificity of gatekeeper adenylation domains in nonribosomal peptide synthetases (NRPSs), comparable strategies for other components of these megaenzymes have not been described. Here we report a high-throughput approach for engineering condensation (C) domains responsible for peptide elongation. We show that a 120-kDa NRPS module, displayed in functional form on yeast, can productively interact with an upstream module, provided in solution, to produce amide products tethered to the yeast surface. Using this system to screen a large C-domain library, we reprogrammed a surfactin synthetase module to accept a fatty acid donor, increasing catalytic efficiency for this noncanonical substrate >40-fold. Because C domains can function as selectivity filters in NRPSs, this methodology should facilitate the precision engineering of these molecular assembly lines.

Biosynthetic Functionalization of Nonribosomal Peptides.

Nonribosomal peptides (NRPs) are a therapeutically important class of secondary metabolites that are produced by modular synthetases in assembly-line fashion. We previously showed that a single Trp-to-Ser mutation in the initial Phe-loading adenylation domain of tyrocidine synthetase completely switches the specificity toward clickable analogues. Here we report that this minimally invasive strategy enables efficient functionalization of the bioactive NRP on the pathway level. In a reconstituted tyrocidine synthetase, the W227S point mutation permitted selective incorporation of Phe analogues with alkyne, halogen, and benzoyl substituents by the initiation module. The respective W2742S mutation in module 4 similarly permits efficient incorporation of these functionalized substrate analogues at position 4, expanding this strategy to elongation modules. Efficient incorporation of an alkyne handle at position 1 or 4 of tyrocidine A allowed site-selective one-step fluorescent labeling of the corresponding tyrocidine analogues by Cu(I)-catalyzed alkyne-azide cycloaddition. By combining synthetic biology with bioorthogonal chemistry, this approach holds great potential for NRP isolation and molecular target elucidation as well as combinatorial optimization of NRP therapeutics.