High-Throughput Reprogramming of an NRPS Condensation Domain

Abstract

Engineered biosynthetic assembly lines could revolutionize the sustainable production of bioactive natural product analogues. While 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. Since C domains can function as selectivity filters in NRPSs, this methodology should facilitate precision engineering of these molecular assembly lines.

High-Throughput Engineering of Nonribosomal Extension Modules

“…For example, condensation reactions can also be assayed in high throughput. We recently showed that a displayed C–A–T domain equipped with a short docking domain could productively interact with an upstream module in solution to produce amide products tethered to the yeast surface, an activity we exploited to alter the substrate specificity of the displayed C domain . Extension to other bio-orthogonal labeling methods to enable variation of substrate side chain preferences or harnessing the power of iterative directive evolution , would further expand the range of problems that can be tackled with this assay platform.…”

Directed Evolution of Piperazic Acid Incorporation by a Nonribosomal Peptide Synthetase**

“…Interestingly, all mutants maintained similar or even higher turnover rates (k cat ) for Piz between 2.8 and 5.0 min À 1 that are all well in the range of typical peptide formation rates of NRPSs. [51,52] However, the Michaelis-constant (K M ) dramatically increased for Pro from 0.14 mM (GrsB1) to 10 mM (GrsB1-AYA) after the first round of directed evolution, and then became undetectable in subsequent rounds. In contrast, the K M for Piz dropped from 9.2 mM in GrsB1 to 2.4 mM in GrsB1-MWG.…”

Controlling Substrate- and Stereospecificity of Condensation Domains in Nonribosomal Peptide Synthetases

“…32,33 In a separate study, the corresponding residues were modified in the surfactin NRPS enzyme SrfAC to incorporate non-native fatty acid substrates. 34 However, the mechanisms underlying the substrate specificity of typical Cdomains from elongation modules with amino acyl or peptidyl substrates remain unknown. Additionally, how D C L and L C L domains distinguish between L-or D-configured donor substrates remains elusive.…”