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

Abstract

Engineering of biosynthetic enzymes is increasingly employed to synthesize structural analogues of antibiotics. Of special interest are nonribosomal peptide synthetases (NRPSs) responsible for the production of important antimicrobial peptides. Here, directed evolution of an adenylation domain of a Pro‐specific NRPS module completely switched substrate specificity to the non‐standard amino acid piperazic acid (Piz) bearing a labile N−N bond. This success was achieved by UPLC‐MS/MS‐based screening of small, rationally designed mutant libraries and can presumably be replicated with a larger number of substrates and NRPS modules. The evolved NRPS produces a Piz‐derived gramicidin S analogue. Thus, we give new impetus to the too‐early dismissed idea that widely accessible low‐throughput methods can switch the specificity of NRPSs in a biosynthetically useful fashion.

High-Throughput Engineering of Nonribosomal Extension Modules

“…Robust methods for modifying nonribosomal assembly lines could provide biosynthetic access to novel peptide analogues with improved properties or altered bioactivities. To that end, diverse strategies for engineering NRPSs have been developed, including module insertion, module deletion, module exchange, and subdomain swaps. − ,− As a less invasive alternative, the substrate specificity of individual A domains can also be altered through mutagenesis and screening. − ,− Because of its high throughput, the FACS-based approach that we recently introduced to monitor the catalytic activity of A domains could greatly increase the utility of the latter approach.…”

Novel Biocatalysts from Specialized Metabolism

“…[30] Using these natural adenylation domain active sites for Piz as a template, a Proadenylation domain was reprogrammed for Piz in a directed evolution experiment. [32] The discovery of the KtzI/KtzT system for Piz biosynthesis has paved the way for efficient biotechnological production in the engineered fungal strain Aureobasidium melanogenum at a gram-per-liter scale. [33] Some mechanistically related pathways also begin with N-hydroxylated amino acids, but NÀ N bond formation is preceded by esterification of the N-hydroxy group to an ATP-activated amino acid (Scheme 2).…”

Novel Biocatalysts from Specialized Metabolism

“…To incorporate Piz into peptides, the free acid is selectively recognized by adenylation domains of nonribosomal peptide synthetases, which can apparently distinguish Piz from the almost isosteric Pro [30] . Using these natural adenylation domain active sites for Piz as a template, a Pro‐adenylation domain was reprogrammed for Piz in a directed evolution experiment [32] . The discovery of the KtzI/KtzT system for Piz biosynthesis has paved the way for efficient biotechnological production in the engineered fungal strain Aureobasidium melanogenum at a gram‐per‐liter scale [33] …”