Decreasing the Ring Size of a Cyclic Nonribosomal Peptide Antibiotic by In-Frame Module Deletion in the Biosynthetic Genes

Abstract

Many natural products of therapeutical and biotechnological importance are nonribosomally synthesized peptides. Structural hallmarks of this class of compounds are the occurrence of unusual amino acids, mostly cyclic peptide backbones, and numerous further modifications such as acylation, heterocyclic ring formation, and glycosylation. Because of their structural complexity, chemical synthesis is usually an unattractive route to these molecules. In contrast, genetic engineering of the biosynthesis genes emerges as a potentially powerful approach to the combinatorial biosynthesis of useful analogues of the lead compounds. Nonribosomal peptide synthetases (NRPSs) carry out a sequential multistep assembly and modification of the peptides in a thiotemplate process described by the multiple carrier model. The modular architecture of NRPSs suggests straightforward methods for the reprogramming of these enzymes by exchange of catalytic subunits. However, many of the reported engineering attempts faced low product yields or even inactive hybrid enzymes. Using a new approach to obtain hybrid NRPSs, we show here that the deletion of an entire module in an NRPS assembly line caused the secretion of the predicted peptide antibiotic variant with a decreased ring size. Furthermore, a module exchange resulted in a significantly higher product yield than that observed in previous studies.

Biosynthetic Gene Cluster for the Polyenoyltetramic Acid α-Lipomycin

“…The stepwise aminoto-carboxy-terminal elongation of the thioesterified intermediates is catalyzed by the condensation domain (C domain). These domains form the three core domains of every elongation module (33). LipNrps consists of only one module with these three core domains (Fig.…”

In Vivo Production of Artificial Nonribosomal Peptide Products in the Heterologous Host Escherichia coli

“…Consequently, the artificial engineering of NRPS assembly lines is an attractive route to prepare analogs of complex natural products (32,40). The practicability of this general concept has been demonstrated in various examples, ranging from targeted modifications of existing products to the constitution of entirely artificial NRPSs from scratch (9,24,29,31,38,44,51). For example, an artificial NRPS that catalyzes formation of the dipeptide precursor of the high-intensity sweetener aspartame was constructed de novo (10).…”

Progress toward re-engineering non-ribosomal peptide synthetase proteins: a potential new source of pharmacological agents

“…5a) resulted in the generation of novel surfactin derivatives substituted at the 7th position (Fig. 5b), albeit with lower catalytic efficiency and product yield (0.1-0.5% [Mootz et al, 2002]. The surfactin derivatives lysed erythrocytes, although with a reduced activity compared to the wild type peptide.…”

“…Mootz et al [2000] created trimodular systems that exhibited the expected biochemical activity and produced the expected product in vitro, although not with exact specificity. In a final example of module swapping, Mootz et al [2002] again used the fusion sites identified in previous work to eliminate the second module from surfactin synthetase. The d2 surfactin synthetase produced the expected product, which was isolated in 19% yield relative to the parent Bacillus subtilis strain that produces surfactin.…”