Non-ribosomal peptides are a diverse and medically important group of natural products. They are biosynthesised by modular non-ribosomal peptide synthetase (NRPS) assembly-lines in which domains from each module act in concert to incorporate a specific amino acid into a peptide. This modular biosynthesis has driven efforts to generate new peptide analogues by substituting amino acid specifying domains. Rational NRPS engineering has increasingly focused on using evolutionarily favoured recombination sites for domain substitution. Here, we present an alternative approach inspired by evolution, which involves large-scale diversification and screening. By adopting a metagenomic approach of amplifying amino acid specifying domains from metagenomic DNA derived from soil, we were able to substitute over 1,000 unique domains into a pyoverdine NRPS. To identify functional domain substitutions, we employed fluorescence and mass spectrometry screening techniques, followed by sequencing. This comprehensive screening process successfully identified more than 100 functional domain substitutions, resulting in the production of 16 distinct pyoverdines as major products. The significance of this metagenomic approach lies in its ability to shift the focus of engineering non-ribosomal peptide biosynthesis. Instead of relying on a high success rate of individual domain substitution, we have developed effective methods that enable the exploration of a broader range of substitutions. This opens new possibilities for the discovery and production of novel non-ribosomal peptides with diverse biological activities.

/pdf/metagenomic-domain-substitution-for-the-high-throughput-2l8k29yd.pdf

Metagenomic domain substitution for the high-throughput modification of non-ribosomal peptide analogues

A Practical Guideline to Engineering Nonribosomal Peptide Synthetases.

Nonribosomal peptides (NRPs) have gained attention due to their diverse biological activities and potential applications in medicine and agriculture. The natural diversity of NRPs is a result of evolutionary processes that have occurred over millions of years. Recent studies have shed light on the mechanisms by which nonribosomal peptide synthetases (NRPSs) evolve, including gene duplication, recombination, and horizontal transfer. Mimicking natural evolution could be a useful strategy for engineering NRPSs to produce novel compounds with desired properties. Furthermore, the emergence of antibiotic-resistant bacteria has highlighted the urgent need for new drugs, and NRPs represent a promising avenue for drug discovery. This review discusses the engineering potential of NRPSs in light of their evolutionary history.

Biomimetic engineering of nonribosomal peptide synthesis.

Nonribosomal peptides (NRPs) have gained attention due to their diverse biological activities and potential applications in medicine and agriculture. The natural diversity of NRPs is a result of evolutionary processes that have occurred over millions of years. Recent studies have shed light on the mechanisms by which nonribosomal peptide synthetases (NRPSs) evolve, including gene duplication, recombination, and horizontal transfer. Mimicking natural evolution could be a useful strategy for engineering NRPSs to produce novel compounds with desired properties. Furthermore, the emergence of antibiotic-resistant bacteria has highlighted the urgent need for new drugs, and NRPs represent a promising avenue for drug discovery. This review discusses the engineering potential of NRPSs in light of their evolutionary history. 

Biomimetic engineering of nonribosomal peptide synthesis

Many clinically used drugs are derived from or inspired by bacterial natural products that often are biosynthesised via non-ribosomal peptide synthetases (NRPS), giant megasynthases that activate and join individual amino acids in an assembly line fashion. Since NRPS are not limited to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would allow the biotechnological generation of complex peptides including linear, cyclic and further modified natural product analogues, e.g. to optimise natural product leads. Here we describe a detailed phylogenetic analysis of several bacterial NRPS that led to the identification of a new recombination breakpoint within the thiolation (T) domain that is important for natural NRPS evolution. From this, an evolution-inspired eXchange Unit between T domains (XUT) approach was developed which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.

/pdf/evolution-inspired-engineering-of-megasynthetases-2k0qtiwy.pdf

Leonard Präve

Papers

Evolution Inspired Engineering of Megasynthetases

A Practical Guideline to Engineering Nonribosomal Peptide Synthetases.