Nonribosomal peptide synthetases (NRPSs) are large multienzyme machineries that assemble numerous peptides with large structural and functional diversity. These peptides include more than 20 marketed drugs, such as antibacterials (penicillin, vancomycin), antitumor compounds (bleomycin), and immunosuppressants (cyclosporine). Over the past few decades biochemical and structural biology studies have gained mechanistic insights into the highly complex assembly line of nonribosomal peptides. This Review provides state-of-the-art knowledge on the underlying mechanisms of NRPSs and the variety of their products along with detailed analysis of the challenges for future reprogrammed biosynthesis. Such a reprogramming of NRPSs would immediately spur chances to generate analogues of existing drugs or new compound libraries of otherwise nearly inaccessible compound structures.

Nonribosomal Peptide Synthesis-Principles and Prospects.

New developments in RiPP discovery, enzymology and engineering

Structural and functional aspects of the nonribosomal peptide synthetase condensation domain superfamily: discovery, dissection and diversity☆

Non-ribosomal peptide synthetases (NRPSs) are giant enzyme machines that activate amino acids in an assembly line fashion. As NRPSs are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would enable microbial production of a diverse range of peptides; however, the structural requirements for reprogramming NRPSs to facilitate the production of new peptides are not clear. Here we describe a new fusion point inside the condensation domains of NRPSs that results in the development of the exchange unit condensation domain (XUC) concept, which enables the efficient production of peptides, even containing non-natural amino acids, in yields up to 280 mg l−1. This allows the generation of more specific NRPSs, reducing the number of unwanted peptide derivatives, but also the generation of peptide libraries. The XUC might therefore be suitable for the future optimization of peptide production and the identification of bioactive peptide derivatives for pharmaceutical and other applications. Non-ribosomal peptide synthetases have now been modified and de novo non-ribosomal peptide synthetases constructed using new assembly points within condensation domains. This approach enabled the production of new-to-nature peptides, including some carrying synthetic amino acids, as well as the generation of peptide libraries.

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Modification and de novo design of non-ribosomal peptide synthetases using specific assembly points within condensation domains.

Evolutionary dynamics of natural product biosynthesis in bacteria

Biochemical Dissection of the Natural Diversification of Microcystin Provides Lessons for Synthetic Biology of NRPS

Filamentous cyanobacteria belong to the most prolific producers of structurally unique and biologically active natural products, yet the majority of biosynthetic gene clusters predicted for these multicellular collectives are currently orphan. Here, we present a systems analysis of secondary metabolite gene expression in the model strain Nostoc punctiforme PCC73102 using RNA-seq and fluorescence reporter analysis. Our data demonstrate that the majority of the cryptic gene clusters are not silent but are expressed with regular or sporadic pattern. Cultivation of N. punctiforme using high-density fermentation overrules the spatial control and leads to a pronounced upregulation of more than 50% of biosynthetic gene clusters. Our data suggest that a combination of autocrine factors, a high CO2 level, and high light account for the upregulation of individual pathways. Our overarching study not only sheds light on the strategies of filamentous cyanobacteria to share the enormous metabolic burden connected with the production of specialized molecules but provides an avenue for the genome-based discovery of natural products in multicellular cyanobacteria as exemplified by the discovery of highly unusual variants of the tricyclic peptide microviridin.

Daniel Dehm

Papers

Biochemical Dissection of the Natural Diversification of Microcystin Provides Lessons for Synthetic Biology of NRPS

Unlocking the Spatial Control of Secondary Metabolism Uncovers Hidden Natural Product Diversity in Nostoc punctiforme.

Characterizing seamless ligation cloning extract for synthetic biological applications