Hydroxy Acid Activation in Fungal Non-Ribosomal Peptide Synthesis Assessed by Site-Directed Mutagenesis.

The fungal cyclodepsipeptides (CDPs) enniatin, beauvericin, bassianolide, and PF1022 consist of alternating N-methylated l-amino and d-hydroxy acids. They are synthesized by non-ribosomal peptide synthetases (NRPS). The amino acid and hydroxy acid substrates are activated by adenylation (A) domains. Although various A domains have been characterized thus giving insights into the mechanism of substrate conversion, little is known about the utilization of hydroxy acids in NRPSs. Therefore, we used homology modelling and molecular docking of the A1 domain of enniatin synthetase (EnSyn) to gain insights into the mechanism of hydroxy acid activation. We introduced point mutations into the active site and used a photometric assay to study the substrate activation. The results suggest that the hydroxy acid is selected by interaction with backbone carbonyls rather than by a specific side chain. These insights enhance the understanding of non-amino acid substrate activation and could contribute to the engineering of depsipeptide synthetases.

Probing Exchange Units for Combining Iterative and Linear Fungal Nonribosomal Peptide Synthetases.

A considerable number of complex peptides are synthesized by nonribosomal peptide synthetases (NRPSs). Due to their multimodular architecture and widely understood basic biosynthetic reactions, these synthetases represent a promising target for compound diversification by active reprogramming. Nevertheless, the limited knowledge about mechanistic details such as C domain specificity hampers rational synthetase engineering. Here, we present a systematic investigation of three fungal NRPS exchange units (C-A-Mt-T, CCTD-A-Mt-T, and A-Mt-T) focusing on the influence of C domains at heterologous domain junctions. By functionally integrating units from linear cyclosporine synthetase into iterative cyclodepsipeptide synthetases in vivo, we demonstrate that fungal NRPSs of different assembly types can be combined using different swapping sites, while respecting the C domain integrity and specificity. Based on 24 hybrid synthetases, we suggest exchange rules for efficient fungal NRPS engineering. The findings are of importance for rational synthetase design and provide a new set of options for combinatorial reprogramming.

Desymmetrization of Cyclodepsipeptides by Assembly Mode Switching of Iterative Nonribosomal Peptide Synthetases.

Nonribosomal peptide synthetases assemble a considerable number of structurally complex peptides of pharmacological importance. This turns them into important biosynthetic machineries for peptide diversification by engineering approaches. To date, manifold reprogramming approaches focus on employing module and domain exchanges, or the engineering of domains responsible for amino acid recognition. In this work, we present an engineering strategy for the manipulation of product assembly modes by fusing iterative fungal cyclodepsipeptide synthetases. The reassignment of terminal condensation domains as canonical condensation domains induces a switch from an exclusively iterative into a mixed linear/iterative peptide assembly mode. In the heterologous host E. coli we thus produced in vivo novel hybrid cyclodepsipeptides with altered structural symmetry. Our findings contribute a new experimental set of nonribosomal peptide synthetase reprogramming to the engineering toolbox for peptide structure diversification.

Harnessing fungal nonribosomal cyclodepsipeptide synthetases for mechanistic insights and tailored engineering.

Nonribosomal peptide synthetases represent potential platforms for the design and engineering of structurally complex peptides. While previous focus has been centred mainly on bacterial systems, fungal synthetases assembling drugs like the antifungal echinocandins, the antibacterial cephalosporins or the anthelmintic cyclodepsipeptide (CDP) PF1022 await in-depth exploitation. As various mechanistic features of fungal CDP biosynthesis are only partly understood, effective engineering of NRPSs has been severely hampered. By combining protein truncation, in trans expression and combinatorial swapping, we assigned important functional segments of fungal CDP synthetases and assessed their in vivo biosynthetic capabilities. Hence, artificial assembly line components comprising of up to three different synthetases were generated. Using Aspergillus niger as a heterologous expression host, we obtained new-to-nature octa-enniatin (4 mg L-1) and octa-beauvericin (10.8 mg L-1), as well as high titers of the hybrid CDP hexa-bassianolide (1.3 g L-1) with an engineered ring size. The hybrid compounds showed up to 12-fold enhanced antiparasitic activity against Leishmania donovani and Trypanosoma cruzi compared to the reference drugs miltefosine and benznidazole, respectively. Our findings thus contribute to a rational engineering of iterative nonribosomal assembly lines.