Stanisic, A. | bioRxiv Science

bioRxiv2022

Defining a Nonribosomal Specificity Code for Design

Nonribosomal peptide synthetases (NRPSs) assemble bioactive peptides from an enormous repertoire of building blocks. How binding pocket residues of the nonribosomal adenylation domain, the so-called specificity code, determine which building block becomes incorporated has been a landmark discovery in NRPS enzymology. While specificity codes enable the prediction of substrate specificity from protein sequence, design strategies based on rewriting the specificity code have been limited in scope. An important reason for failed NRPS design has been that multispecificity has not been considered, for a lack of suitable assay formats. Here, we employ a multiplexed hydroxamate specificity assay (HAMA) to determine substrate profiles for mutant libraries of A-domain in the termination module the SrfAC of surfactin synthetase. A generalist version of SrfAC is developed and the functional flexibility of the adenylation reaction is probed by fully randomizing 15 residues in and around the active site. We identify mutations with profound impact on substrate selectivity and thus reveal a remarkable evolvability of A-domains. Statistical analysis of the specificity divergence caused by point mutations has determined the impact of each code position on specificity, which will serve as a roadmap for NRPS engineering. The shortness of evolutionary pathways between NRPS specificities explains the rich natural substrate scope and suggests directed evolution guided by A-domain promiscuity as a promising strategy.

bioengineering↗

bioRxiv2020

Bacterial-like nonribosomal peptide synthetases produce cyclopeptides in the zygomycetous fungus Mortierella alpina

Fungi are traditionally considered as reservoir of biologically active natural products. However, an active secondary metabolism has long not been attributed to early diverging fungi such as Mortierella spec. Here, we report on the biosynthesis of two series of cyclic pentapeptides, the malpicyclins and malpibaldins, as products of Mortierella alpina ATCC32222. The molecular structures of malpicyclins were elucidated by HR-MS/MS, Marfeys method, and 1D and 2D NMR spectroscopy. In addition, malpibaldin biosynthesis was confirmed by HR-MS. Genome mining and comparative qRT-PCR expression analysis pointed at two pentamodular nonribosomal peptide synthetases (NRPS), malpicyclin synthetase MpcA and malpibaldin synthetase MpbA, as candidate biosynthetic enzymes. Heterologous production of the respective adenylation domains and substrate specificity assays proved promiscuous substrate selection and confirmed their respective biosynthetic roles. In stark contrast to known fungal NRPSs, MpbA and MpcA contain bacterial-like dual epimerase/condensation domains allowing the racemization of enzyme-tethered O_SCPLOWLC_SCPLOW-amino acids and the subsequent incorporation of O_SCPLOWDC_SCPLOW-amino acids into the metabolites. Phylogenetic analyses of both NRPS genes indicate a bacterial origin and a horizontal gene transfer into the fungal genome. This is the first report of nonribosomal peptide biosynthesis in basal fungi which highlights this paraphylum as novel and underrated resource of natural products. IMPORTANCEFungal natural compounds are industrially produced with application in antibiotic treatment, cancer medications and crop plant protection. Traditionally, higher fungi have been intensively investigated concerning their metabolic potential, but re-identification of already known compounds is frequently observed. Hence, alternative strategies to acquire novel bioactive molecules are required. We present the genus Mortierella as representative of the early diverging fungi as an underestimated resource of natural products. Mortierella alpina produces two families of cyclopeptides, denoted malpicyclins and malpibaldins, respectively, via two pentamodular nonribosomal peptide synthetases (NRPSs). These enzymes are much closer related to bacterial than to other fungal NRPSs, suggesting a bacterial origin of these NRPS genes in Mortierella. Both enzymes are the first biochemically characterized natural product biosynthesis enzymes of basal fungi. Hence, this report establishes early diverging fungi as prolific natural compound producers and sheds light on the origin of their biosynthetic capacity.

biochemistry↗