Bacterial MbtH-like Proteins Stimulate Nonribosomal Peptide Synthetase-Derived Secondary Metabolism in Filamentous Fungi

Abstract

Filamentous fungi are known producers of bioactive natural products, low molecular weight molecules that arise from secondary metabolism. MbtH-like proteins (MLPs) are small (∼10 kDa) proteins, which associate noncovalently with adenylation domains of some bacterial nonribosomal peptide synthetases (NRPS). MLPs promote the folding, stability, and activity of NRPS enzymes. MLPs are highly conserved among a wide range of bacteria; however, they are absent from all fungal species sequenced to date. We analyzed the interaction potential of bacterial MLPs with eukaryotic NRPS enzymes first using crystal structures, with results suggesting a conservation of the interaction surface. Subsequently, we transformed five MLPs into Penicillium chrysogenum strains and analyzed changes in NRPS-derived metabolite profiles. Three of the five transformed MLPs increased the rate of nonribosomal peptide formation and elevated the concentrations of intermediate and final products of the penicillin, roquefortine, chrysogine, and fungisporin biosynthetic pathways. Our results suggest that even though MLPs are not found in the fungal domain of life, they can be used in fungal hosts as a tool for natural product discovery and biotechnological production.

Nonribosomal peptide synthetases and their biotechnological potential in Penicillium rubens

“…MLPs have been studied extensively in recent years, and yet the exact function remains still unknown. Growing evidence suggests that they can increase the activity of A domains as well as enhance the soluble expression of their partner NRPSs (Boll et al., 2011 ; Felnagle et al., 2010 ; Zwahlen et al., 2019 ), although in some cases the deletion of the MLP gene from a cluster does not have any effect on the biosynthesis of the NRP (Stegmann et al., 2006 ). An interesting feature of MLPs is that they can activate non-cognate NRPSs as well, both in vivo and in vitro (Boll et al., 2011 ; Lautru et al., 2007 ; Wolpert et al., 2007 ; Zhang et al., 2010 ; Zwahlen et al., 2019 ).…”

“…Growing evidence suggests that they can increase the activity of A domains as well as enhance the soluble expression of their partner NRPSs (Boll et al., 2011 ; Felnagle et al., 2010 ; Zwahlen et al., 2019 ), although in some cases the deletion of the MLP gene from a cluster does not have any effect on the biosynthesis of the NRP (Stegmann et al., 2006 ). An interesting feature of MLPs is that they can activate non-cognate NRPSs as well, both in vivo and in vitro (Boll et al., 2011 ; Lautru et al., 2007 ; Wolpert et al., 2007 ; Zhang et al., 2010 ; Zwahlen et al., 2019 ). In the most recent of these works, it has been shown that the heterologous expression of bacterial MLPs can boost the production of NRPS-related secondary metabolites in the filamentous fungus Penicillium chrysogenum (Zwahlen et al., 2019 ).…”

“…An interesting feature of MLPs is that they can activate non-cognate NRPSs as well, both in vivo and in vitro (Boll et al., 2011 ; Lautru et al., 2007 ; Wolpert et al., 2007 ; Zhang et al., 2010 ; Zwahlen et al., 2019 ). In the most recent of these works, it has been shown that the heterologous expression of bacterial MLPs can boost the production of NRPS-related secondary metabolites in the filamentous fungus Penicillium chrysogenum (Zwahlen et al., 2019 ).…”

A roadmap for metagenomic enzyme discovery

“…Although STRING is not specifically targeted towards metagenomics or natural product biosynthesis, it can be used to predict–protein interactions such as for MbtH-like proteins in NRPS systems. 147 A more specific tool, CO-ED, is useful for network analysis and identification of unusual co-occurring domains in multi-domain proteins including megasynthases commonly involved in natural product biosynthesis. 148 CO-ED relies on PFAM information as inputs which can be extracted from (meta)genomes using PfamScan.…”

Functional Cross-Talk of MbtH-Like Proteins During Thaxtomin Biosynthesis in the Potato Common Scab Pathogen Streptomyces scabiei

“…CloY from the clorobiocin BGC of S. roseochromogenes (Table 3; Pojer et al, 2002) is essential for production of the aminocoumarin antibiotic (Wolpert et al, 2007), as it is required for the solubility and adenylation activity of its corresponding NRPS partner, CloH (Boll et al, 2011). The ComB-encoding gene is situated within a glycopeptide-like complestatin NRP BGC from S. lavendulae (Chiu et al, 2001) and was recently shown to stimulate the production of several NRPs in the mold Penicillium chrysogenum, which does not harbor any MLP-encoding genes in its genome (Zwahlen et al, 2019). YbdZ has been extensively investigated in recent studies and is required for the biosynthesis of the ENT siderophore in E. coli (Schomer and Thomas, 2017;Schomer et al, 2018).…”