Substrate plasticity of a fungal peptide α-N-methyltransferase

This work was financially supported by the Commission for Technology and Innovation (CTI/Innosuisse Grant No. CTI 25951.2), the Swiss National Science Foundation (Grant No. 31003A_173097), Wellcome Trust (Grant No. 094476/Z/10/ Z), and BBSRC (Grant No. BB/R018189/1).The methylation of amide nitrogen atoms can improve the stability, oral availability, and cell permeability of peptide therapeutics. Chemical N-methylation of peptides is challenging. Omphalotin A is a ribosomally synthesized, macrocylic dodecapeptide with nine backbone N-methylations. The fungal natural product is derived from the precursor protein, OphMA, harboring both the core peptide and a SAM-dependent peptide α-N-methyltransferase domain. OphMA forms a homodimer and its α-N-methyltransferase domain installs the methyl groups in trans on the hydrophobic core dodecapeptide and some additional C-terminal residues of the protomers. These post-translational backbone N-methylations occur in a processive manner from the N- to the C-terminus of the peptide substrate. We demonstrate that OphMA can methylate polar, aromatic, and charged residues when these are introduced into the core peptide. Some of these amino acids alter the efficiency and pattern of methylation. Proline, depending on its sequence context, can act as a tunable stop signal. Crystal structures of OphMA variants have allowed rationalization of these observations. Our results hint at the potential to control this fungal α-N-methyltransferase for biotechnological applications.Publisher PDFPeer reviewe

The current strategies and parameters for the enhanced microbial production of 2,3-butanediol

2,3-Butanediol (2,3-BD) is a propitious compound with many industrial uses ranging from rubber, fuels, and cosmetics to food additives. Its microbial production has especially attracted as an alternative way to the petroleum-based production. However, 2,3-BD production has always been hampered by low yields and high production costs. The enhanced production of 2,3-butanediol requires screening of the best strains and a systematic optimization of fermentation conditions. Moreover, the metabolic pathway engineering is essential to achieve the best results and minimize the production costs by rendering the strains to use efficiently low cost substrates. This review is to provide up-to-date information on the current strategies and parameters for the enhanced microbial production of 2,3-BD.ISSN:2215-017

Enzyme-mediated backbone N-methylation in ribosomally encoded peptides

Backbone N-methylation as a posttranslational modification was recently discovered in a class of ribosomally encoded peptides referred to as borosins. The founding members of the borosins are the omphalotins (A-I), backbone N-methylated, macrocyclic dodecapeptides produced by the mushroom Omphalotus olearius. Omphalotins display a strong and selective toxicity toward the plant parasitic nematode Meloidogyne incognita. The primary product omphalotin A is synthesized via a concerted action of the omphalotin precursor protein (OphMA) and the dual function prolyloligopeptidase/macrocyclase (OphP). OphMA consists of α-N-methyltransferase domain that autocatalytically methylates the core peptide fused to its C-terminus via a clasp domain. Genome mining uncovered over 50 OphMA homologs from the fungal phyla Ascomycota and Basidiomycota. However, the derived peptide natural products have not been described yet, except for lentinulins, dendrothelins and gymnopeptides produced by the basidiomycetes Lentinula edodes, Dendrothele bispora and Gymnopus fusipes, respectively. In this chapter, we describe methods used to isolate and characterize these backbone N-methylated peptides and their precursor proteins both in their original hosts and in the heterologous hosts Escherichia coli and Pichia pastoris. These methods may pave the path for both the discovery of novel borosins with interesting bioactivities. In addition, understanding of borosin biosynthetic pathways may allow setting up a biotechnological platform for the production of pharmaceutical leads for orally available peptide drugs.ISSN:0076-687

Identification, heterologous production and bioactivity of lentinulin A and dendrothelin A, two natural variants of backbone N-methylated peptide macrocycle omphalotin A

Backbone N-methylation and macrocyclization improve the pharmacological properties of peptides by enhancing their proteolytic stability, membrane permeability and target selectivity. Borosins are backbone N-methylated peptide macrocycles derived from a precursor protein which contains a peptide α-N-methyltransferase domain autocatalytically modifying the core peptide located at its C-terminus. Founding members of borosins are the omphalotins from the mushroom Omphalotus olearius (omphalotins A-I) with nine out of 12 L-amino acids being backbone N-methylated. The omphalotin biosynthetic gene cluster codes for the precursor protein OphMA, the protease prolyloligopeptidase OphP and other proteins that are likely to be involved in other post-translational modifications of the peptide. Mining of available fungal genome sequences revealed the existence of highly homologous gene clusters in the basidiomycetes Lentinula edodes and Dendrothele bispora. The respective borosins, referred to as lentinulins and dendrothelins are naturally produced by L. edodes and D. bispora as shown by analysis of respective mycelial extracts. We produced all three homologous peptide natural products by coexpression of OphMA hybrid proteins and OphP in the yeast Pichia pastoris. The recombinant peptides differ in their nematotoxic activity against the plant pathogen Meloidogyne incognita. Our findings pave the way for the production of borosin peptide natural products and their potential application as novel biopharmaceuticals and biopesticides. © 2021, The Author(s).ISSN:2045-232

Author Correction: Identifcation, heterologous production and bioactivity of lentinulin A and dendrothelin A, two natural variants of backbone N-methylated peptide macrocycle omphalotin A

An amendment to this paper has been published and can be accessed via a link at the top of the paper