Promiscuity of Omphalotin A Biosynthetic Enzymes Allows de novo Production of Non-Natural Multiply Backbone N-Methylated Peptide Macrocycles in Yeast.

Multiple backbone N-methylation and macrocyclization improve the proteolytic stability and oral availability of therapeutic peptides. Chemical synthesis of such peptides is challenging, in particular for the generation of peptide libraries for screening purposes. Enzymatic backbone N-methylation and macrocyclization occur as part of both non-ribosomal and ribosomal peptide biosynthesis, exemplified by the fungal natural products cyclosporin A and omphalotin A, respectively. Omphalotin A, a 9fold backbone N-methylated dodecamer isolated from the agaricomycete Omphalotus olearius, can be produced in Pichia pastoris by coexpression of the ophMA and ophP genes coding for the peptide precursor protein harbouring an autocatalytic peptide α-N-methyltransferase domain, and a peptide macrocyclase, respectively. Since both OphMA and OphP were previously shown to be relatively promiscuous in terms of peptide substrates, we expressed mutant versions of ophMA, encoding OphMA variants with altered core peptide sequences, along with wildtype ophP and assessed the production of the respective peptide macrocycles by the platform by high-performance liquid chromatography, coupled with tandem mass spectrometry (HPLC-MS/MS). Our results demonstrate the successful production of fifteen non-natural omphalotin-derived macrocycles, containing polar, aromatic and charged residues, and, thus, suggest that the system may be used as biotechnological platform to generate libraries of non-natural multiply backbone N-methylated peptide macrocycles.

Conformations of Macrocyclic Peptides Sampled by Nuclear Magnetic Resonance: Models for Cell-Permeability.

The biological activities and pharmacological properties of peptides and peptide mimetics are determined by their conformational states. Therefore, a detailed understanding of the conformational landscape is crucial for rational drug design. Nuclear magnetic resonance (NMR) is the only method for structure determination in solution. However, it remains challenging to determine the structures of peptides using NMR because of very weak nuclear Overhauser effects (NOEs), the semiquantitative nature of the rotating frame Overhauser effect (ROE), and the low number of NOEs/ROEs in N-methylated peptides. In this study, we introduce a new approach to investigating the structures of modified macrocyclic peptides. We utilize exact NOEs (eNOEs) in viscous solvent mixtures to replicate various cellular environments. eNOEs provide detailed structural information for highly dynamic modified peptides. Structures of high precision were obtained for cyclosporin A, with a backbone atom rmsd of 0.10 Å. Distinct conformational states in different environments were identified for omphalotin A (OmphA), a fungal nematotoxic and multiple backbone N-methylated macrocyclic peptides. A model for cell-permeation is presented for OmphA, based on its structures in polar, apolar, and mixed polarity solvents. During the transition from a polar to an apolar environment, OmphA undergoes a rearrangement of its H-bonding network, accompanied by a cis to trans isomerization of the ω torsion angle within a type VIa ß-turn. We hypothesize that the kinetics of these conformational transitions play a crucial role in determining the membrane-permeation capabilities of OmphA.

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.

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.

Selecting cells expressing high levels of recombinant proteins using the GPI-anchored protein with selenocysteine system.

Most biopharmaceutical proteins are produced in mammalian cells because they have the advantageous capacity for protein folding, assembly, and posttranslational modifications. To satisfy the increasing demand for these proteins for clinical purposes and studies, traditional methods to improve protein productivity have included gene amplification, host cell engineering, medium optimization, and screening methods. However, screening and selection of high-producing cell lines remain complex and time consuming. In this study, we established a glycosylphosphatidylinositol (GPI)-anchored protein with a selenocysteine (GPS) system to select cells producing high levels of target secretory proteins. Recombinant lysosomal acid lipase (LIPA) and α-galactosidase A (GALA) were fused with a GPI attachment signal sequence and a selenocysteine insertion sequence after an in-frame UGA codon. Under these conditions, most of the recombinant proteins were secreted into the culture medium, but some were found to be GPI-anchored proteins on the cell surface. When sodium selenite was supplied into the culture medium, the amount of GPI-anchored LIPA and GALA was increased. High-expressing cells were selected by detecting surface GPI-anchored LIPA. The GPI-anchored protein was then eliminated by knocking out the GPI biosynthesis gene PIGK, in these cells, all LIPA was in secreted form. Our system provides a promising method of isolating cells that highly express recombinant proteins from large cell populations.

Substrate Plasticity of a Fungal Peptide α-N-Methyltransferase.

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.

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.

Molecular switching system using glycosylphosphatidylinositol to select cells highly expressing recombinant proteins.

Although many pharmaceutical proteins are produced in mammalian cells, there remains a challenge to select cell lines that express recombinant proteins with high productivity. Since most biopharmaceutical proteins are secreted by cells into the medium, it is difficult to select cell lines that produce large amounts of the target protein. To address this issue, a new protein expression system using the glycosylphosphatidylinositol (GPI)-anchor was developed. PGAP2 is involved in processing GPI-anchored proteins (GPI-APs) during transport. In PGAP2 mutant cells, most GPI-APs are secreted into the medium. Here, we established a HEK293 cell line where endogenous PGAP2 was knocked out and exogenous PGAP2 was inserted with a piggyBac transposon in the genome. Using these cells, human lysosomal acid lipase (LIPA) and α-galactosidase A (GLA) were expressed as GPI-anchored forms (LIPA-GPI and GLA-GPI) and cells expressing high levels of LIPA-GPI or GLA-GPI on the cell surface were enriched. Removal of the PGAP2 gene by piggyBac transposase or FLP recombinase converted LIPA-GPI and GLA-GPI from membrane-bound to the secreted forms. Thus, cells expressing LIPA or GLA in large amounts could be enriched using this approach. The GPI-based molecular switching system is an efficient approach to isolate cells expressing recombinant proteins with high productivity.

Current Immunoassay Methods for the Rapid Detection of Aflatoxin in Milk and Dairy Products.

The presence of mycotoxins in foodstuff causes serious health problems to consumers and economically affects the food industry. Among the mycotoxins, aflatoxins are very toxic and highly carcinogenic contaminants which affect the safety of many foods, and therefore endanger human health. Aflatoxin M1 (AFM1 ) found in milk results from the biotransformation of aflatoxin B1 . Many efforts have been made to control the source of AFM1 from farmers to dairy product companies. However, AFM1 escapes ordinary methods of food treatment such as cooking, sterilization, and freezing, hence it appears in milk and dairy products. The presence of high levels of AFM1 constitutes an alarming threat as milk and dairy products contain essential nutrients for human health, especially for infants and children. For this reason, there is a pressing need for developing a fast and reliable screening method for detecting trace aflatoxins in food. Several analytical methods based on high-performance liquid chromatography (HPLC) and mass spectroscopy have been used for aflatoxin detection; however, they are expensive, time-consuming, and require many skills. Recently, immunoassay methods, including enzyme-linked immunosorbent assay (ELISA), immunosensors, and lateral flow immunoassay (LFIA), have been preferred for food analysis because of their improved qualities such as high sensitivity, simplicity, and capability of onsite monitoring. This paper reviews the new developments and applications of immunoassays for the rapid detection of AFM1 in milk.