Roquefortine and isofumigaclavine A, metabolites fromPenicillium roqueforti

OHMOMO S., SATO T., UTAGAWA T., ABE M.

Bach N.J., Boaz H.E., Kornfeld E.C., Chang C., Floss H.G., Hagaman E.W., Wenkert E.

Hart N., Johns S., Lamberton J., Summons R.

Psychotridine, the major alkaloid from Psychotria beccarioides Wernh. has the molecular composition C55H62N10. It has been shown to be related to hodgkinsine, and a structure (4) derived from five Nb-methyltryptamine units is proposed. Mass spectral evidence for the structure is supported by the conversion of psychotridine through a pentamethiodide into a penta-indolenine base, analogous to the tri-indolenine base derived from hodgkinsine Nb,Nb',Nb?-trimethiodide. The structure is also supported by comparison of the 13C N.M.R. spectra of hodgkinsine and psychotridine.

Nagel D.W., Pachler K.G., Steyn P.S., Wessels P.L., Gafner G., Kruger G.J.

The structure of oxaline, the main alkaloid of Penicillium oxalicum has been established as (1a) by X-ray methods.

Ahond A., Janot M.M., Langlois N., Lukacs G., Potier P., Rasoanaivo P., Sangare M., Neuss N., Plat M.

Minato H., Matsumoto M., Katayama T.

Hauser D., Weber H.P., Sigg H.P.

The structure and absolute configuration of Chaetocin, a metabolite of the fungus Chaetomium minutum with antibacterial and cytostatic activity has been elucidated by chemical and X-ray methods.

Jamieson W.D., Rahman R., Taylor A.

Chromatography of crude extracts of cultures of Pithomyces chartarum on silicic acid has been shown to separate the biologically active sporidesmins from a group of inactive metabolites. One of these inactive compounds has been purified and shown to be the dimethyl thioether analogue of sporidesmin, named spordesmin-D. Evidence is presented for the structure of another inactive metabolite, sporidesmin-F, which has not been obtained in crystalline form. Sporidesmin has been converted into sporidesmin-D by treatment with sodium borohydride and methyl iodide.

Spilsbury J.F., Wilkinson S.

Metin B.

Penicillium roqueforti is a fungal starter culture used for the production of blue-veined cheeses, such as Roquefort, Gorgonzola, Stilton, Cabrales, and Danablue. During ripening, this species grows in the veins of the cheese, forming the emblematic blue-green color and establishing the characteristic flavor owin to its biochemical activities. P. roqueforti synthesizes a diverse array of secondary metabolites, including the well-known compounds roquefortine C, clavine alkaloids, such as isofumigaclavine A and B, mycophenolic acid, andrastin A, and PR-toxin. This review provides an in-depth exploration of P. roqueforti’s secondary metabolites, focusing on their biosynthetic pathways, the gene clusters responsible for their production, and their bioactivities. The presence of these compounds in blue cheeses is also reviewed. Furthermore, the silent clusters and the potential of P. roqueforti for producing secondary metabolites were discussed. The review highlights recently identified metabolites, including sesterterpenoids; tetrapeptides, D-Phe-L-Val-D-Val-L-Tyr, and D-Phe-L-Val-D-Val-L-Phe; cis-bis(methylthio)silvatin; and the 1,8-dihydroxynaphthalene (DHN)-melanin precursor, scytalone. Additionally, a gene cluster for DHN–melanin biosynthesis is presented. Finally, a revised cluster for roquefortine C biosynthesis comprising three rather than four genes is proposed.

Chávez R., Vaca I., García-Estrada C.

Filamentous fungi are an important source of natural products. The mold Penicillium roqueforti, which is well-known for being responsible for the characteristic texture, blue-green spots, and aroma of the so-called blue-veined cheeses (French Bleu, Roquefort, Gorgonzola, Stilton, Cabrales, and Valdeón, among others), is able to synthesize different secondary metabolites, including andrastins and mycophenolic acid, as well as several mycotoxins, such as Roquefortines C and D, PR-toxin and eremofortins, Isofumigaclavines A and B, festuclavine, and Annullatins D and F. This review provides a detailed description of the biosynthetic gene clusters and pathways of the main secondary metabolites produced by P. roqueforti, as well as an overview of the regulatory mechanisms controlling secondary metabolism in this filamentous fungus.

Robinson B.

In his report (Christison 1855) to the Royal Society of Edinburgh on 5th February 1855 of his pioneering studies upon the toxicology of the Calabar bean by self-experimentation, Robert Christison (see footnote 14 in Chap. 1 ) stated “that its active properties may be concentrated in an alcoholic extract, which constitutes 2.7 per-cent of the seed”. Although experimental details are not presented (Christison 1855), this deficiency was rectified some 8 years later by Thomas Fraser (see footnote 17 in Chap. 1 ), formerly Christison’s assistant at the University of Edinburgh (see footnote 14 in Chap. 1 ), who published the following method for the preparation of the tincture that he used in connection with his investigation into the therapeutic properties of the Calabar bean (Fraser TR 1863)

García-Domínguez P., Lorenzo P., Álvarez R., de Lera A.R.

The total synthesis of the suggested structure of (-)-novofumigatamide, a natural product containing a C3-reverse prenylated N-acetyl-exo-hexahydropyrrolo[2,3-b]indole motif fused to a 10-membered ring lactam, was achieved using the macrolactam formation in advance of a diastereoselective bromocyclization and reverse prenylation steps. Since the NMR data of the synthetic sample did not match those of the natural product, the endo-bromo precursor of a N-Boc analogue and additional diastereomers derived from l-Trp were also synthesized. Five alternative synthetic routes, which differed in the order of final key steps used for the construction of the 10-membered ring lactam and the hexahydropyrrolo[2,3-b]indole framework within the polycyclic skeleton and also in the amide bond selected for the ring-closing of the macrolactam, were thoroughly explored. Much to our dismay, the lack of spectroscopic correlations between the proposed structure of natural (-)-novofumigatamide and the synthetic products suggested a different connectivity between the atoms. Additional synthetic efforts to assemble alternative structures of the natural product and isomers thereof (see accompanying paper; DOI: 10.1021/acs.joc.2c01228) further highlighted the frustrating endeavors toward the identification of a natural product.

Rehagel C., Akineden Ö., Geisen R., Cramer B., Plötz M., Usleber E.

The present study aimed to develop a novel enzyme immunoassay (EIA) for the tremorgenic mycotoxin, penitrem A (PTA). This competitive EIA, based on rabbit anti-PTA polyclonal antibodies and a PTA-bovine serum albumin conjugate, yielded a mean standard curve detection limit of 220 ng/mL. The PTA-EIA, together with two EIAs for paxilline (PAX) and ergoline alkaloids, were used to study the toxin profiles in culture of fungi isolated from some naturally infected food sample materials, including dairy products, citrus fruits, and walnuts. PTA was produced by eight out of 27 mycelium extracts from malt extract agar and Sabouraud glucose chloramphenicol selective agar (2–200 μg/mL). Ten isolates from all types of food were also positive in the PAX-EIA (0.004–4 μg/mL). The EIA for ergoline alkaloids yielded positive results (0.0002–0.4 μg/mL) in isolates from dairy products and from walnuts, but not from citrus fruits. Control analyses of selected fungal extracts by HPLC-MS/MS for PTA and PAX qualitatively confirmed the EIA results, poor quantitative agreement could be attributed to the presence of penitrems other than PTA, and to PAX analogues, respectively. Selected PTA-positive fungal isolates were subjected to sequencing of the internal transcribed spacer region and the β-tubulin gene, and were identified as Penicillium polonicum and P. crustosum . In conclusion, Penicillium spp. capable to produce PTA, PAX, and ergoline alkaloids, either alone or in combination, appear to be quite common as contaminants on foods at the retail or production level. Some are capable to produce PTA in culture at high levels, which would have to be considered as toxic if present in food. • First report on antibodies against the tremorgenic mycotoxin penitrem A. • PTA immunoassay detection limit was 200 ng/mL. • PTA immunoassay detected toxin production in fungal cultures isolated from foods. • First report on PTA production by Penicillium polonicum , in addition to P. crustosum. • LC-MS/MS and immunoassay results: isolates from citrus fruit produce penitrems and paxillines.

Maragos C.M.

Harmful secondary metabolites produced by fungi, mycotoxins, are found worldwide in a multitude of products. Roquefortine C (ROQ-C) is a mycotoxin produced by Penicillium roqueforti, the major fungus used to ripen blue-veined cheeses. To facilitate the screening of cheeses for ROQ-C, a method based upon the ambient ionization technique of direct analysis in real time-mass spectrometry (DART-MS) was developed. The method involved extraction with acetonitrile/water, removal of interferences with a cleanup column, and application to test strips. Thermal desorption was used to facilitate volatilization and ionization of the ROQ-C. The limits of detection and quantitation were 0.011 and 0.036 µg/mL, respectively, equivalent to 0.06 µg/g and 0.18 µg/g in cheese. Average recoveries at concentrations from 0.5 to 5 µg/g ranged from 80.2 to 87.4%. The assay was validated as a screening assay, with a cut-off value of 0.31 µg/g and a t-statistic of 25.95 at this level, indicating a very low probability of false positives (p < .00001). Results from the DART-TD-MS method were compared to results from an LC-PDA-MS method for 64 naturally contaminated cheeses. The two methods were in good agreement, with r2 = 0.9152, suggesting that the DART-MS method can be used to screen for ROQ-C in blue cheeses.

Maragos C.M.

Certain fungi can produce secondary metabolites that are toxic, mycotoxins. Two groups of cheeses where fungi are used for ripening are the blue-veined cheeses (Penicillium roqueforti) and the "soft-ripened" cheeses (P. camemberti). An enzyme-linked immunosorbent assay was used to screen for the mycotoxin roquefortine C (ROQC) in 202 samples of cheeses sold in the United States. Of these 152 were blue-veined cheeses, 46 were soft-ripened cheeses and 4 were other varieties of mould-ripened cheeses. ROQC was not detected in any of the soft-ripened cheeses, at a limit of detection of 1.8 µg/kg. ROQC was found in 151 of 152 blue-veined cheeses. The maximum level found was 6,630 µg/kg (median 903 µg/kg, average of positives 1430 µg/kg, limit of quantitation 6.9 µg/kg). These levels are consistent with the levels found previously in blue-veined cheeses in the United Kingdom and Europe, which have generally been considered non-hazardous for human consumption.

Agrawal K., Verma P.

At present, all sectors are diverting their interest toward biologically synthesized products via fungi as they are nonexhaustive and have immense applicability. Fungi are ubiquitous in nature and have tremendous capability to produce various secondary metabolites that have both deleterious and beneficial effects. The production of beneficial secondary metabolites is in great demand in industry and in various biotechnological sectors. However, the cultures remain silent under laboratory conditions and production on a large scale to meet existing demand has been an issue. This can be overcome by the use of genomics, which will help in the detection as well as the production of high titers of the desired metabolites. Thus, this chapter deals with the various metabolites produced by fungi and their various biotechnological applications. In addition, limitations and future prospects are incorporated that will further broaden the understanding of fungal metabolites.

Maragos C.M.

Roquefortine, also known as roquefortine C (ROQC) is a fungal secondary metabolite (mycotoxin) that is produced by some of the same Penicillia as the tremorgen penitrem-A (PEN-A). The two mycotoxin...

Jakubczyk D., Dussart F.

Fungal natural products and their effects have been known to humankind for hundreds of years. For example, toxic ergot alkaloids produced by filamentous fungi growing on rye poisoned thousands of people and livestock throughout the Middle Ages. However, their later medicinal applications, followed by the discovery of the first class of antibiotics, penicillins and other drugs of fungal origin, such as peptidic natural products, terpenoids or polyketides, have altered the historically negative reputation of fungal “toxins”. The development of new antimicrobial drugs is currently a major global challenge, mainly due to antimicrobial resistance phenomena. Therefore, the structures, biosynthesis and antimicrobial activity of selected fungal natural products are described here.

Singh A.K., Rana H.K., Pandey A.K.

The kingdom Fungi represents an incredibly rich and untapped source of bioactive natural products and seems to be an ideal agent for providing unique chemical compounds against various diseases. They are present in almost every ecological niche, making them the second largest kingdom after bacteria. It has been reported that earth is approximately estimated to have 1.5 million species and only 10% of it is known to scientific community. Several fungal secondary metabolites are useful for mankind, for example, penicillin a β-lactam antibiotic was isolated first time from Penicillium sp. Now, it is one of the widely used antibiotics worldwide. Fungal kingdom produces a variety of secondary metabolites, including all important classes like terpenes, terpenoids, alkaloids, and sugar derivatives. Though many fungal-derived natural products are known today, the production potential of fungus is significantly low because the expression of gene and corresponding secondary metabolites are cryptic/very less under laboratory condition. Therefore, scientific community around the world is searching for a chemical method to synthesize the secondary metabolite in laboratory at higher yield. Moreover, total in vitro chemical synthesis does not always signify a cost-effective method for producing fungal-derived natural compound, particularly when synthesizing compounds with complex chemistry. However, this issue can be overcome by utilizing heterologous production of secondary metabolites. Current chapter describes in detail the variety of secondary metabolites produced, their synthesis strategies via chemical and heterologous mode, as well as their biological applications.

Cantor M.D., van den Tempel T., Hansen T.K., Ardö Y.

Blue or blue-veined cheeses are characterized by growth of the mold Penicillium roqueforti, giving them their typical appearance and flavor. Blue cheese is produced in many countries all over the world, where their own types of blue cheeses have been developed, each with different characteristics and involving different manufacturing methods. This chapter aims to review the knowledge on different aspects of blue cheese ripening, emphasizing changes in the microenvironment, for example, pH and salt gradients within the cheese matrix; the microorganisms that contribute to ripening and their interactions, that is, lactic acid bacteria, mold, and yeasts; and the various biochemical changes, that is, lipolysis, proteolysis, and aroma formation and the effect on the texture and consistency of the ripened cheese. Potential mycotoxin production is also covered. Finally, thoughts on the selection of appropriate starter and mold cultures, as well as new, possible adjunct cultures, will be discussed.

Waldman A.J., Ng T.L., Wang P., Balskus E.P.

Natural products that contain functional groups with heteroatom-heteroatom linkages (X-X, where X = N, O, S, and P) are a small yet intriguing group of metabolites. The reactivity and diversity of these structural motifs has captured the interest of synthetic and biological chemists alike. Functional groups containing X-X bonds are found in all major classes of natural products and often impart significant biological activity. This review presents our current understanding of the biosynthetic logic and enzymatic chemistry involved in the construction of X-X bond containing functional groups within natural products. Elucidating and characterizing biosynthetic pathways that generate X-X bonds could both provide tools for biocatalysis and synthetic biology, as well as guide efforts to uncover new natural products containing these structural features.

Martín J.F., Liras P.

Several filamentous fungi grow on the surface or inside different types of cheese, produce secondary metabolites, and contribute to the organoleptic characteristics of mature cheese. Particularly relevant is the contribution of Penicillium roqueforti to the maturation of blue-veined cheeses (Roquefort, Danablu, Cabrales, etc.). P. roqueforti is inoculated into these cheeses as a secondary starter. This fungus is closely related taxonomically to Penicillium carneum and Penicillium paneum, but these two species are not used as starters because they produce the potent toxin patulin. P. roqueforti Thom has the capability to produce about 20 secondary metabolites of at least seven different families, but it seems that only some of them are produced in microaerobic conditions and accumulate inside the cheese (e.g., andrastins). This article focuses on the biosynthetic pathways, gene clusters, and relevance of the known metabolites of P. roqueforti including roquefortines, PR-toxin and eremofortins, andrastins, mycophenolic acid, clavines (agroclavine and festuclavine), citreoisocoumarin, and orsellinic acid. In addition the biosynthesis of patulin (a P. paneum and P. carneum product) is discussed. Penicillium camemberti grows on the surface of Camembert, Brie, and related white rind cheeses, and the penetration of secondary metabolites inside the cheese is relevant. One of the P. camemberti metabolites, cyclopiazonic acid, is important because of its neurotoxicity and its biosynthesis is reviewed. The removal of toxic metabolites gene clusters by precise gene excision while preserving all other characteristics of the improved starter strains, including enzymes involved in cheese ripening and aroma formation, is now open. A possible strain improvement application to the cheese industry is of great interest.

Martín J.F., Coton M.

Blue cheeses undergo complex fermentation and maturation processes mainly mediated by lactic acid bacteria and fungi. Penicillium roqueforti is a common secondary starter culture for blue-veined cheese manufacture and largely contributes to the characteristic blue cheese flavor and color of the final product, although fungi other than P. roqueforti may occur in artisanal style blue-veined cheeses. Penicillium roqueforti is particularly well adapted to the conditions encountered during blue cheese manufacture including low oxygen levels and temperatures. This species is also actively involved in lipolysis and proteolysis and produces many volatile and nonvolatile aroma compounds among which methylketones are the most abundant. Penicillium roqueforti produces about a dozen secondary metabolites some of which, like PR-toxin, are toxic and may represent a problem for human health. Others, like roquefortines C and D, are well known, but they do not pose health problems. Andrastins are antitumor molecules and are present in significant concentrations in blue-veined cheeses and mycophenolic acid is an antifungal agents, clinically used as an immunosuppressant. Advances in molecular genetics and metabolite biosynthesis allow us to understand how they are synthesized and secreted by P. roqueforti. The possibility of using high-quality P. roqueforti strains unable to produce toxic secondary metabolites in cheese is of great interest.