Author's personal copy Provided for non-commercial research and educational use only. Not for reproduction, distribution or commercial use. This chapter was originally published in the book Encyclopedia of Food Microbiology. The copy attached is provided by Elsevier for the author's benefit and for the benefit of the author's institution, for non-commercial research, and educational use. This includes without limitation use in instruction at your institution, distribution to specific colleagues, and providing a copy to your institution's administrator. All other uses, reproduction and distribution, including without limitation commercial reprints, selling or licensing copies or access, or posting on open internet sites, your personal or institution’s website or repository, are prohibited. For exceptions, permission may be sought for such use through Elsevier’s permissions site at: http://www.elsevier.com/locate/permissionusematerial From Nigam, P.S., Singh, A., 2014. Metabolic Pathways: Production of Secondary Metabolites - Fungi. In: Batt, C.A., Tortorello, M.L. (Eds.), Encyclopedia of Food Microbiology, vol 2. Elsevier Ltd, Academic Press, pp. 570–578. ISBN: 9780123847300 Copyright © 2014 Elsevier, Ltd unless otherwise stated. All rights reserved. Academic Press Author's personal copy Production of Secondary Metabolites – Fungi PS Nigam, University of Ulster, Coleraine, UK A Singh, Technical University of Denmark, Lyngby, Denmark Ó 2014 Elsevier Ltd. All rights reserved. This article is a revision of the previous edition article by Poonam Nigam, Dalel Singh, volume 2, pp 1319–1328, Ó 1999, Elsevier Ltd. Introduction Metabolic Pathway for Secondary Metabolites Secondary metabolites usually accumulate during the later stage of fermentation, known as the idiophase, which follows the active growth phase called the trophophase. Compounds produced in the idiophase have no direct relationship to the synthesis of cell material and normal growth of the microorganisms. Secondary metabolites are formed in a fermentation medium after the microbial growth is completed. Comparatively, a few microbial organisms produce the majority of secondary metabolites and a single microbial type has the capacity to produce very different metabolites, for example, Streptomyces griseus and Bacillus subtilis each can produce more than 50 different secondary metabolites. The production of economically valuable secondary metabolites (e.g., antibiotics) is one of the major activities of the bioprocess industry. The most common secondary metabolites are antibiotics; others include mycotoxins, ergot alkaloids, the widely used immunosuppressant cyclosporin, and fumagillin, an inhibitor of angiogenesis and a suppressor of tumor growth. For the production of a desired secondary metabolite, it is essential to ensure that appropriate conditions for metabolic pathways are provided during the trophophase to maximize growth of the microbial species. It is important that the conditions are altered properly at the appropriate time of fermentation to obtain the best product yield. Secondary metabolites are produced by a branch off the pathways from primary metabolism. Microorganisms cultured under ideal conditions for primary metabolism without environmental limitations attempt to maximize the microbial biomass formation. Under conditions of balanced growth, however, the microbial cell minimizes the accumulation of any particular cellular building blocks in amounts beyond those required for growth. Hence, the metabolic pathway of a particular microorganism can be manipulated for the production of a large excess of the desired metabolite. The production of secondary metabolites starts as growth is limited due to the unavailability of one of the key nutrients – for example, nitrogen, carbon, phosphorus, and so on. Most secondary metabolites are complex organic molecules that require a large number of specific enzymatic reactions for synthesis. One characteristic of a secondary metabolite is that the enzymes involved in the production of the secondary metabolite are regulated separately from the enzymes of primary metabolism. In some cases, specific inducers of secondary metabolite production have been identified. The metabolic pathways of these secondary metabolites start from primary metabolism, because the starting materials for the secondary metabolism come from the major biosynthetic pathways. Many structurally complex secondary metabolites originate from structurally quite similar precursors. Thus, the secondary metabolite generally is produced from several intermediate products that accumulate in the fermentation medium or in microbial cells during primary metabolism. Characteristics of Secondary Metabolites In secondary metabolism, the desired product usually is not derived from the primary growth substrate, but rather a product formed from the primary growth substrate acts as a substrate for the production of a secondary metabolite and usually is suppressed by high specific growth rates of the secondary metabolites producing cultures. Secondary metabolites have the following characteristics: l l l l l l l l 570 Secondary metabolites can be produced only by a few microorganisms. They tend to be produced at the end of exponential growth or during substrate-limited conditions. They are produced from common metabolic intermediates but use specialized pathways encoded by a specific gene. These compounds are not essential for the organism’s own growth, reproduction, and normal metabolism. Secondary metabolites have unusual chemical linkages, for example, lectam rings, cyclic peptides, unsaturated bonds of polyacetylenes and polyenes, large macrolide rings, and so on. Growth conditions, especially the composition of the medium within a fermentation system, control the formation of secondary metabolites. These compounds are produced as a group of closely related structures. Secondary metabolic compounds can be overproduced. Transformation within Cells There are several hypotheses about the role of secondary metabolites. Besides the five phases of the cell’s own metabolism – intermediary metabolism, regulation, transport, differentiation, and morphogenesis – secondary metabolism is the activity center for the evolution of further biochemical development. This development can proceed without damaging primary metabolite production. Secondary metabolites form a heterogeneous class of structurally highly diverse compounds (the mode of action of such compounds is highly complicated), having specific chemical reactivity, and known to affect the intracellular redox homeostasis by increasing levels of reactive oxygen species and subsequently inducing apoptosis in Encyclopedia of Food Microbiology, Volume 2 http://dx.doi.org/10.1016/B978-0-12-384730-0.00202-0 Encyclopedia of Food Microbiology, Second Edition, 2014, 570–578 Author's personal copy METABOLIC PATHWAYS j Production of Secondary Metabolites – Fungi target cells. Genetic changes leading to the modification of secondary metabolites would not be expected to have any major effect on normal cell function. If a genetic change leads to the formation of a compound that may be beneficial, then this genetic change would be fixed in the cell’s genome and become essential, with the result that this secondary metabolite would be converted into a primary metabolite. Antibiotics 571 belong to the b-lactam group of antibiotics, so called because their structure consists of a b-lactam ring system (Figure 1). All of these are medically useful antibiotics produced by fungi. Penicillins The basic structure of the penicillins is 6-aminopenicillanic acid, which consists of a thiazolidine ring with a condensed b-lactam ring. The types of penicillins are presented in Table 2. Natural penicillins: The fermentation is carried out without the addition of side-chain precursors. l Biosynthetic penicillins: Out of more than 100 biosynthetic types, only benzylpenicillin, phenoxymethylpenicillin, and allylmercaptomethylpenicillin (penicillins G, V, and O) are produced commercially. l Antibiotics are chemical substances produced by certain microorganisms as products of secondary metabolism. These substances possess activity to inhibit growth processes or kill other microorganisms, even used at low concentrations. Growth inhibition of one organism by another organism in mixed culture has been known for a long time. The most famous example is the growth inhibition observed by Alexander Fleming in 1929. He noticed that staphylococcal growth on plate culture was inhibited by a contaminant, Penicillium notatum, which produced the antibiotic penicillin. Medicinally useful antibiotics have shown their impact on the treatment of infectious diseases. Some less-effective antibiotics work after a chemical modification, making them semisynthetic antibiotics. The sensitivity of microorganisms and other chemotherapeutic agents varies. Gram-positive bacteria are more sensitive to antibiotics than are Gram-negative bacteria. Broad-spectrum antibiotics act on Gram-positive as well as Gram-negative bacteria and therefore are used more widely in medicine than narrow-spectrum antibiotics, which are effective for only a single group of microorganisms. Antibiotics are produced by bacteria (about 950 types of antibiotics), actinomycetes (about 4600 types), and fungi (about 1600 types). This article deals only with secondary metabolites that are produced by fungal cultures (Table 1). The antibiotics produced by the Aspergillaceae and Moniliales are of practical importance. Only 10 of the known fungal antibiotics are produced commercially and only the penicillins, cephalosporin C, griseofulvin, and fusidic acid are clinically important. Penicillins, cephalosporins, and cephamycins Table 1 O R H N C 1 6 5 H Acyl residue CH 3 S H 2 CH 3 3 N 4 O H β-lactam ring COO (Na⊕, K⊕ ) Thiazolidine ring 6-Aminopenicillanic acid R3 H S R1 N CH 2 O R2 COOH β-lactam ring Dihydrothiazine ring Cephalosporin Figure 1 b-Lactam antibiotics from fungi. Antibiotics produced by fungal cultures Antibiotic group Produced by Spectrum of action Cell target Cephalosporin Fumagillin Griseofulvin Cephalosporium acremonium Aspergillus fumigatus Penicillium griseofulvum Penicillium nigricans Penicillium urticae Penicillium chrysogenum Aspergillus nidulans Cephalosporium acremonium Streptomyces venezuelae Broad spectrum Amebae Fungi Cell wall Gram-positive bacteria Cell wall Gram-positive and Gram-negative bacteria Gram-positive bacteria and most Gram-negative bacteria Gram-positive bacteria Inhibit translation during protein synthesis Inhibit protein synthesis Prokaryotes and eukaryotes Ribosomal translocation Penicillins Chloramphenicol Macrolides (erythromycin, oleandomycin) Pleuromutilin Fusidic acid Streptomyces erythreus Pleurotus mutilus Pleurotus passeckerianus Fusidium coccineum Acremonium fusidioides Encyclopedia of Food Microbiology, Second Edition, 2014, 570–578 Microtubules in fungi Mycoplasms Author's personal copy 572 Table 2 METABOLIC PATHWAYS j Production of Secondary Metabolites – Fungi Three main types of penicillins Natural Biosynthetic Semisynthetic Benzylpenicillin (penicillin G) Benzylpenicillin (penicillin G) Acid labile low activity against Gram-negative bacteria b-Lactamase sensitive Propicillin Acid stable b-Lactamase sensitive 2-Pentenylpenicillin (penicillin F) n-Amylpenicillin (penicillin-dihydro F) Methylpenicillin n-heptylpenicillin (penicillin K) p-hydroxybenzylpenicillin (penicillin X) Methicillin Acid stable b-Lactamase resistant Oxacillin Acid stable b-Lactamase resistant Ampicillin Broadened spectrum of activity (against Gram-negative bacteria) Acid stable b-Lactamase sensitive Carbenicillin Broadened spectrum of activity (against Pseudomonas aeruginosa) Acid stable Ineffective orally b-Lactamase sensitive Phenoxymethylpenicillin (penicillin V) Acid stable b-Lactamase sensitive Low activity against Gram-negative bacteria Allylmercaptomethyl penicillin (penicillin O) Reduced allergenic properties Penicillin N (synnematin B) (D-4-amino-4-carboxy-n-butylpenicillin) Isopenicillin N (L-4-amino-4-carboxy-n-butylpenicillin) l Semisynthetic penicillins: Benzylpenicillin and phenoxymethylpenicillin (penicillins G and V) are used in their synthesis; these penicillins have a broadened spectrum of activity and improved characteristics, such as acid stability, resistance to plasmid or chromosomally coded b-lactamases, and expanded antimicrobial effectiveness; and, therefore, they are used extensively in therapy. HOOC L H 2N CH SH CH CH 2 CH 2 CH 2 L-α-aminoadipic COOH COOH acid (α-AAA) L-cysteine CH 3 CH H 2N CH α-AAA-Cys CH 3 L-valine L SH Synthetic Pathway and Regulation of Penicillin Formation CH 2 NH 2 COOH α-AAA NH CH (D) CO CH 3 C CH 2 NH δ-(L-α-aminoadipyl) CH 3 cysteinyl-D-valine CH (LLD-tripeptide) COOH The b-lactam–thiazolidine ring of penicillin is constructed from L-cysteine and L-valine in a nonribosomal process by means of a dipeptide composed of L-a-aminoadipic acid (L-aAAA) and L-cysteine. Subsequently, L-valine is connected by an epimerization reaction, resulting in the formation of the tripeptide. The first product of the cyclization of tripeptide is isopenicillin N. Benzylpenicillin is produced in the exchange of L-a-AAA with activated phenylacetic acid (Figure 2). Penicillin biosynthesis is affected by phosphate concentration, shows a distinct catabolite repression by glucose, and is regulated by ammonium ion concentration. Cyclization in 2 steps α-AAA S NH CH Isopenicillin N N COOH O CO 2 CH 2 CoA Penicillin transacetylase α-AAA CoA SH S Industrial Production of Penicillin CH 2 CO CH NH Benzylpenicillin Benzylpenicillin and phenoxymethylpenicillin (penicillins G and V) are produced in a submerged process (Figure 3) in fermenters from 40 000 to 200 000 l in size. The process is highly aerobic with a volumetric oxygen absorption rate of 0.4–0.8 mmol l1 min1, an aeration rate of 0.5–1.0 volume per volume per minute (vvm), and an optimal temperature range 25–27 C. A typical penicillin fermentation medium consists of corn-steep liquor; an additional N O Figure 2 COOH Biosynthesis of penicillin in Penicillium chrysogenum. nitrogen source, such as yeast extract, whey, or soy meal; and a carbon source, such as lactose; the pH is maintained at 6.5 and phenylacetic acid or phenoxyacetic acid is fed continuously as a precursor. Encyclopedia of Food Microbiology, Second Edition, 2014, 570–578 Author's personal copy METABOLIC PATHWAYS j Production of Secondary Metabolites – Fungi Glucose feedings L-α-AAA-L-Cys I 1.45 mg l –1 h –1 II 1.31 mg l –1 h –1 III 1.15 mg l –1 h –1 573 + L-Val LLD-Tripeptide 120 I 3 glucose feedings Nitrogen feeding 100 II III 18 mg l –1 h –1 Isopenicillin N (L-α-AAA-APA) Biomass (g l –1) Carbohydrate, ammonia, penicillin (g l –1 × 10) Racemase Lactose Penicillin Penicillin N (D-α-AAA-APA) 80 (D) 60 α-AAA Ring formation stimulated by Fe 2+, ascorbate, ATP S H N N O CH 3 COOH 40 Deacetoxycephalosporin C Biomass O2 20 (D) α-AAA Ammonia 0 Dioxygenase stimulated by Fe 2+, ascorbate, α-ketoglutarate H N S N 0 20 40 60 80 100 120 140 O 160 CH 2 COOH Fermentation time (h) OH Deacetylcephalosporin C Figure 3 Penicillin fermentation with Penicillium chrysogenum. CH 3 Cephalosporins (D) α-AAA Cephalosporins are b-lactam antibiotics containing a dihydrothiazine ring with D-a-aminoadipic acid. Cephalosporins are produced by Cephalosporium acremonium (Acremonium chrysogenum), Emericellopsis, and Paecilomyces spp. Cephalosporins are less toxic and have a broader spectrum of action than ampicillin. Thirteen therapeutically important semisynthetic cephalosporins are produced commercially. Synthetic Pathway of Cephalosporins in Fungi Cephalosporin biosynthesis (Figure 4) proceeds from d-(aaminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. In the next stage, penicillin N is produced by the transformation of the L-a-AAA side chain into the D-form, by the action of a labile racemase. After ring expansion to deacetoxycephalosporin C by the expandase reaction, hydroxylation via a dioxygenase to deacetylcephalosporin C occurs. The acetylation of cephalosporin C by an acetyl-CoA-dependent transferase is the end point of the pathway in fungi. Industrial Production of Cephalosporins Fermentations are carried out as batch-fed processes with semicontinuous addition of nutrients at pH 6.0–7.0, temperature H N S C CoA O N O CH 2 O CO CH 3 COOH Cephalosporin C Figure 4 Biosynthesis of cephalosporin C by Cephalosporium acremonium. 24–28 C in complex media with corn-steep liquor, meat meal, sucrose, glucose, and ammonium acetate. The biosynthesis is affected by phosphate, nitrogen, and carbohydrate catabolite regulation. Rapidly metabolizable carbon sources, such as glucose, maltose, or glycerol, reduce the production. The repression of expandase is the most significant effect. Lysine in low concentrations and methionine stimulate the synthesis. Fusidic Acid The antibiotic fusidic acid was first isolated in 1960 from fermentations of the imperfect fungus Fusidium coccineum (Moniliaceae) or Acremonium fusidioides. In addition, the production of fusidic acid by strains of Cephalosporium, various dermatophytes and Isaria kogane has been reported. Ramycin, an antibiotic mixture, has been isolated from the Encyclopedia of Food Microbiology, Second Edition, 2014, 570–578 Author's personal copy 574 METABOLIC PATHWAYS j Production of Secondary Metabolites – Fungi culture fluid of the zygomycete Mucor ramannianus and the identity of one of the components with fusidic acid was proved later. Fusidic acid belongs chemically to the group of tetracyclic triterpenoids with a fusidane skeleton. This type of hydrocarbon skeleton also is present in many natural steroids and triterpenes and contains a cyclopentanoperhydrophenanthrene ring connected at C17 with an a,b-unsaturated carboxylic acid side chain and a b-(16,21) cis-oriented acetoxy group on C16. Naturally occurring antibiotics related to fusidic acid include helvolic acid from cultures of Aspergillus fumigatus and Cephalosporium caerulens; cephalosporin P1 and related derivatives from C. acremonium; and the viridominic acids A, B, and C from a Cladosporium species. Biosynthetic Pathway of Fusidic Acid The biosynthesis of fusidic acid follows the general pathway for the formation of sterols and polycyclic triterpenes. The isolation of common intermediates, such as several protosterols in the biosynthesis of fusidic acid and helvolic acid from the mycelium of F. coccineum and C. caerulens, indicates that the biogenetic pathways leading to these antibiotics are identical. Besides the total syntheses of fusidic acid, several 100 semisynthetic derivatives have been synthesized by chemical or microbial modification to achieve a broader antibacterial spectrum, increased potency, modified pharmacokinetics or better stability in solution. One derivative, 16-deacetoxy-16bacetylthiofusidic acid, is more stable and twice as active as fusidic acid against several Gram-positive bacteria. Large-scale production of fusidic acid is carried out in batch fermentations using a complex medium containing sucrose, glycerol, or glucose as the carbon source; soybean meal, cornsteep liquor, or milk powder as the nitrogen source; vitamins (biotin); and inorganic salts. The fermentation is carried out at 27–28 C for 180–200 h with efficient aeration and vigorous agitation with a high-producing mutant of F. coccineum. Applications of Fusidic Acid Fusidic acid is used for the treatment of multiply-resistant staphylococcal infections or in combination with other antibiotics. Systemic application includes treatment of septicemias, endocarditis, staphylococcal pneumonia, osteomyelitis, and wound infections. Topically applied, fusidic acid is effective in the treatment of staphylococcal and streptococcal skin infections, wounds, burns, and ulcers. Fusidic acid is available in various forms of pharmaceutical presentations (Fucidin), either in the form of tablets containing sodium fusidate, as an aqueous solution for parenterally or intravenous infusion in a sterile buffer, and as an ointment for topical applications. Immunosuppressive activities have been reported for fusidic acid on activated blood mononuclear cells. Fusidic acid inhibits protein biosynthesis in both prokaryotes and eukaryotes. The antibiotic binds to the translocation factors in prokaryotic and eukaryotic cell-free systems. Resistance to fusidic acid mainly has been studied in Staphylococcus aureus and Escherichia coli. Griseofulvin The systemic antifungal antibiotic griseofulvin was first isolated from the mycelium of Penicillium griseofulvum Dierckx. In 1946, a compound named ‘curling factor’ was isolated from the mycelium and the culture filtrate of Penicillium janczewskii Zal. It caused abnormal curling of the hyphae of Botrytis allii and later was identified as griseofulvin. Many other fungi were shown to produce griseofulvin with most of these species belonging to the genus Penicillium (e.g., Penicillium urticae, Penicillium raistrickii, Penicillium raciborskii, Penicillium kapuscinskii, Penicillium albidum, Penicillium melinii, and Penicillium brefeldianum, as well as some mutant strains of Penicillium patulum). In addition, Aspergillus versicolor and Nematospora coryli have been shown to produce the antibiotic. The carbon skeleton of griseofulvin is a tricyclic-spiro system based on grisan and consists of a chlorine-substituted coumaranone and an enone containing a cyclohexane ring adjacent to the asymmetric spirane center. Biosynthesis of Griseofulvin Griseofulvin is formed by linear combination of acetate units with the benzophenone as a possible intermediate. Oxidative coupling followed by saturation of one of the double bonds in the resulting dienone may form griseofulvin, as demonstrated by labeling with [2-3H]- and [14C]acetate. The double-bond saturation in the intermediate dienone occurs via transaddition of hydrogen. Labeling with [1-13C, 18O2]acetate and analysis by 13C nuclear magnetic resonance spectroscopy proved that all oxygen atoms derive from acetate. The occurrence of dechlorogriseofulvin in some producing fungi (e.g., A. versicolor) indicates that the chlorination must occur as a late step in the biosynthesis of griseofulvin, although the exact mechanism of this reaction has not yet been elucidated. Commercial Production Griseofulvin is produced commercially in submerged culture with mutant strains of P. patulum, P. raistrickii, or P. urticae, which have been obtained by mutating the spores with ultraviolet light, chemical mutagens, or sulfur isotopes, in a cornsteep medium in which the factors of pH (through intermittent addition of glucose), aeration, and the concentration of chloride and nitrogen are controlled carefully. A typical griseofulvin titer of 6–8 g l1 is achieved after 10 days of fermentation. Higher yields (up to 12–15.5 g l1) can be obtained by the addition of various methyl donors (choline salts, methyl xanthate, and folic acid) to the medium. Applications of Griseofulvin Griseofulvin is used for the treatment of infections caused by species of certain dermatophytic fungi (Epidermophyton, Trichophyton, and Microsporum), which cannot be cured by topical therapy with other antifungal drugs. In vitro, minimal inhibitory concentrations ranging from 0.18 to 0.42 mg ml1 against various dermatophytes have been reported. The drug has no Encyclopedia of Food Microbiology, Second Edition, 2014, 570–578 Author's personal copy METABOLIC PATHWAYS j Production of Secondary Metabolites – Fungi 575 effect on bacteria, other pathogenic fungi, and yeasts. Griseofulvin is effective in vivo in cutaneous mycoses because, when administered orally, it concentrates in the deep cutaneous layer and the keratin cells. The uptake of griseofulvin into the susceptible fungal cells is an energy-requiring process dependent on concentration, temperature, pH, and an energy source such as glucose. It has been suggested that insensitive fungi and yeasts do not bind sufficient amounts of the antibiotic. Claviceps, and Alternaria), and their biological activity is toxicity against vertebrates. Mycotoxins are a structurally diverse group of generally low-molecular-weight compounds produced by fungi. Although both chemically and biologically diverse, they are all fungal secondary metabolites. As such, the principles of their biosynthesis, physiology, and evolution are similar to those of antibiotics and other pharmacologically active secondary metabolites. Pleuromutilin (Tiamulin) Impact of Mycotoxins The only commercial antibiotic produced by a basidiomycete is the diterpene pleuromutilin. Pleuromutilin was first isolated from Pleurotus mutilus and Pleurotus passeckerianus in a screening for antibacterial compounds. Pleuromutilin is active against Gram-positive bacteria, but its most interesting biological activity is its effectiveness against various forms of mycoplasms. The preparation of more than 66 derivatives of pleuromutilin resulted in the development of tiamulin, which exceeds the activity of the parent compound against Gram-positive bacteria and mycoplasms by a factor of 10–50. The minimal inhibitory concentrations against different strains of mycoplasma were in the range 0.0039–6.25 mg ml1. Production of Pleuromutilin Pleuromutilin can be produced by the fermentation in a medium composed of glucose 50 g, autolyzed brewer’s yeast 50 g, KH2PO4 50 g, MgSO4$7H2O 0.5 g, Ca(NO3)2 0.5 g, NaCl 0.1 g, FeSO4$7H2O 0.5 g, water to 1 l, and pH 6.0. The yield after 6 days of growth in a 1000 l fermenter was reported as 2.2 g l1. It could be demonstrated that during fermentation of pleuromutilin, derivatives differing in the acetyl portions attached to the 14-OH group of mutilin were formed. The biosynthesis of these derivatives was stimulated strongly by the addition of corn oil as the carbon source during fermentation. Pleuromutilin overproducers have been obtained by conventional mutagenesis and selection programs, as well as by protoplast fusion and genetic studies. Applications of Pleuromutilin Studies on the mode of action revealed that pleuromutilin and its derivatives act as inhibitors of prokaryotic protein synthesis by interfering with the activities of the 70S ribosomal subunit. The ribosome-bound antibiotics lead to the formation of inactive initiation complexes, which are unable to enter the peptide chain elongation cycle. In various bacteria, resistance to the drug develops in a stepwise fashion. Because of its outstanding properties, pleuromutilin is used for the treatment of mycoplasma infections in animals. Other Secondary Metabolites Mycotoxins Mycotoxins are natural products produced by filamentous fungi, mainly with five genera (Penicillium, Fusarium, Aspergillus, Mycotoxins cause adverse health effects in human and livestock populations, which range from acute toxicity and death to milder chronic conditions and impairment of reproductive efficiency. In addition, some mycotoxins show insecticidal, antimicrobial, and phytotoxic effects. Mycotoxins cause huge economic losses in agriculture because they contaminate crops in the field, after harvest, or during storage. A few companies produce and sell mycotoxins as analytical standards. Otherwise, the economic impact of mycotoxins is largely negative, and the major biotechnological emphasis in mycotoxin research is on prevention rather than production. These compounds play a major role in agricultural ecosystems. Elimination or minimization of mycotoxin contamination in the raw materials for industrial fermentations is a continuing biotechnological challenge. In addition, several classes of mycotoxins have emerged as models for research in the biosynthesis and molecular biology of fungal secondary metabolism. The genetic engineering of existing mycotoxin biosynthetic pathways ultimately could yield novel products for medicinal use. Major Classes of Mycotoxins The extreme toxicity and carcinogenicity of the aflatoxins and the common occurrence of their producer Aspergillus species means that molds are more than mere agents of deterioration. Reports that even trace levels of aflatoxin in feeds had disastrous consequences for young poultry led to the awareness that other mold metabolites may also have serious consequences for human and veterinary health. Mycotoxins have been discovered in various ways. Aflatoxins were identified after outbreaks of turkey X disease. The symptoms caused by ingestion of ergot alkaloids – gangrenous necrosis, neurological disturbances, and the human disease called St. Anthony’s fire – have been known for centuries. Trichothecenes also have been implicated in several natural intoxications, for example, alimentary toxic aleukia in human beings and a variety of moldy corn toxicoses of domesticated animals. Ochratoxins, on the other hand, were discovered by laboratory screening targeted specifically at finding toxigenic fungi. Patulin and trichothecin originally were discovered as part of screens for new antibacterial compounds from fungi. During the 1960s, they were reclassified from ‘antibiotics too toxic for drug use’ to ‘mycotoxins.’ A list of major classes of mycotoxins and producing fungal species is presented in Table 3. Aflatoxins are the most biologically potent, economically important, and scientifically understood of the mycotoxins. In addition to the acute toxicity leading to such conditions as Encyclopedia of Food Microbiology, Second Edition, 2014, 570–578 Author's personal copy 576 METABOLIC PATHWAYS j Production of Secondary Metabolites – Fungi Table 3 Important mycotoxins and their producer fungi Class Chemical taxonomy Major producing species Aflatoxins Citrinin Ergot Fumonisin Ochratoxin Patulin Rubratoxin Sterigmatocystin Tremorgens Trichothecenes Polyketides Polyketides Amino acid derived Amino acid derived Polyketides Polyketides Zearalenone Polyketide Aspergillus flavus, A. parasiticus, A. nomius Penicillium citrinum, P. verrucosum, numerous Aspergillus and Penicillium spp. Numerous Claviceps spp. Fusarium moniliforme, F. proliferatum, F. napiforme, F. nygamai, other Fusarium spp. Aspergillus ochraceus, Penicillium verrucosum, numerous Aspergillus and Penicillium spp. Penicillium expansum, P. griseofulvum, and Aspergillus spp. Penicillium rubrum Aspergillus versicolor, numerous Aspergillus spp. Penicillium cyclopium, Alternaria tenuis, Phoma sorghina, Pyricularia oryzae Fusarium roseum, F. nivale, several Myrothecium roridum, M. verrucaria, several Fusarium spp. Trichothecium roseum Fusarium graminearum and numerous Fusarium spp. Polyketides Sesquiterpenoids turkey X disease, in laboratory tests, aflatoxin B1 is one of the most potent carcinogens known. There is strong epidemiological evidence linking aflatoxin to human liver cancer. Under appropriate environmental conditions, aflatoxins are produced by toxigenic strains of Aspergillus flavus and Aspergillus parasiticus. The crops at greatest risk for aflatoxin contamination are corn, peanuts, and cottonseed, but rice, nuts, and spices also are susceptible. When animals consume aflatoxin-contaminated feeds, the toxic factor may be transferred to animal products, such as meat and milk. After the aflatoxins, trichothecenes are the next most important group of mycotoxins. Health Impact of Mycotoxins The toxic effects of mycotoxins can be divided into two broad categories: l l Acute effects, which cause rapid, often fatal diseases Chronic effects, which may cause weight loss, immunosuppression, cancer, reduced milk yields, and other sublethal changes. The wide range of pathological effects is listed in Table 4. Diseases caused by mycotoxins – mycotoxicoses – are not only clinically diverse but often are extremely difficult to diagnose owing to the numerous pharmacological effects of mycotoxins. Human diseases associated with mycotoxin ingestion include St. Anthony’s fire (ergot alkaloids), alimentary toxic aleukia (T-2 toxin), and yellow rice disease (citrinin and citreoviridin). Table 4 Most mycotoxicoses are known as veterinary syndromes. Some of the best known include zearalenone as the cause of an estrogenic syndrome in swine, fumonisins as the cause of a brain encephalopathy in horses, and ochratoxins as the cause of a porcine nephropathy. Examples of specific human and veterinary mycotoxicoses are listed in Table 5. There are virtually no effective treatments for any of these mycotoxicoses. Therefore, prevention is of the utmost importance. Fungal Secondary Metabolites in Fermented Foods Most of the mold-fermented foods are considered to be safe, even when they are produced using species of Aspergillus and Penicillium that include strains capable of producing mycotoxins. The inability of Aspergillus oryzae and Aspergillus sojae to produce aflatoxins is not understood; presumably, aflatoxin production offers no selective advantage in the koji environment. Some species used for food fermentations are even able to reduce the mycotoxin concentration in the substrate. For example Rhizopus oligosporus, used for tempeh fermentation, reduces aflatoxin present in the substrate to 40% and is able to inhibit growth, sporulation, and aflatoxin production of A. flavus. Species of Neurospora used to prepare oncom (from peanuts) inhibit aflatoxin-producing strains of A. flavus and A. parasiticus by competition or antagonism. Nevertheless, the application of defined starter organisms will improve the quality and consistency of the products without the production of undesired secondary metabolites. Range of pathological effects of mycotoxins Mycotoxin group Pathological effect Aflatoxin Ergot alkaloid Hepatotoxicity, hematopoiesis, carcinogenicity Vasoconstriction, neurotoxicity, reproductive irregularities Neurotoxicity Hematopoiesis, carcinogenicity Nephrotoxicity Carcinogenicity Neurotoxicity Dermal toxicity Reproductive irregularities Fumonisins Ochratoxin Ochratoxin A Sterigmatocystin Tremorgens Trichothecenes Zearalenone Meat Products The spontaneous mycoflora of mold-ripened salami and ham mainly consists of Penicillium spp. In mold-ripened sausages in Europe, 50% of the Penicillium population was identified as Penicillium nalgiovense; Penicillium verrucosum, Penicillium oxalicum, and Penicillium commune were only minor components of the mycoflora. The Aspergillus spp. Aspergillus candidus, A. flavus, A. fumigatus, Aspergillus caespitosus, Aspergillus niger, Aspergillus sulphureus, and Aspergillus wentii were isolated from Italian hams. Mycelium of the mold penetrates the product, causing some biochemical changes by its metabolism. Encyclopedia of Food Microbiology, Second Edition, 2014, 570–578 Author's personal copy METABOLIC PATHWAYS j Production of Secondary Metabolites – Fungi Table 5 577 Selected human and veterinary diseases associated with mycotoxins Causative toxin Disease Affected species Food/feed T-2 and other Fusarium toxins Fumonisins Sporidesmin Zearalenone Ochratoxin Satratoxin H, roriden, verrucarin Ergot alkaloids Citreoviridin, citrinin Aflatoxins Alimentary toxic aleukia Encephalopathy Facial eczema Hypoestrogenism Nephropathy Stachybotryotoxicosis St. Anthony’s fire Yellow rice disease Turkey X disease Humans Horses Sheep Swine Pigs, poultry Horses, cattle Humans Humans Turkeys, other poultry Overwintered wheat Grains Pasture grass Corn Barley, oats Hay, straw Rye bread Rice Peanut meal, grain Production of Secondary Metabolites in Meat Products Not only products of the fungal primary metabolism are formed but also secondary metabolites, such as mycotoxins and antibiotics. For example, the antibiotic penicillin may be produced from Penicillium chrysogenum, P. nalgiovense, and additional species of the genus growing on fermented meat. The production of penicillin in meat products is not desirable, as it may cause allergic reactions in sensitive people. A continual ingestion of low doses of penicillin or other antibiotics may lead to the development of resistant bacteria in the human digestive tract. This antibiotic-resistant flora is able to transfer its genetic information to pathogenic bacteria and prevent therapy with this antibiotic. Also, for technological reasons, the presence of penicillin is undesirable; although pathogenic bacteria can be suppressed, it also may inhibit the bacterial starter organisms. The production of penicillin is a consistent characteristic of P. nalgiovense when grown on a medium optimal for penicillin production. It seems highly probable that P. nalgiovense cannot produce penicillin on meatbased substrates, but selection of non-penicillin-producing strains is advised. The problem of mycotoxin production in mold-ripened sausages and ham often is discussed. About 70–80% of the Penicillium species of the spontaneous flora of salami are potential producers of mycotoxins, such as ochratoxin A and cyclopiazonic acid. From country-cured ham stored under dry conditions, aspergilli from the species A. flavus and A. parasiticus rarely were identified. No case has been reported of aflatoxin detection in fermented meat products from the market. The same can be said about the presence of sterigmatocystin. Ochratoxin can be produced on ham by Aspergillus ochraceus and P. verrucosum under experimental conditions but no reports are available about the occurrence of ochratoxin in market products of mold-ripened ham and sausages. Cyclopiazonic acid frequently has been isolated from Penicillium strains grown on mold-ripened sausages. Penicillic acid could not be detected after experimental inoculations of sausages with producer strains. It is suggested that this toxin is inactivated by reactions with amino acids in the meat. Although molds isolated from fermented meat products have the potential to produce mycotoxins under appropriate conditions in laboratory media – and, in some cases, even on the fermented product – scant evidence exists that the market-ready products contain dangerous concentrations of mycotoxins; there is usually little carryover of mycotoxins to the muscle tissues of animal. Cheeses Mold-ripened cheeses include the blue-veined cheeses – for example, Roquefort, Blue (France), Gorgonzola (Italy), Brick, Muenster and Monterey (the United States), Limburger (Belgium), and Stilton (United Kingdom) – and the surfaceripened Camembert and Brie (France). Blue-veined cheeses are produced by inoculation of the curd with cultures of Penicillium roqueforti, which produces blue-green spores. Proteolytic enzymes of the mold contribute to the ripening of the cheese and influence texture and aroma; concomitantly, water-soluble lipolytic enzymes produce free fatty acids and mono- and diacylglycerols from milk fat. For the production of Camembert and Brie, white strains of Penicillium camemberti form the surface crust. Secondary Metabolites in Cheese Penicillium camemberti is able to produce the mycotoxin cyclopiazonic acid. From 61 strains tested, all synthesized cyclopiazonic acid. This mycotoxin is isolated from both laboratory media and commercial cheeses. In cheeses, it is produced mainly in the rind and after storage at too-high temperatures. No risk to human health exists according to toxicological data and consumption habits. Mutants of P. camemberti that cannot produce cyclopiazonic acid were isolated. This may be a first step to improve the starter organisms by the methods of genetic engineering. For P. roqueforti, the production of isofumigaclavine A and B, marfortines, mycophenolic acid, PR toxin, and roquefortine C were described for chemotype I and botryodiploidin, mycophenolic acid, patulin, penicillic acid, and roquefortine C for chemotype II. In samples of commercial blue-veined cheeses, roquefortine was observed in all samples, and isofumigaclavine A and traces of isofumigaclavine B were observed in several samples; PR toxin was not detected. Mycophenolic acid is produced by some starter cultures in laboratory media and in cheese. Starter cultures now are available that do not have the ability to produce patulin, PR toxin, penicillic acid, and mycophenolic acid. The toxicity of roquefortine and isofumigaclavines is relatively low. Adequate handling of the cheese during ripening Encyclopedia of Food Microbiology, Second Edition, 2014, 570–578 Author's personal copy 578 METABOLIC PATHWAYS j Production of Secondary Metabolites – Fungi and storage, and screening of strains with low potential for the production of roquefortine and isofumigaclavines or a modification with genetic methods, will improve the production of Roquefort. Secondary Metabolites in Soy Sauce The koji molds are yellow-green aspergilli morphologically characterized as A. oryzae and A. sojae. A clear separation of these strains from the aflatoxin-producing A. flavus and A. parasiticus is difficult, because of the occurrence of intermediate forms. The conidia of the domesticated A. oryzae are larger and germinate faster than those of the wild A. flavus. The domesticated strains of A. oryzae and A. sojae appear to have lost the ability to produce aflatoxins. Because of the relatedness of the koji strains to the aflatoxin-producing strains of A. flavus, there is a fundamental interest in the mycotoxin-producing abilities of the koji strains. No aflatoxin production has been demonstrated in A. oryzae, A. sojae, and Aspergillus tamarii. Other mycotoxins are reported to be produced by these strains under special conditions. Aspergillus oryzae produces cyclopiazonic acid, kojic acid, 3-nitropropionic acid, and maltoryzine; A. sojae produces aspergillic acid and kojic acid; and A. tamarii produces cyclopiazonic acid and kojic acid. Nevertheless, A. oryzae has ‘generally recognized as safe’ status and is used for the production of enzymes. There is only scant evidence that these mycotoxins exist in industrial products. Generally, the koji fermentation lasts 48–72 h, whereas toxin production needs a longer incubation (5–8 days). In addition, the soybean may be an unsuitable substrate for the production of mycotoxins, and the subsequent fermentation by bacteria and yeasts may inactivate any mycotoxins. The large industries use well-defined, nontoxigenic koji molds as starters, but some small-scale manufacturers continue to use the house flora, for which the risk exists of contamination by aflatoxin-producing strains of A. flavus and A. parasiticus. See also: Aspergillus; Aspergillus: Aspergillus oryzae; Aspergillus: Aspergillus flavus; Cheese: Mold-Ripened Varieties; Fermented Foods: Origins and Applications; Fermented Meat Products and the Role of Starter Cultures; Fermented Foods: Fermentations of East and Southeast Asia; Fermented Milks: Range of Products; Mycotoxins: ClassificationNatural Occurrence of Mycotoxins in Food; Mycotoxins: Detection and Analysis by Classical Techniques; Mycotoxins: Immunological Techniques for Detection and Analysis; Mycotoxins: Toxicology; Penicillium andTalaromyces: Introduction; Penicillium/Penicillia in Food Production. Further Reading Bery, J., 1986. Further antibiotics with practical applications. In: Rehm, H.J., Reed, G. (Eds.), Biotechnology, vol. 4. VCH Verlagsgesellschaft, Weinheim, p. 465. Bhatnagar, D., Lillehoj, E.B., Arora, A., 1992. Handbook of Applied Mycology. In: Mycotoxins in Ecological Systems, vol. 5. Marcel Dekker, New York. Buckland, B.C., Omstead, D.R., Santamaria, V., 1985. Novel b-lactam antibiotics. In: Moo-Young, M. (Ed.), Comprehensive Biotechnology, vol. 3. Pergamon, Oxford, p. 49. Cole, R.J., 1986. Modern Methods in the Analysis and Structural Elucidation of Mycotoxins. Academic Press, San Diego. Crueger, W., Crueger, A., 1989. Antibiotics. In: Brock, T.D. (Ed.), Biotechnology: A Textbook of Industrial Microbiology, second ed. Sinauer Associates Inc., USA, MA, p. 229. Ellis, W.O., Smith, J.P., Simpson, B.K., et al., 1991. Aflatoxins in food: occurrence, biosynthesis, effects on organisms detection, and methods of control. Critical Reviews on Food Science and Nutrition 30, 403–439. Hesseltine, C.W., 1986. Global significance of mycotoxins. In: Steyn, P.S., Vleggaar, R. (Eds.), Mycotoxins and Phycotoxins. Elsevier, Amsterdam, p. 1. Jacob, C., Jamier, V., Aicha Ba, L., 2011. Redox active secondary metabolites. Current Opinion Chemical Biology 15, 149–155. Page, M.I. (Ed.), 1992. The Chemistry of b-Lactams. Chapman & Hall, London. Tim, A. (Ed.), 1997. Fungal Biotechnology. Chapman & Hall, London. Vaishnav, P., Demain, A.L., 2011. Unexpected applications of secondary metabolites. Biotechnology Advances 29, 223–229. Vining, L.C., Stuttard, C. (Eds.), 1994. Genetics and Biochemistry of Antibiotic Production. Butterworth-Heinemann, Oxford. Encyclopedia of Food Microbiology, Second Edition, 2014, 570–578