Production of Secondary Metabolites – Fungi

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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
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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
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Microtubules in fungi
Mycoplasms
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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.
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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
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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
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METABOLIC PATHWAYS j Production of Secondary Metabolites – Fungi
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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
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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.
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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
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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.
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