Fungal Biology - Chapter 7: Fungal Metabolism and Products

Fungal Metabolism and Products

7.1 Energy from Glucose and Non-Sugar Substrates

• Fungi obtain energy by oxidizing a variety of compounds, but glucose is the primary carbon source.
• Like all eukaryotes, fungi break down glucose into carbon dioxide, metabolic water, and ATP through aerobic respiration.
• The net result of aerobic respiration is:
  $C<em>6H</em>{12}O<em>6 + 6O</em>2 \rightarrow 6CO<em>2 + 6H</em>2O + 38 ATP$

Figure 7.2: Embden-Meyerhof Pathway and Tricarboxylic Acid Cycle

• The Embden-Meyerhof pathway and tricarboxylic acid cycle are the major pathways for generating energy from sugars.
• The pentose phosphate pathway provides some energy but is mainly used for biosynthesis, including the synthesis of 5-carbon sugars for nucleic acids.
• Important cofactors include:
  • Flavin adenine dinucleotide (FADH2)
  • Nicotinamide adenine dinucleotide phosphate (NADPH) - used to donate electrons and hydrogens in enzyme-catalyzed reactions

Cellular Respiration

• Cellular respiration is a four-stage process where glucose is oxidized to carbon dioxide, and oxygen is reduced to water.
• The energy released is stored as ATP; 36 to 38 ATP molecules are produced per glucose molecule.
• The four stages are:
  1. Glycolysis: Partial oxidation of glucose to form 2 molecules of pyruvate in the cytosol.
  2. Formation of Acetyl CoA: Pyruvate enters the mitochondrial matrix and undergoes oxidative decarboxylation to form 2 molecules of Acetyl CoA, catalyzed by pyruvate dehydrogenase.
  3. Krebs Cycle (TCA or Citric Acid Cycle): Acetyl CoA enters the tricarboxylic acid cycle.
     • Glucose is fully oxidized.
     • Acetyl CoA combines with oxaloacetate (4-carbon compound) to form citrate (6-carbon).
     • Two molecules of $CO_2$ are released, and oxaloacetate is recycled.
     • Energy is stored in ATP, NADH, and FADH2.
  4. Electron Transport System and Oxidative Phosphorylation: ATP is generated when electrons are transferred from NADH and FADH2 (produced in glycolysis, citric acid cycle, and fatty acid oxidation) to molecular $O_2$ by a series of electron carriers.
     • $O<em>2$ is reduced to $H</em>2O$.
     • Occurs in the inner membrane of mitochondria.

Glycolysis and the Citric Acid Cycle

• Glycolysis (ten enzyme-catalyzed reactions) of one glucose molecule generates two acetyl CoA molecules.
• The glycolytic pathway and citric acid cycle produce six $CO_2$ molecules, 10 NADH molecules, two FADH2 molecules, and 38 ATP per glucose molecule.
• NADPH: A cofactor that donates electrons and hydrogens in enzyme-catalyzed reactions.
• FADH2: A redox cofactor created during the Krebs cycle and used in the electron transport chain.
• ATP: Carries energy in its phosphate bonds; breaking a phosphate bond releases energy.

Gluconeogenesis: Generating Sugars from Non-Sugar Substrates

• Sugars are needed for the synthesis of fungal walls, nucleic acids, and storage compounds.
• Gluconeogenesis is the reversal of the Krebs cycle, used when fungi grow on non-sugar substrates.
• Instead of starting from glucose, a product like glyoxylate is converted to oxaloacetate, and metabolism proceeds in reverse (Figure 7.5).
• The glyoxylate cycle is a short-circuited form of the TCA cycle.

Figure 7.5: Role of the Glyoxylate Cycle

• The glyoxylate cycle generates sugars for biosynthesis when fungi are grown on non-sugar substrates such as acetate or organic acids.

Secretion of Organic Acids as Commercial Products

• Fungi are important commercial sources of organic acids.
• Aspergillus niger converts most sugar to citric acid, used in beverage, food, pharmaceutical, and cosmetic industries.
• Approximately 1.6 million tons of citric acid are produced each year, with China accounting for 35-40% of worldwide production.
• Other Aspergillus species produce gluconic, malic, itaconic, and gallic acids.
• Rhizopus nigricans produces large amounts of fumaric acid.
• Rhizopus oryzae produces fumaric and kojic acids.
• Other Rhizopus species produce lactic acid.
• Approximately 7,000,000 tons of acetic acid are produced annually, with only 190,000 tons produced by microbes.
• Examples include Aspergillus for citric acid and Lactobacillus for lactic acid production.
• Gluconic acid is naturally found in fruits and honey and is produced by Aspergillus niger.
• Gluconic acid imparts a refreshing sour taste and is a common additive in food and drinks and is used in pickling.

Organic Acids Production

• Lactic acid production is approximately 150,000 tons annually; global consumption is expected to rise to 500,000 tons a year due to use in polymers and plastics.
• Lactic acid can be polymerized to polylactic acid (PLA), forming a sustainable bioplastic.
• Lactic acid is used in the food industry as a preservative and flavoring, in the cosmetic industry in moisturizers and skin-rejuvenation agents, and in the pharmaceutical industry in I.V solutions and controlled drug delivery systems.
• Itaconic acid production is 15,000 tons annually, mainly by Aspergillus terreus.

Primary & Secondary Metabolites

• Primary metabolites: Intermediates or end products of common metabolic pathways (sugars, amino acids, organic acids, glycerol, etc.) essential for normal cellular functions.
• Secondary metabolites: A diverse range of compounds formed by specific pathways of particular organisms; not essential for growth but can confer an advantage (e.g., antibiotics, fungal toxins).

Secondary Metabolites: Penicillin

• Penicillin was discovered by Alexander Fleming in 1929 from Penicillium chrysogenum (originally misidentified as P. notatum), which prevented the growth of Staphylococcus spp.
• It is a broad-based antibiotic active against Gram-positive bacteria.
• • Penicillin is still a front-line antibiotic after more than 60 years of use.
• Penicillins are susceptible to breakdown by plasmid-encoded β-lactamases from enteric bacteria, neutralizing penicillins and causing allergic reactions in some patients.
• In such cases, cephalosporins from Cephalosporium acremonium are used.
• Cephalosporin is now commercially obtained from strains of Streptomyces spp.

Mycotoxins

• Mycotoxins are poisonous secondary metabolites produced by many filamentous fungi in the phylum Ascomycota.
• Mycotoxins are toxic to humans and animals depending on their toxicity levels and concentration.
• Mycotoxin problems result from improper storage of food and feed products, grains, and nuts.
• These compounds can be produced during preharvest and postharvest of crops.
• Different types of toxins include aflatoxin, ochratoxin, ergot, phallotoxins, and amatoxins (by Amanita spp.).

Major Mycotoxins and US/EU Limits

Ergot Toxin

• The ergot fungus, Claviceps purpurea, produces sclerotia fruiting bodies that develop in place of the grain in infected cereals and grasses; the sclerotia are termed ergots.
• Ergots develop alkaloids called ergot.
• Ergot toxin causes ergotism:
  • Convulsive ergotism affects the nervous system, causing violent convulsions.
  • Gangrenous ergotism causes blood capillaries to contract, leading to oxygen starvation and serious tissue damage.
• Ergot alkaloids have medical uses, such as relieving certain migraines and controlling hemorrhaging after childbirth.
• Ergotamine is lysergic acid, which can be chemically altered to produce the hallucinogenic drug LSD (lysergic acid diethylamide).

Ergotism

• Historically known as "holy fire" or "St. Anthony's fire."
• Outbreaks occurred in: France (1093), Russia (1926), Ireland (1929), France (1953), India (1958), and Ethiopia (1973).

Aflatoxin

• Aflatoxins are mainly produced by Aspergillus flavus and A. parasiticus, normally present in soil and various organic materials.
• A. flavus strains produce aflatoxins B1 (AFB1) and B2 (AFB2), while A. parasiticus strains produce AFB1, AFB2, G1 (AFG1), and G2 (AFG2).
• Stored grains and oil-rich crops, such as peanuts and cottonseed, are favorable for aflatoxin production.
• Aflatoxin-producing fungi grow on cereals (maize, rice, barley, oats, and sorghum), peanuts, ground nuts, pistachio nuts, almonds, walnuts, and cotton seeds.
• AFs have carcinogenic, teratogenic, hepatotoxic, mutagenic, and immunosuppressive effects, primarily affecting the liver.

Aflatoxins Contamination and Effects

• Aflatoxins absorbed from the gut pass to the liver, causing liver cancer.
• Milk can be contaminated with aflatoxin M1 (AFM1), detectable 12–24 hours after a cow consumes feed contaminated with AFB1; the concentration of AFM1 correlates to AFB1 levels in feedstuffs.
• AFM1 can also be detected in dairy products like cheese.
• Acute aflatoxicosis in humans is characterized by vomiting, abdominal pain, pulmonary and cerebral edema, coma, convulsions, and death.
• In animals, effects include gastrointestinal dysfunction, reduced reproduction, lowered milk and egg production, and anemia.

Sporidesmin

• Sporidesmin, found in spores of Pithomyces chartarum, is a saprotroph growing on dead leaf sheaths at the bases of pasture grasses.
• Common in New Zealand, Australia, and South Africa, causing facial eczema in sheep and cattle.
• Infected grazing cattle show blistering sores on exposed body parts (face, udders) and damage to internal organs.

Patulin

• Patulin is produced by many species of Penicillium and Aspergillus, including the common apple-rot fungus Penicillium expansum.
• P. expansum causes a soft, watery rot when spores enter the apple skin through wounds.
• Patulin can cause edema and hemorrhaging when ingested and is carcinogenic in experimental animals.
• It is unwise to eat any part of a rotted apple.

Ochratoxins

• Ochratoxins (OTA) were discovered in 1965 in South Africa; they are produced by Aspergillus ochraceus, Penicillium verrucosum, and other Penicillium species.
• The most important toxin is ochratoxin A.
• Found in agricultural commodities such as corn, wheat, barley, flour, coffee, rice, oats, rye, beans, peas, and mixed feeds; also present in wine, grape juice, and dried vine fruits.
• Can contaminate animal-derived products like meat and milk and can be found in human milk.
• Coffees and wines are major contributors to OTA intake.
• OTA is acutely nephrotoxic and hepatotoxic and is linked to Balkan Endemic Nephropathy (BEN), a chronic tubulointerstitial disease affecting south-eastern Europeans.

Roquefort Cheese and Sick Building Syndrome

• Roquefort cheese and other blue-veined cheeses are produced from goats’ milk and inoculated with the fungus Penicillium roqueforti.
• Roquefort cheese contains low levels of the mycotoxin roquefortine, but these levels are not considered hazardous.
• Sick building syndrome is associated with dampness and condensation, encouraging the growth of several mould fungi, including Stachybotrys chartarum.
• Stachybotrys chartarum produces the toxin trichothecene.
• This fungus was implicated in the death of thousands of horses in the Soviet Union in the 1930s when the animals were fed on contaminated hay.