Day 3 part 1

Applied Molecular Cellular Biology

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🌲🍄 Fungi: Theoretical Summary (Fun + Detailed!)

1. Fungi in Evolution 🧬🍄

In the evolutionary tree, fungi share a common ancestor with animals and are placed very high—sometimes humorously argued as the “top of evolution.”
They are among the largest organisms on Earth. Example: a Armillaria (“honey fungus”) individual in Canada spans ~3.5 miles, genetically identical across the entire distance.

Why fungi get so large

The underground network of fungal hyphae forms huge interconnected systems.
Fungi connect trees via mycorrhizal networks, transporting water, nutrients, and even pigments between trees.

2. Ecological Roles of Fungi 🌍

✔ Friends

Nutrient cycling: Fungi are expert decomposers, breaking down cellulose and lignin using strong extracellular enzymes.
Bioactive compounds: They produce important natural products:
- Penicillin (antibiotic)
- Statins (cholesterol-lowering drugs)
- Many other yet-undiscovered compounds

✔ Enemies

Mycotoxins: Toxic secondary metabolites harmful to humans and animals. Effects include liver damage, cancer, hormonal disruption, DNA damage, immune suppression.
Plant pathogens:
- Phytophthora infestans caused the Irish potato famine.
- Fusarium species infect major crops (wheat, maize, bananas).
Food contamination: Mycotoxins accumulate in grains, apples, juice, etc., because fungi grow inside plant tissue.

3. Penicillin and Chance 🍞🔬

Alexander Fleming’s discovery was extremely lucky:
- Only 4 species of Penicillium can produce penicillin.
- A spore from the “right” species had to randomly fall on his plate.
In practice, recreating the experiment succeeds only ~5–10% of the time.

Why so few species make penicillin?

Fungi only maintain antibiotic clusters if they need to fight bacteria in their environment.
Other species likely produce other antimicrobials we haven't discovered yet.

4. Statins: Fungal Chemical Weapons ❤️🍄

Fungi produce molecules that inhibit cholesterol synthesis (targeting the mevalonate pathway).
Fungi don’t use cholesterol; they use ergosterol, making this pathway a convenient target to attack competitors.
Humans repurposed these compounds as statin drugs.

5. Fungal Pathogens and Global Impact 🍌🌾🐴

Potato late blight

Caused widespread famine, shaped historical migration.

“Hole-in-the-head” disease in horses

Caused by fusarium toxins in contaminated oats.

Banana crisis 🍌

Cavendish bananas are genetically identical clones, making them vulnerable.
Fusarium oxysporum Tropical Race 4 infects and kills entire plantations.
Cavendish bananas may disappear within ~10 years and need replacement cultivars.

6. Fusarium: Friend and Foe 🦠

Nearly every plant species has a Fusarium species specialized to infect it.
Climate change allows Fusarium to thrive in new areas (e.g., maize in Denmark).
Farmers manage toxin levels by mixing contaminated harvests to get levels below allowed limits.

But humans also eat Fusarium!

Quorn is made from Fusarium venenatum.
Strain can produce toxins, but industrial fermentation conditions silence toxin production.

7. Food Safety and Mycotoxins 🍎🥛

Apples: Fungi grow inside, so juice production often grinds fungi into the product.
Blue cheese: Made from Penicillium roqueforti, capable of producing many toxins—but strain selection and growth conditions make commercial varieties safe.

8. OSMAC Principle 🔄

"One Strain, Many Compounds."

Changing culture conditions (light, pH, nitrogen, carbon source, temperature) alters fungal metabolite production dramatically.
Example: A single strain produces different pigments and compounds depending on the nitrogen source.
Important idea:
- Fungi respond strongly to environmental cues
- Secondary metabolites change accordingly

9. Environmental Factors Influencing Fungal Growth 🌞🌡️💧

Light: regulates germination timing; prevents spores from germinating in winter.
Temperature: most fungi grow only at 4–28°C.
- This protects humans from most fungal infections.
- Birds evolved 42°C body temperature possibly as antifungal protection.
Carbon source: glucose preferred; complex sources require enzyme secretion.
Interactions with microbes: fungi produce toxins to kill plant cells; bacteria/fungi competition drives compound evolution.
O₂/CO₂ levels: influence growth and metabolism.

10. Genome Sizes and Fungal Genomics 📏🧬

Approximate genome sizes discussed:

Plants > Humans > C. elegans > Fungi > Yeast > Bacteria
Some Fusarium species have 4 chromosomes, others have 15, causing complications in genetic manipulation due to gene duplication.

11. Genetic Manipulation Techniques 🧬🛠️

All methods rely ultimately on homologous recombination, even CRISPR.

A. Classical Gene Knockout via Homologous Recombination

Amplify flanking regions of target gene (PCR).
Clone flanks + selectable marker (e.g., hygromycin resistance gene) into a vector.
Transform fungus → recombination replaces gene with marker.

B. CRISPR-Cas9 ✂️

Guide RNA directs Cas9 to make a double-strand break.
Homologous recombination inserts new sequence.

Limitations:

Requires a compatible PAM site.
Patent restrictions for commercial use.
Companies modify Cas versions to extend patents.

C. RNA Interference (RNAi) 🔇

Introduce sequence complementary to mRNA → forms dsRNA → cell degrades it.
Good for gene knockdown, not complete knockout.
Cheap and fast.

D. Viral Vectors 🦠

Viruses inject DNA into cells to introduce new sequences.
Used less today due to biosafety concerns.

E. Mobile DNA Elements (Transposons) 🔀

“Jumping genes” integrate randomly.
Useful for discovering gene function via random mutagenesis.
Avoids GMO regulations since mutations occur “naturally.”

F. Random Mutagenesis (UV, chemicals, plasmids) ☢️

Generates large numbers of mutants.
Screen for desired phenotype.
Example: white Chlorella (chloroplast-free algae) used in food products.

12. GMO vs. Non-GMO Strategy 🧪⚖️

Companies often prefer random mutagenesis to avoid GMO classification.
CRISPR introduces precise one-gene changes but is regulated as GMO (and patent-restricted).
Random mutagenesis can make many unintended mutations, but remains legal and commercially preferred.

Quiz

Score: 0/30 (0%)

Q0. Why was Alexander Fleming’s discovery of penicillin considered extremely lucky?

Most Penicillium species can produce penicillin

Only a few Penicillium species have the gene cluster to produce penicillin

Penicillin production happens spontaneously on all agar plates

Bacterial contamination enhances penicillin production

Q1. What is the primary ecological reason fungi can grow over extremely large areas underground?

They photosynthesize efficiently

Their spores are transported by insects

They form extensive networks of hyphae that connect plant roots

They rely on bacterial symbiosis exclusively

Q2. What is the main mechanism fungi use to degrade plant materials like cellulose?

Intracellular digestion

Secretion of extracellular enzymes

Phagocytosis

Oxidative DNA cleavage

Q3. Why do fungi make good producers of statins?

Fungi require cholesterol for survival

Fungi naturally produce molecules that inhibit cholesterol synthesis

Statins strengthen fungal cell walls

Statins help fungi degrade cellulose

Q4. What is the biggest agricultural threat to the Cavendish banana?

Viral wilt

Insect predation

Fusarium oxysporum Tropical Race 4

Salt accumulation in soils

Q5. Which factor most strongly limits the number of fungi capable of infecting humans?

Lack of oxygen inside the body

Human immune system antibodies

Human body temperature of 37°C

High salinity of human tissues

Q6. Which Fusarium-related issue arises in Denmark due to changing climate conditions?

Increased banana imports

Higher incidence of maize infections and mycotoxin contamination

Reduced fungal biodiversity

Enhanced frost damage

Q7. In classical homologous recombination knockouts, what is the purpose of flanking regions?

To inhibit transcription of nearby genes

To guide recombination to the correct genomic locus

To enhance Cas9 binding

To increase PCR amplification efficiency

Q8. What key limitation may prevent the use of CRISPR-Cas9 on a specific gene?

Lack of sequencing data

Absence of a PAM site near the target

Cas9 cannot function in eukaryotes

DNA is too GC-rich

Q9. Which method produces gene knockdown rather than complete knockout?

CRISPR-Cas9

RNA interference

Classical homologous recombination

UV-induced mutagenesis

Q10. What is the purpose of using viral vectors in gene manipulation?

To increase fungal growth rate

To deliver DNA or RNA into cells efficiently

To suppress immune responses

To sterilize contaminated cultures

Q11. What characterizes random mutagenesis compared to other genetic modification techniques?

Precise insertion of a desired allele

Generation of unpredictable genome-wide mutations

Requirement of homologous arms

Complete silencing of essential genes

Q12. Why do fungal researchers apply the OSMAC principle?

To reduce fungal metabolism to minimal levels

To generate diverse metabolites by varying culture conditions

To enhance antibiotic resistance

To synchronize fungal cell cycles

Q13. What is one practical reason researchers isolate their own fungal strains?

Wild fungi grow faster

To avoid CRISPR-Cas9 patent restrictions for commercial applications

To eliminate contamination risks

To simplify culturing requirements

Q14. Which organism group has the largest genome size according to the lecture?

Fungi

C. elegans

Humans

Flowering plants

Q15. Fungi can transport nutrients between trees using underground mycelial networks.

True

False

Q16. Most Penicillium species naturally produce penicillin.

True

False

Q17. Statins work because fungi use cholesterol in their membranes.

True

False

Q18. Birds may maintain higher body temperatures partly to prevent fungal infections.

True

False

Q19. Fusarium species are typically generalists and infect many unrelated plants equally well.

True

False

Q20. Blue cheese contains a Penicillium species capable of producing many mycotoxins.

True

False

Q21. Random mutagenesis is often used commercially to avoid GMO regulations.

True

False

Q22. RNAi is capable of producing a complete and permanent gene knockout.

True

False

Q23. CRISPR-Cas9 requires a PAM site near the target sequence to function.

True

False

Q24. Classical homologous recombination uses flanking regions to guide DNA insertion.

True

False

Q25. The OSMAC principle states that different fungal strains always produce identical metabolite profiles.

True

False

Q26. Fungal contamination in crops can be reduced by mixing contaminated harvests with clean grain to fall below legal toxin thresholds.

True

False

Q27. Transposon-based mutagenesis introduces mutations in a targeted and predictable manner.

True

False

Q28. CRISPR editing is always legally unrestricted for commercial use.

True

False

Q29. Only a few fungi can grow efficiently at human body temperature.

True

False