🌍 Introduction — Fungi: The Hidden Giants

• Fungi are ancient, highly evolved organisms, sometimes forming the largest living networks on Earth — connected underground by hyphae called mycelium.
• They act as nature’s clean-up crew, decomposing dead organic matter and recycling nutrients.
• But they can be both allies and enemies.

🍄 Fungi — Friends or Foes?

🧫 Friends

Fungi produce many bioactive compounds:

• Penicillin G (antibiotic, NRPS)
• Cyclosporin A (immunosuppressant)
• Lovastatin (cholesterol-lowering, PKS)
• Ergotamine, Rapamycin, Bleomycin, Griseofulvin – various medical or industrial uses. 👉 NRPS = Non-Ribosomal Peptide Synthase; PKS = Polyketide Synthase.

🌾 Foes

• Plant pathogens, like Fusarium or Phytophthora infestans, cause diseases such as potato blight (responsible for the Irish famine, 1845–1849).
• Mycotoxins: toxic fungal metabolites contaminating food (e.g. aflatoxins, trichothecenes).

🧠 Fungal Compounds and Mycotoxins

• Fusarium species infect cereals like barley and wheat, causing Fusarium Head Blight (scab).
• Some Fusarium species produce toxic secondary metabolites, but others (like F. venenatum) are used to produce Quorn — a fungal-based food source.
• Around 50 bioactive compounds are known, but only ~15 identified.

🧪 OSMAC — “One Strain, Many Compounds”

Environmental changes like: 🌞 light, 🌡️ temperature, 💧 oxygen, 💨 pH, 🍽️ nutrients → can switch on/off secondary metabolite genes, leading to new compounds. This is key in discovering new drugs and toxins from fungi.

🧬 Fungal Molecular Biology

Researchers can:

• Knock out or overexpress genes
• Create GFP-tagged constructs
• Use long-read sequencing to link genes → metabolites Goal: understand and manipulate secondary metabolism.

📏 Genome Size Comparison

💡 Genome size doesn’t equal complexity! Amoebas just have many chromosome copies.

🔬 Gene Knockout Methods

1. Homologous recombination
2. RNA interference (RNAi)
3. CRISPR/Cas9
4. Virus-mediated knockouts
5. Mobile DNA elements / random mutagenesis

Each has pros and cons:

• Homolog recombination = precise but slow.
• CRISPR = fast and targeted.
• RNAi = temporary knockdown.

🧫 Example Study: Fusarium graminearum PKS9 Cluster

🧾 Paper Summary

Authors: Jens Laurids Sørensen et al. Journal: Environmental Microbiology (IF 5.476, 65 citations)

Findings:

• 3 novel Fusarielins (F, G, H) discovered.
• Developed a new ectopic expression system.
• Linked PKS9 gene cluster → Fusarielin production.
• Tested toxicity of new compounds.

🧬 Activating Silent Gene Clusters

Silent genes = no product under normal conditions. To activate:

• Change conditions (OSMAC)
• Add chromatin-modifying agents
• Modify transcription factors (like LaeA, AreA)
• Overexpress local regulators → e.g., PKS9 TF

🧫 Experimental Workflow

1. Identify gene cluster → NCBI & antiSMASH
2. Build mutants (overexpress TF or delete PKS9)
3. Verify expression by RT-PCR
4. Analyze metabolites via LC-MS, HPLC, NMR
5. Test toxicity using oCelloScope (real-time cell assay)

🧩 USER-Friendly Cloning (Main Technique)

“USER” = Uracil-Specific Excision Reagent

• Uses uracil-containing primers to create long overhangs for seamless DNA assembly.
• Enzymes:
  • Uracil DNA glycosylase
  • Endonuclease VIII
• Allows precise gene insertion/replacement.

Steps:

1. PCR amplify with uracil primers
2. Treat with enzymes → sticky ends
3. Insert into vector → transform into host
4. Verify integration via PCR or Southern blot.

🧬 Gene Overexpression Workflow

1. Clone TF (FSL7) into vector (USER cloning)
2. Transform E. coli → Agrobacterium → Fusarium
3. Select transformants using kanamycin / hygromycin
4. Verify correct insertion by PCR
5. Analyze metabolite output

⚗️ Analytical Results

• LC-MS showed new peaks → new metabolites
• NMR confirmed new Fusarielins (F, G, H)
• Only Fusarielin H found in wild type → PKS9 cluster confirmed responsible.

💬 Discussion

• PKS12 locus used as safe harbor for insertions.
• The new metabolites suggest F. graminearum can produce unexpected toxins under certain conditions.
• Demonstrates fungi’s latent biosynthetic potential — “sleeping factories” of chemistry!

🧫 Follow-Up: CRISPR/Cas9 & Protoplasts

Used for faster, more efficient fungal transformations and gene knockouts.

🧵 Fungal Biotechnology Spin-Off: Mycosealium

AAU project – Engineering nature’s raincoat!

🌍 Problem

Leather tanning uses PFAS and plastics → toxic, polluting, non-biodegradable. EU plans to ban PFAS by 2027.

💡 Solution

Create fungal leather that is:

• Naturally water-repellent (from fungal peptides)
• Plastic-free, biodegradable, vegan
• Made from agricultural waste (straw, corn stalks)

🧫 Method

Optimize growth conditions (temp, CO₂, pH, media) Enhance fungal strains’ water-repellent genes Vertical farming → scalable, sustainable process.

💰 Market & Business Model

• Leather market: USD 42M (2024) → 221M (2032) 📈
• Mycosealium: aims for 60 DKK/m² production cost (vs competitors’ 2000 DKK/m²)
• Applications: footwear, fashion, furniture, automotive.
• Competes with MycoWorks, Hermès, Adidas, etc.
• Advantage: inherent repellency & biodegradability.

🧱 Timeline

Grant: Danish Spin-Outs 2025 — funding one researcher salary.

🧩 Exercise

Final slide: “USER cloning exercise on Moodle — 20 minutes next lecture.”