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Day 3 part 2

Applied Molecular Cellular Biology
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🌟 Fun & Full Master’s-Level Summary

Polyketide Synthases, Gene Clusters, Transformation Systems & Fungal Biotechnology

(Theory Only β€” methods explained conceptually)

🧬 1. Background: Fusarium, Secondary Metabolites, and PKS Gene Clusters

Fusarium graminearum is a red-pigmented fungus that produces many secondary metabolites, including toxins. These are often synthesized by polyketide synthases (PKSs) β€” some of the largest multi-modular enzymes known.

At the start of the study:

  • Fusarium had ~50 potential toxins,
  • 12 PKS genes,
  • Only 5 PKS clusters were previously linked to their products β€” meaning 7 PKS clusters were completely unknown.

πŸ‘‰ Goal: Identify what the gene PKS9 produces, and develop a system to activate its cluster.

🎨 Why does Fusarium produce pigments?

Pigments serve two purposes:

  • UV protection (like fungal sunscreen)
  • Warning coloration (signals toxicity to animals)

🧬 2. Identifying Gene Clusters Using Genome Databases

With the genome sequence available in NCBI, the researchers used:

πŸ§ͺ antiSMASH

A tool that scans the fungal genome β†’ predicts biosynthetic gene clusters (BGCs) such as PKS, NRPS, terpene clusters.

For PKS9, the cluster contained:**

  • A PKS core gene
  • Accessory enzymes (lipase, epimerase, oxygenase, reductase, MFS transporter)
  • A local transcription factor (TF)

πŸ‘‰ Local TFs often have zinc fingers or leucine zippers β†’ these protein motifs indicate DNA-binding activity.

πŸ“ˆ 3. Microarray Data: Proving the Genes Belong Together

To confirm that the predicted genes form a functional cluster, researchers checked microarray/ expression data.

Logic: If multiple genes respond together (up/downregulated by the same stimulus), they likely belong to the same cluster.

In this case:

  • Genes in the PKS9 cluster were co-regulated during infection, supporting their clustering.

Limitation: Microarray chips only exist for model organisms, not rare fungi β†’ so this method works only when chips exist.

πŸ”§ 4. Strategy: How to Activate a Silent PKS Cluster

Three theoretical options:

  1. Put constitutive promoters in front of all cluster genes – Too time-consuming.
  2. Clone and insert the entire gene cluster repeatedly – Impossible due to size (~20–30 kb).
  3. Overexpress the cluster’s transcription factor βœ”οΈ – Easiest and most effective.

πŸ‘‰ They chose option 3.

🧬 5. Gene Engineering Concept: The Double-Marker System

The fungus naturally has a red pigment gene (PKS12). The researchers used this gene locus as an β€œinsertion slot.”

When PKS12 is disrupted β†’ fungus turns white. So white colonies = successful integration.

The double-marker logic:

  • Loss of red pigment β†’ correct genomic insertion
  • Presence of hygromycin resistance β†’ transformant contains plasmid cassette

πŸ”§ 6. Concept: USER Cloning (Uracil-Specific Excision Reagent)

A method for fast, modular assembly of DNA fragments.

Theoretical basis:

  • Primers contain a uracil base β†’ unusual in DNA
  • A USER enzyme mix removes uracil β†’ forms predictable single-stranded overhangs
  • These overhangs guide the DNA fragments together in one step

This bypasses:

  • Multiple digestions
  • Multi-day ligation strategies
  • Assembly problems with large constructs

For this study, USER cloning allowed:

  • Insertion of two homology arms
  • Addition of a constitutive promoter (from Aspergillus)
  • Overexpression of the cluster TF

🌿 7. Concept: Agrobacterium-Mediated Transformation (ATMT)

Agrobacterium tumefaciens naturally inserts DNA into plant genomes. Scientists repurpose this system to transform fungi.

Theory of how it works:

  • Agrobacterium recognizes a host β†’ builds a transfer channel β†’ injects T-DNA
  • The T-DNA integrates semi-randomly into host nuclear DNA

Advantages of ATMT:

  • Works with fungi that are hard to electroporate
  • Favors single-copy insertions
  • High efficiency

Disadvantages (important theory!):

  • Unpredictable insertion numbers: ~1/3 single insertion, 1/3 double, 1/3 messy
  • Off-target T-DNA can disrupt unrelated genes

πŸ‘‰ Even when PCR looks correct, hidden insertions can still exist.

πŸ§ͺ 8. Theory: How PCR Confirms Correct Insertion

They used different primer combinations to test:

  • Presence of insert
  • Orientation
  • Correct genomic location

The theoretical logic:

  • Primers flanking the entire cassette β†’ large band = cassette inserted
  • Primers inside cassette + outside homology arm β†’ confirms correct site
  • Reverse combinations check orientation

But PCR cannot detect:

  • Hidden off-target insertions
  • Extra copies elsewhere in the genome

This is a known ATMT limitation.

πŸ’₯ 9. Secondary Metabolite Analysis (HPLC-MS + Mutants)

The team compared:

  • Wild-type (red)
  • TF-overexpression mutant (white)
  • PKS9 knockout mutant

Using HPLC-MS, they observed:

  • Overexpression β†’ new peaks appear
  • Knockout β†’ these peaks disappear

This proves:

  • The PKS9 cluster produces the compounds
  • The TF controls the cluster

The compounds were purified and structures solved using NMR spectroscopy β†’ resulting in three novel polyketides (Fusarium F, G, H).

πŸ§ͺ 10. RT-PCR for Expression Verification

Theoretical basis:

  • Extract RNA
  • Reverse-transcribe β†’ cDNA
  • Perform PCR with gene-specific primers
  • Compare cluster gene expression across strains

Housekeeping genes (TEF1, Ξ²-tubulin) used to normalize RNA amounts.

Findings:

  • Overexpression strain β†’ all cluster genes upregulated
  • Knockout β†’ PKS9 not expressed
  • Wild-type β†’ baseline expression

This confirms functional activation of the silent cluster.

🧫 11. Cytotoxicity: Experimental Rationale

The purified compounds were tested on human cell lines:

  • Caco-2 (intestinal)
  • HT29 (colorectal cancer)

Using impedance-based real-time monitoring (xCELLigence concept), the compounds slowed cell growth.

Conclusion: β†’ The PKS9 polyketides are mycotoxins.

Later findings: β†’ One compound stimulates breast cancer cell proliferation β†’ potential carcinogen.

🧡 12. Broader Fungal Biotechnology Concepts

The lecture ends with examples of fungi in industry:

  • Brewing (yeast fermentation)
  • Enzyme production (Novozymes)
  • Citric acid (Aspergillus)
  • Biobased materials (fungal acoustic panels, mycelium composites)
  • Fungal leather (water-repellent materials)
  • Redox batteries using fungal pigments
  • Asphalt bound by fungal mycelium

The key theoretical insights:

  • Fungi are biomanufacturing platforms
  • Gene clusters enable diverse chemical outputs
  • Genome engineering β†’ unlocks new industrial applications

Quiz

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