Questions & explanations
1. Compare the orsellinic acid cluster to a housekeeping gene cluster (like for making amino acids).
The orsellinic acid cluster is a secondary metabolite cluster: its products are not essential for growth, but they help the fungus compete or defend itself. Housekeeping gene clusters, like the trp cluster for tryptophan, are essential for survival and are expressed all the time. Secondary metabolite clusters are often silent under lab conditions and need special triggers (like stress or a specific nutrient) to turn on. Also, secondary metabolite genes are usually physically clustered on the chromosome, while housekeeping genes can be scattered. The orsellinic acid cluster is regulated by a pathway-specific transcription factor, whereas housekeeping genes are controlled by general regulators.
2. Why is the orsellinic acid gene cluster a good model for studying fungal secondary metabolism?
It is small (only a few genes) and produces a simple, well-known compound, making it easy to manipulate. The cluster is present in a genetically tractable species, Aspergillus nidulans, which has many tools for gene deletion and expression. Also, orsellinic acid is a precursor to many other important fungal polyketides, so understanding its cluster helps decipher more complex clusters. The cluster's regulation by both cluster-specific and global factors makes it a good system to study how fungi control secondary metabolism. Finally, its products have biological activities, such as antimicrobial properties, that are relevant for drug discovery.
3. How can you tell whether a yeast strain is [PSI+] or [psi-] (normal)?
You can use a reporter gene with a premature stop codon, like the ADE1 gene that makes a red pigment when broken. In [psi-] cells, the stop codon stops translation, so the ADE1 protein is not made, and colonies are red because of a buildup of a red intermediate. In [PSI+] cells, readthrough of the stop codon produces functional ADE1, so colonies are white or pink. Another test is growth on medium lacking adenine: [PSI+] cells can grow because they make enough Ade1 protein, while [psi-] cells cannot. The [PSI+] state is also curable by growing cells on low concentrations of guanidine hydrochloride, which dissolves the prion aggregates.
4. Compare how secondary metabolite genes are regulated in a fungus versus a bacterium.
In both fungi and bacteria, secondary metabolite genes are often grouped in clusters and controlled by specific regulatory proteins. However, fungal regulation is more complex because fungi have multiple layers of control, including chromatin remodeling and pathway-specific transcription factors. Bacteria often use simpler systems like two-component regulators. Additionally, fungal secondary metabolism is frequently linked to development, such as spore formation, while bacterial production is more often triggered by nutrient stress. Both can be silenced under lab conditions, requiring special methods to activate them.
5. How can you test whether two genes in the RAD52 group work in the same pathway or in parallel pathways?
You can do an epistasis test: make a double mutant lacking both genes and compare its sensitivity to DNA damage (e.g., radiation) to each single mutant. If the double mutant is no more sensitive than the most sensitive single mutant, the genes likely work in the same pathway (epistasis). If the double mutant is much more sensitive (additive or synergistic), they work in parallel pathways. For example, rad52 rad51 double mutant has the same sensitivity as rad52 alone, showing they act in the same HR pathway. In contrast, rad52 yku70 double mutant is more sensitive, because they act in HR and NHEJ separately.
6. How does the scale-up of a fungal bioreactor from lab to industrial size affect oxygen transfer?
As bioreactor size increases, the surface-area-to-volume ratio decreases, making oxygen transfer less efficient. Larger reactors require higher agitation and aeration rates to maintain adequate dissolved oxygen for fungal growth. However, increased agitation can cause shear damage to fungal hyphae. Engineers use dimensionless parameters like the volumetric oxygen transfer coefficient (kLa) to predict and maintain oxygen supply during scale-up. They also consider mixing time and power input per volume to ensure uniform conditions. In practice, scale-up often requires iterative testing to optimize conditions.
7. Give an example of a fungal product that requires a specific bioreactor design to avoid product inhibition.
Citric acid production by Aspergillus niger is often done in stirred-tank bioreactors with controlled pH and aeration. However, high citric acid concentrations can inhibit fungal growth. To avoid this, fed-batch or continuous fermentation is used, where substrate is added gradually to keep acid levels low. Some designs incorporate in-situ product removal, like membrane bioreactors, that separate citric acid from the broth continuously. This maintains a low concentration in the reactor, boosting yield. Another example is penicillin production, where glucose is fed slowly to prevent catabolite repression.
8. How does the RAD52 epistasis group differ from the non-homologous end joining (NHEJ) repair pathway?
Homologous recombination (HR) uses a sister chromatid or homologous chromosome as a template to copy the missing information, so it is accurate but only works when a template is available (usually after DNA replication). Non-homologous end joining (NHEJ) simply glues the broken ends together without a template, often causing small deletions or insertions. In yeast, the RAD52 group is the main HR pathway, while NHEJ uses different genes like YKU70 and YKU80. Cells lacking RAD52 are deficient in HR but can still use NHEJ, though less accurately. HR is preferred in yeast when a template is present.
9. How can you test whether a fungal strain produces mycotoxins?
Mycotoxin testing involves growing the fungus under conditions that promote toxin production, then analyzing the culture extract. Common methods include high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA) to detect specific toxins. For example, aflatoxins are detected by HPLC with fluorescence detection. The strain is tested on different media and at various temperatures to see if toxins are produced. If no toxins are detected under relevant conditions, the strain is considered non-toxigenic. Regular monitoring is needed because toxin production can vary.
10. Compare the effects of a loss-of-function frq mutation versus a mutation that makes FRQ protein more stable.
A loss-of-function frq mutation (like frq knockout) destroys the circadian rhythm — the fungus shows no regular 24-hour pattern in growth or spore formation. In contrast, a mutation that makes FRQ more stable (e.g., by removing phosphorylation sites) lengthens the period because FRQ stays active longer, delaying the feedback loop. For example, a stable FRQ mutant might have a 30-hour rhythm instead of 22 hours. Both types show that the clock's period is controlled by FRQ's stability, not just its presence. The loss-of-function proves FRQ is required; the stable mutant shows how timing is set.
11. How does light reset the Neurospora circadian clock through the frq gene?
Light activates a protein called WC-1 and WC-2 that bind to the frq gene's promoter and turn on frq transcription quickly. This sudden increase in FRQ protein shifts the clock's phase — for example, if light is given at night, it makes the clock think it is morning. The amount of FRQ produced depends on the time of day: a light pulse in the early night causes a big phase delay, while in late night it causes a phase advance. This resetting allows the fungus to synchronize its internal clock with the day-night cycle. The response is fast because frq mRNA rises within minutes of light exposure.
12. What is the orsellinic acid gene cluster in Aspergillus and what does it produce?
The orsellinic acid gene cluster is a group of genes in the fungus Aspergillus nidulans that work together to make a chemical called orsellinic acid. This cluster includes a polyketide synthase (PKS) gene that builds the carbon backbone, plus genes that modify it. Orsellinic acid is a small aromatic compound that is a building block for many other fungal natural products, such as antibiotics and toxins. The cluster is regulated by a specific transcription factor that turns on all the genes at the same time. Studying this cluster helps scientists understand how fungi produce useful chemicals.