Comprehensive guide to extracting and expressing fungal secondary metabolites: Aspergillus fumigatus as a case study

Abstract

Fungal secondary metabolites (SM) have captured the interest of natural product researchers in academia and industry for decades. In recent years, the high re-discovery rate of previously characterized metabolites is making it increasingly difficult to uncover novel compounds. Additionally, a vast majority of fungal SMs reside in genetically intractable fungi or are silent in normal laboratory conditions in genetically tractable fungi. The fungal natural products community has broadly overcome these barriers by altering the physical growth conditions of the fungus and heterologous/homologous expression of biosynthetic gene cluster regulators or proteins. The protocols described here summarize vital methodologies needed when researching secondary metabolite production in fungi. We have also summarized the growth conditions, genetic backgrounds, and extraction protocols for every published secondary metabolite in Aspergillus fumigatus, enabling readers to easily replicate the production of previously characterized SMs. Readers will also be equipped with the tools for developing their own strategy for expressing and extracting SMs from their given fungus or suitable heterologous model system.

INTRODUCTION:

Beginning with the discovery of penicillin in 1929 by Alexander Fleming, widespread attention has been given to mining fungal metabolites for pharmaceutical applications (Bennett and Chung, 2001; Keller et al., 2005). Secondary metabolites (SM), which are found in fungi, bacteria, and plants, can be broadly defined as organic compounds not directly involved in the reproduction, growth, or development of an organism (Keller et al., 2005). They often provide important beneficial fitness adaptations that are highly tuned to an organism’s ecological niche (Macheleidt et al., 2016). SMs can provide fungi with antimicrobial defenses, protection from UV damage, in addition to playing important roles in many development structures (Keller, 2019). Out of the 195,000 SMs currently publicly available in the dictionary of natural products, roughly 10% are produced by fungi (CRC Press, 2021). Out of the SMs with known biological activity, fungi are among the largest sources for antimicrobial, antineoplastic, antiviral, and anti-inflammatory agents (Ntie-Kang and Svozil, 2020). The potential for fungal SMs use in medicine, compounded with the estimated 2.2–3.8 million fungal species that exists, only 120,000 which have been characterized, shows strong promise for fungal SM research into the future (Hawksworth and Lücking, 2017).

The methods described in this publication detail the entire workflow of growing, transforming, and extracting secondary metabolites from Aspergillus fumigatus. From start to finish the user will go from inoculating from a glycerol stock to extracting a metabolic crude sample. While focused on A. fumigatus, the methods can be easily adapted to other fungi with amendments based on specific growth conditions. Additionally, while it is recommended to use alternative molecular biology methods for the budding-yeast subphylum, the metabolic extraction protocols will be identical. Included are comments, troubleshooting, and warnings for difficult steps in the protocols, in addition to overall guides for common workflows in SM research. Furthermore, we have created a summary methods table for every characterized SM in A. fumigatus (see Table 1 and Sup. Table 1). This should allow readers to quickly reference other laboratories’ successful growth and extraction conditions for previously published SMs. This species was chosen as representing, along with A. nidulans (Caesar et al., 2020; Romsdahl and Wang, 2019), the most thoroughly studied filamentous fungus with regard to SM characterization and ecological function of these metabolites (Boysen et al., 2021; Raffa and Keller, 2019; Wang et al., 2021b).

Table 1:

Growth, conditions, and extraction methods for secondary metabolites in A. fumigatus

Metabolite	Strain	Media 2	Growth Conditions	Method of extr.	References
Dihydroxyna-phthalene	B-5233 (patient isolate)	AMM (see Sup. Table 1 for recipe)	AMM at 37 C for 5 days.	MeOH extraction followed by 1:1 CH2Cl2/MeOH extraction.	Tsai et al., 1999; Chianga et al., 2011; Chamilos and Carvalho, 2012
Endocrocin	Cea17	GMM	Point inoculate GMM with 1 × 104 spores/ml, 29 C, 10 days.	Homogenize and EtOAc extraction.	Lim et al., 2012
Ferricrocin	ATCC 46645	GMM (Liquid)	Inoculate 108 conidia into 250 mL of GMM, 200 rpm, 37 C, 4 days.	Passed through Amberlite XAD-16 resin (see Sup. Table 1 for full details).	Diekmann and Krezdorn, 1975; Konetschny-Rapp et al., 1988; Oberegger et al., 2001; Blatzer et al., 2011
Fumagillin	KACC 41191	Czapek-Dox with 5 g/L of yeast extract (CYA)	CYA, 3 days at 28 C. Move over 6 mm diameter piece to fresh CYA plate. Incubate at 28 C, 3 days. Again move a 6 mm dimater piece to fresh CYA plate, 12 days.	EtOAc extraction.	Lin et al., 2013; Kang et al., 2013
Fumigaclavine	Af293	Malt extract agar	Inoculate malt extract agar with 2.5 × 105 spores/ml from a spore suspension. Grow for 2–4 weeks.	MeOH extraction.	Panaccione and Arnold, 2017
Fumigermin	ATCC 46645	AMM (Liquid) for A. fumigatus and M79 (Liquid) for S. rapamycinicus	Grow fumigatus pre-culture ~16 hrs, filter with miracloth. Frow S. rapamycinicus preculture on M79 medium. Place the mycelium in fresh AMM and incoluate with 1/20 volume of streptomycete culture.	EtOAc extraction.	Cristina Stroe et al., 2020; Langfelder et al., 1998
Fumihopaside A and B	CEA17	PDB (Liquid)	Grow in 100mL of PDB at 37 C for 4 days.	Mycelial isolation and filtering followed by EtOAc extraction (see Sup. Table 1 for full details).	Ma et al., 2019
Fumiquinazoline	GA-L7	potato dextrose broth + valproic acid (Liquid; 0.05% v/v; valproic acid)	Incubate in the broth for 2 days at 28 C, 200 rpm. Take 2% of the seed culture and inoculate 25 L. Grow at 0.5 vvm, 0.33 bar, 100 rpm, and 28C for 12 days.	10% MeOH added to broth, followed by DCM extraction.	Magotra et al., 2017; Ames et al., 2011; McCloud, 2010
Fumisoquin	Af293	GMM (Liquid)	Inoculate 1.0 × 106 spores/ml in 1 L of GMM, grow at 37 C, 220 rpm, 4 days.	MeOH extraction.	Baccile et al., 2016
Fumitremorgin	BM939	Complete medium	Grow at 28 C for 3–5 days.	EtOAc extraction.	Kato et al., 2009
Gliotoxin	ATCC 26933	Czapek-Dox	Grow at 37 C for 3 days.	Chloroform extraction.	Reeves et al., 2004; Dolan et al., 2017
Helvolic Acid	Marine isolate from authors	PDA and liquid medium (see Sup. Table 1 for recipe)	Grow at 28 C for 5 days. Transfer plugs to 300 mL of the liquid medium. Grow 30 days at RT.	EtOAc extraction, myeclium extracted with acetone followed by EtOAc extraction.	Kong et al., 2018
Hexadehydro-astechrome	Af293 & Cea17	GMM (Liquid)	Inoculate 1.0×10^6 spores/mL of A. fumigatus, grow for 3 days, 25 C, 250 rpm.	10% MeoH:Ethyl Acetate extraction.	Yin et al., 2013
Neosartoricin	Heterologous expression in A. nidulans	GMM (Liquid) with 0.5 μM pyridoxine HCl	Add 105 spores per 10 cm (diameter) of plate, grow in the dark for two days, 200 rpm.	EtOAac)/MeOH/ AcOH (89:10:1) extraction.	Yin et al., 2013
Nidulanin A	Soil isolate from authors	GMM	Innoculate 107 spores per 10 cm (diameter) of plate and grow for 5 days in the dark.	MeOH extraction followed by 1:1 CH2Cl2/MeOH extraction.	Oakley et al., 2017
Pseurotin	Af293	Xylose minimal medium (Liquid; 4% xylose)	Pre-culture on MEA medium. Move small agar plugs onto XMM and continue growing at 30 C for 72 hrs, 150 rpm.	EtOAc extraction twice with 6 M HCl added between extractions.	Yu et al., 2018; Abdelwahed et al., 2020
Pyomelanin	CEA17	AMM (Liquid)	Innoculate 200 mL of AMM with 107 conidia. Grow at 37 C, 200 RPM. After 20 hours add 10 mM L-tyrosine. Grow for 55 hours.	Filter the liquid culture through mirocloth and save the flowthrough.	Schmaler-Ripcke et al., 2009; Keller et al., 2011
Pyripyropene A	FO-1289	Custom seed medium and production medium (see Sup. Table 1 for recipe)	Fermentation growth: Three rounds of growth and transfering at 27C (see Sup. Table 1 for full details).	EtOAc extraction.	Tomoda et al., 1994
Rubrofusarin B 1	Isolote from authors	PDA and PDB	Three rounds of growth transfering between PDA and PDB (see Sup. Table 1 for full details).	EtOAc extraction.	Hua et al., 2020
Triacetylfusa-rinine C	Tü 142	Low-iron medium (see Sup. Table 1 for recipe)	Grow at 27 C until extensive conidiation.	Passed through Amberlite XAD-16 resin (see Sup. Table 1 for full details).	Diekmann and Krezdorn, 1975; Blatzer et al., 2011; Oberegger et al., 2001; Konetschny-Rapp et al., 1988
Trypacidin	Af293	GMM	Inoculate 5 μL of 2 × 106 spores/ml spore suspension. Grow at 29 C, 120 hrs, dark.	EtOAc extraction (see Sup. Table 1 for full details).	Throckmorton et al., 2016
Xanthocillin	Af293	GMM	Inoculate 10 mL of top agar with 1×106 spores and pour on bot agar. Incubate 37 C, 5 days.	Ethyl acetate:methanol (9:1) extraction.	Raffa et al., 2021

¹Rubrofusarin B, Alternariol 9-O-methyl ether, Fonsecinone, and Asperpyrone were all grown and collected the same way (Hua et al., 2020).

²Glucose minimal medium (GMM), Aspergillus minimal medium (AMM), Potato Dextrose Agar/Broth (PDA/PDB), Condensed information for growing and extracting every characterized secondary metabolite in A. fumigatus. This table does not represent a comprehensive list of every possible way to culture said SMs. See Supplementary Table 1 for a more detailed summary of the growth and extraction steps, genetic backgrounds, and other pertinent data.

Basic Protocols 1 and 2 will explain creating and activating strains from spore suspension glycerol stocks. These are core techniques for working with sporulating filamentous fungi. Support Protocol 1 explains how to create glycerol stocks in non-sporulating filamentous fungi. Basic Protocol 3 and Alternate Protocols 1–3 provide four commonly used metabolic extraction techniques. The protocols detail extraction from solid media or liquid media with organic solvents that are miscible/immiscible with H₂O, and high-volume methods. Basic Protocols 4-6 are core methodologies to conducting genetic transformations in filamentous fungi. Protocol 4 details extracting genomic DNA, while protocol 5 and Alternate Protocol 4 explain the methods for creating DNA constructs. Basic Protocol 6 explains the transformation process. Lastly, Basic Protocol 7 details co-culturing methods with bacteria and fungi. For recipes see the Reagents and Solutions section, and for tips on troubleshooting see the italicized text within the protocols, in addition to the Troubleshooting table at the end of the publication.

CAUTION: To prevent contamination, always work under a sterile biosafety cabinet when working with fungal cultures. Always use sterile equipment, and parafilm your plates to limit accidental contamination. For more sterile best practices see the “I am having frequent contamination issues” section under Troubleshooting.

CAUTION: Aspergillus fumigatus, in addition to other human pathogenic fungi, is a Biosafety Level 2 pathogen. Make sure to follow the proper guidelines at your institution if you are using or handling any BSL 2 pathogens.

STRATEGIC PLANNING (optional):

When designing experiments for secondary metabolism studies, much care needs to be taken into choosing the environmental conditions and strains. SM metabolite expression can be strongly influenced by slight changes in medium volume, light, temperature, pH, and nutrients (Keller, 2019). Additionally, different strains of the same species will not always produce the same SMs. (Drott et al., 2021)

While documenting conditions for SM discovery in non-A. fumigatus strains goes beyond the scope of this methods paper, most media and growth conditions broadly work for filamentous fungi. Check the literature on your species for commonly used growth conditions and media that work well for your fungus.

There are two commonly seen strategies to studying secondary metabolites (SM) in fungi, plants, or bacteria (see Figure 1). The first involves a chemical-based approach, where the researcher identifies the full spectrum of metabolites produced to discover targets of interest. Depending on if the researcher is interested in the function, there may be a screening process to identify fractions from a metabolic extraction that exhibit a given biological activity. Once the fractions of interest have been identified, the compounds must be separated and purified, typically through a series of high-performance liquid chromatography runs. After purification, the compound is structurally identified via a combination of mass spec, UV, crystallography, or NMR. This approach was how many of the first natural product discoveries were made in what is sometimes coined as “The Golden Age” of natural product discovery (Konetschny-Rapp et al., 1988; Katz and Baltz, 2016). Today, the strategy is still frequently utilized by more chemistry-oriented labs, where the compounds themselves are the primary focus of research. It is also commonly used on organisms that lack publicly available sequenced genomes and/or are difficult to genetically manipulate. For excellent reviews on fungal identification and compound purification see (Sticher, 2008; Bucar et al., 2013; Raja et al., 2017).

Figure 1:

Broad outline of the two common secondary metabolite discovery pipelines. Created with BioRender.com.

The second major approach is driven by genome mining and targeted biosynthetic gene cluster activation. This is dependent on genome sequences and gene annotations, both of which are rapidly growing in number. Typically, a biosynthetic gene cluster (BGC) will be identified through existing genome mining software (Ren et al., 2020; Chavali and Rhee, 2018). To tie a metabolite to the given BGC, genetic manipulations are required typically through gene knockouts, overexpression of regulatory factors and/or manipulation of the epigenome (Keller, 2019). Mutant strains are chemically assessed as above. As more fungal genomes become publicly available, this genome mining approach can help inform hypotheses on chemical structure and function before any chemical data is obtained thus allowing for targeted experiments that are more likely to give rise to exciting novel discoveries (Robey et al., 2020). For a summary of the known biosynthetic gene clusters and pathway specific transcription factors in A. nidulans and A. fumigatus see (Wang et al., 2021b).

BASIC PROTOCOL 1

Basic protocol title:

Making glycerol stocks from spore suspensions

Introductory paragraph:

Glycerol stocks are commonly used in microbiology labs to safely store strains for long periods of time. Unlike bacteria and budding yeast which are commonly grown in liquid medium for glycerol stock creation, filamentous fungal conidia are collected directly from agar plates. Our laboratory commonly uses GMM for both activation and creation of glycerol stocks, however, any medium that your fungal strain grows on will suffice.

If making glycerol stocks for unicellular fungi such as yeast, the procedure is identical to making glycerol stocks with bacteria. Simply substitute the growth medium for an alternative like YPD. For more details see Creation of bacterial glycerol stocks in Basic Protocol 7 or Basic Protocol 3 in the methods paper by (Cosetta and Wolfe, 2020).

Common glycerol concentrations in mycology labs range from 33% and 50%. Any quantity in this range will work as well for long-term spore preservation. Which you use depends on how viscous you desire your glycerol stock to be.

CAUTION: Take extreme care whenever creating or opening glycerol stocks. It is very easy to have accidental contamination from spores in the air if not working in a very clean environment. Contaminated glycerol stocks can cause serious downstream issues and can be very difficult to detect. We highly recommend using only sterile filter tips and doing all work in a biosafety cabinet equipped with UV light. When activating multiple strains, or creating multiple glycerol stocks, to ensure cross contamination does not occur it is best to turn on the UV light and wait 15 min between working with each strain.

Materials:

Plate with growing fungus

Sterilized 50% glycerol (V/V) (e.g., Fisher, cat. no. 327255000)

Cryogenic tube (e.g., VWR, cat. no. 89004–320)

Fine point permanent marker

Biological safety hood (e.g., Baker SterilGARD III Advance SG403, or equivalent)

Cell spreader (e.g., VWR, cat. no. 76207–748)

1000 μL Filter tips (e.g., Phenix, cat. no. TS-059BR)

−80°C Storage space

Protocol steps with step annotations:

1. Clearly label a cryogenic tube with the name of the fungus using a fine point permanent marker.

2. Take the plate of your growing fungus which has begun sporulation (typically 4–5 days old but can vary with fungus) and place into sanitized biological safety hood.

   Typically, this is GMM but any medium that your fungus sporulates on will work.

3. Add 1–2 mL of sterile glycerol directly onto the plate.

4. Rub the spores with the cell spreader until they are suspended in the glycerol solution.

   Be cautious to not touch any surface with the cell spreader when removing from package.

   It is helpful to use cell spreaders that have grooves built into them. This makes it easier to disperse the spores from the fungus. Additionally, take care to not use excessive force. If one presses too hard or stirs too vigorously, the mycelium can be broken up and accidentally suspended in glycerol with the spores.

5. Using a P1000 with filter tips, put 1 mL of spores into the labeled cryotube and close the lid.

6. Repeat for as many strains as you need to store long term.

   If concerned about contamination, this is where you would turn on the UV light and wait at least 15 min before starting each subsequent strain.

7. Invert the closed cryotubes several times to make sure spores are properly resuspended and place into storage box.

8. Store in −80°C freezer.

   Usually during the first 24 h, cell numbers will decrease by 25%. Please recount the cells after the incubation to make sure you use the correct amount in the following step.

SUPPORT PROTOCOL 1

Support protocol title:

Creating glycerol stocks from non-sporulating filamentous fungi

Introductory paragraph:

Not every fungal strain will produce spores in laboratory conditions. Additionally, certain mutations prevent sporulation from occurring. When such is the case, it is still important to create glycerol stocks for long term storage. The protocol is very similar, except agar plugs imbedded with mycelia used are in replacement of spore suspensions.

Additional Materials:

Sterile razor blade or equivalent

Protocol steps with step annotations:

1. Grow up the culture until there is visible mycelial growth.

2. Add 600 μL of glycerol to a labeled 2 mL cryotube.

3. Using a sterile razor blade cut out small agar circles or squares that are imbedded with mycelia.

   Cut small enough to insert ~4–5 small portions per 2 mL cryotube.

4. When ready to grow, thaw and place in liquid medium.

BASIC PROTOCOL 2

Basic protocol title:

Activating spore suspension glycerol stocks

Introductory paragraph:

Assuming the strain is being stored in a glycerol stock spore suspension, this is the first important step in extracting secondary metabolites from Aspergillus fumigatus and other sporulating fungi. As with creating glycerol stocks, proper sterile technique is essential to prevent accidental contamination. The medium that is used for the initial activation can be substituted if your strain will grow on it. If applicable, using selective media can help ensure only your desired strain grows but may delay the sporulation time.

Materials:

Glycerol stocks of fungal strains

Ice

GMM solid medium (or equivalent, see recipe in Reagents and Solutions)

Sterile Tween water (see recipe in Reagents and Solutions)

70 % EtOH (V/V) (e.g., Decon Laboratories, cat. no. 64–17-5)

200 μl filter pipette tips (e.g., Fisherbrand SureOne Pipet Tips, cat. no. 02–707-420)

Incubation Chamber or room (e.g., MaxQ 4000, cat. no. SHKE4000)

Biological safety hood (e.g., Baker SterilGARD® III Advance SG403, or equivalent)

Cell spreader (e.g., VWR, cat. no. 76207–748)

Motorized pipet filler (e.g., Thermo Scientific S1 Pipet Fillers, cat. no. 14–387-165)

25 mL Disposable serological pipet (e.g., Dot Scientific, cat. no. 457225)

50 mL Falcon conical tube (e.g., Corning, cat. no. 14–432-22)

1.5 mL microcentrifuge tube (e.g., VWR, cat. no. 111564)

Hemocytometer (e.g., Bright-Line, SKU Z359629–1EA)

Optical microscope (e.g., Nikon Eclipse E200)

Tally Counter (e.g., Fisherbrand Hand Tally Counters, cat. no. 07–905)

Kimwipes (e.g., Kimberly-Clark, cat. no. 34155)

4°C refrigerator

Protocol steps with step annotations:

Growing from the glycerol stock

• 1Remove the glycerol stocks from the −80°C freezer and thaw on ice for roughly 15–30 min.
• 2Using a sterile 200 μl filter pipette tip, take 20 μl of the thawed glycerol stock and create three lines on the left half of the GMM plate. Repeat this again on the right half to make six total streaks.

  Slowly and evenly dispense the 20 μl while making the streaks. This method ensures maximum spore growth with minimal stock being used. An alternative method is to chip a small glycerol fragment from the stock with a sterile toothpick and place directly onto medium.

• 3Grow the strain at 37°C (or optimal temp for your strain) until sporulation has occurred.
• 4Place the grown fungal plate into a biosafety cabinet.
• 5Add 15–20 mL of sterile tween water directly onto the agar plate.
• 6Rub the spores with a sterile cell spreader until they are fully suspended in tween water.

  Same recommendations apply as in Basic Protocol 1, Step 4.

• 7Using a 25 mL serological pipet, slowly move the suspended spores into a 50 mL falcon tube.

Counting the spores using a hemocytometer

• 8Dilute the spore suspension x10 by combining 900 μL of tween water and 100 μL of the spore suspension in a 1.5 mL microcentrifuge tube.

  In order to ensure the spore suspension and dilutions are homogenized always vortex before using them. Spores will start to settle on the bottom of the falcon tube if left alone for several min.

• 9Further dilute the spore suspension x100 by combining 900 μl of tween water and 100 μl of the x10 dilution in a 1.5 mL microcentrifuge tube. Repeat the process to obtain x1000 dilution if spore concentration is high.
• 10Set up the hemocytometer by cleaning it with 70% ethanol and Kimwipes. Place a clean coverslip over the hemocytometer.

  Moistening the coverslip with water can help affix it to the hemocytometer. If you see Newton’s refraction rings this is an indication of proper adhesion.

• 11Gently fill each side of the hemocytometer with 10 μl of one of the dilutions. The spore suspension will be sucked in by capillary action.

  Avoid injecting bubbles into the chamber.

  It is important to not over or underfill the chamber in the hemocytometer. The amount that one should add might vary depending on the brand and type of hemocytometer being used.

• 12Place the filled hemocytometer under the microscope and allow the slide to sit for 2 min to allow the spores to settle.
• 13Use the 10x objective to localize the view around the larger 3×3 grid.
• 14Switching to the 40x objective, count all spores inside the corner and center squares (see Figure 2). While counting use the smaller internal squares as a guide, starting from the top left internal square snaking down to the bottom right internal square. If the spore touches the left or bottom line do not count it. If it falls on the right or top line count it.

  We highly recommend using a tally counter when doing this work. For increased accuracy, redo the count 1–2 times and average amongst your counts.

  The goal is to get anywhere between 25–300 spores within the five grids. If you have less than 25 spores, use a higher concentration, while if you have over 300 spores, use a lower concentration.

• 15Clean the hemocytometer and coverslip with 70% EtOH and Kimwipes before putting away.
• 16
  To calculate the number of spores per mL, use the following equation:

  $$\left(\frac{totalcount}{5}\right)\times dilutionfactorx{10}^4=\frac{spores}{mL}$$

  There are alternative ways to gather total counts that will also work fine. If using an alternative method make sure to use the correct equation. The large squares in most hemocytometers (assuming you have the proper cover slip in place) will have a total volume of 0.1 mm³ = 10⁻⁴ cm³. Additionally, 1 cm³ is equal to 1 mL.

• 17Dilute the spore suspension to your desired concentration and store spore suspension in 4°C refrigerator.

  Spore suspensions are typically good for ~2 weeks if kept in a 4°C refrigerator. While they can be kept longer, older spore suspensions are not recommended for use in metabolomic studies

Figure 2:

Microscopic view of the hemocytometer through compound light microscope. (A) 100X view centered around the large 3×3 grid inside the hemocytometer. Outlined in green are the squares to count. Outlined in blue is what is zoomed in on in B. (B) 400x view of the blue square in A. Circled in green are the spores that would be counted as within the square, red represents those that are outside of the square.

BASIC PROTOCOL 3:

Basic protocol title:

Extracting secondary metabolites from Aspergillus spp grown on solid medium

Introductory paragraph:

When extracting secondary metabolites from filamentous fungi, the general rule of thumb is that fungi will produce more metabolites when grown on solid media than in liquid shake media (Frisvad, 2012). Additionally, undefined media that uses vegetative matter (e.g., crushed corn seed, V8, oatmeal) tends to result in the highest production of metabolites but can lead to more variability. To reduce variability and increase the metabolite extraction yield, it is recommended to first try defined solid media. If one’s metabolite of interest is not produced or is produced in low quantity, try using undefined media or liquid shake (Frisvad, 2012).

Fungal secondary metabolite profiles can be altered depending on the concentration of spores added, method of inoculation, growth conditions, media, and length of time grown (Boruta, 2018). To extract a previously studied secondary metabolite in the same species it is recommended to follow previously published conditions as close as possible (see Table 1).

If studying a new secondary metabolite, it is recommended to try varying the inoculation method. A point inoculation with a low concentration (10³) may yield different results than a point inoculation with higher concentrations (10⁶ spores/mL). Additionally, one can overlay the agar plates with spores to create a lawn of growth (see Support protocol 2).

This basic protocol will detail an extraction using a point inoculation with a low concentration of spores and chloroform. While chloroform is good for extracting organic soluble metabolites, alternative solvents must be used to extract water-soluble compounds. The alternative protocols detail larger scale metabolite extractions, extractions from liquid media, and other commonly used organic solvents. It is important to note that these are not the only ways to extract secondary metabolites. Overall, the organic solvent one should use depends on what the compound of interest dissolves in. If the organic solvent is miscible with water, the sample must be lyophilized before the extraction. If the solvent is immiscible, one can separate out the organic and aqueous layers. If using different species, alter the incubation temperature and time to what is most used in the literature.

CAUTION: Chloroform is acutely toxic and should only be worked with while inside chemical fume hoods. Take your time during any steps involving it, and always make sure you are aware of the nearest eye wash station and shower in case accidental skin exposure occurs.

Materials:

Spore suspension of fungus

Sterile toothpicks

GMM solid medium (or equivalent, see recipe in Reagents and Solutions)

ddH₂O

Chloroform (e.g., Fisher, cat. no. C298–4)

Biological safety hood (e.g., Baker SterilGARD III Advance SG403, or equivalent)

Motorized pipet filler (e.g., Thermo Scientific S1 Pipet Fillers, cat. no. 14–387-165)

25 mL Disposable serological pipet (e.g., Dot Scientific, cat. no. 457225)

Petri Dishes 100 × 15 mm (e.g., VWR, cat. no. 25384–302)

Incubation Chamber or room (e.g., MaxQ 4000, cat. no. SHKE4000)

5 mL pipette tips (e.g., USA scientific, cat. no. 1050–0000 or equivalent)

20 mL glass vials (e.g., Wheaton, cat. no. 03–341-25E)

Homogenizer (e.g., Fisherbrand Homogenizer 150, cat. no. 15–340-168)

Chemical fume hood (e.g., Hamilton Pioneer Fume Hood or equivalent)

Centrifuge (e.g., Thermo Sorvall ST 40R, cat. no. 75–004-525 or equivalent)

Glass Pasteur pipettes (e.g., Fisherbrand, cat. no. 13–678-8B)

Weight balance

−20°C or −80°C freezers

Protocol steps with step annotations:

Inoculation

• 1Add 20mL of molten GMM medium to a 100 × 15 mm petri dish with a motorized pipet filler. Once hardened, inoculate 5 μL of your strain’s spore suspension (10³ spores/mL) via point inoculation in the center of petri dish.

  If unsure what medium to use GMM is always a good first choice, especially for the Aspergillus species. Furthermore, if you wanted to utilize a different growth/plating technique, this is the step to change.

• 2Incubate in the dark at 37°C for 3–5 days if using A. nidulans or A. fumigatus. If using A. flavus grow at 28°C for 3–5 days.

  While it will take longer to grow, A. fumigatus produces more metabolites at 25°C. Altering the temperature to something higher or lower than what your species grows quickest in can be a way to troubleshoot low yields or lack of a specific SM production.

  Some secondary metabolites are growth dependent, meaning they will only be created at specific stages of the growth cycle. Take care to follow the length of time used on previously published methods if reproducing work.

  Our lab has noted an increase in the production of most metabolites in various Aspergillus spp when the fungus is grown in the dark. This is also a variable that should be deliberately considered when designing SM experiments. pH and nitrogen/carbon sources also are key components that can induce synthesis of different SMs.

Extraction

• 3Remove six plugs (around 15 mm in diameter) from the GMM plates using a 5 mL pipette tip or a cork borer.

  The exact size of the borer is not important. The more plugs you get, the higher the yield but the more time consuming the protocol. We have found 4–6, 15 mm diameter plugs to be sufficient for most detection methods. If wanting to increase the yields use the entire plate.

• 4Place the plugs into a 20-mL glass vial.

  Using plastic instead of glass will result in small contamination during chloroform treatment. This is fine if running thin layer chromatography (TLC) procedures but not for HPLC/MS/NMR procedures.

• 5Add 3 mL of H₂O into the tube and homogenize completely using a homogenizer. Usually takes about 30 seconds.
• 6Add 3 mL of chloroform and vortex for 10 seconds.
• 7Let the sample sit for 30 min, shaking it every 5–10 min.
• 8Centrifuge for 20 min at 750 RCF.
• 9Transfer the chloroform layer (bottom layer) using glass Pasteur pipettes to pre-weighed glass vials and allow it to evaporate in a fume hood for 2–3 days at room temperature.

  If trying to speed up the evaporation or working with a volatile compound that may break down if not put into cold storage, you can speed up the evaporation process by bubbling nitrogen gas through the extract or by using a rotary evaporator.

• 10Once dry, obtain the new weight of the glass vial. Subtract from the base weight of the vial to obtain the total yield (mg).
• 11Store the dried extract in −20°C or −80°C freezers.
• 12When ready for use, re-suspend in solvent to desired mg/ml concentration.

ALTERNATE PROTOCOL 1

Alternate protocol title:

Extracting secondary metabolites from Aspergillus spp using ethyl acetate

Introductory paragraph:

Chloroform and ethyl acetate have similar polarity indexes meaning they will extract similar metabolites. However, while chloroform is acutely toxic, an irritant, and a suspected carcinogen, ethyl acetate is only a flammable irritant (National Center for Biotechnology Information, 2021a, 2021b). These safety concerns for chloroform might opt some to use ethyl acetate over chloroform. Broadly the protocols are the same, the only difference being some steps during the extraction. Follow the inoculation steps in Basic Protocol 3 (steps 1 and 2) before starting this alternate protocol.

Additional Materials:

Ethyl Acetate (e.g., Fisher, cat. no. E195–4)

Spatula

Ultrasonic bath Sonicator (e.g., VWR, cat. no. 97043–960)

Protocol steps with step annotations:

1. Remove six plugs (around 15 mm in diameter) from the GMM plates using 5 mL pipette tips or a cork borer.

   If scaling up this procedure, you will have to use alternative glassware and a separation funnel during step 4.

2. Place the plugs into a 20-mL glass vial and cut into small pieces using a spatula.

3. Add 10 mL of ethyl acetate and sonicate in an ultrasonic water bath for 180 min.

   Make sure you are using glass vials that have lids to prevent unwanted things from entering your vial during the sonication.

4. Add 10 mL of water and shake vigorously for 5 seconds. Leave upright for 10 min to allow for separation of the two layers.

   Sometimes there will not be a clear separation between the two layers. If this happens, add more ethyl acetate, mix thoroughly, and again let it sit upright until separation occurs.

5. Move the ethyl acetate layer (upper layer) to a new pre-weighed glass vial to evaporate until dry.

   As with the chloroform extraction, you can speed up the evaporation process with nitrogen gas or a rotary evaporator.

6. Store the dried extract in −20°C or −80°C freezers.

7. When ready for use, re-suspend in solvent to desired mg/ml concentration.

ALTERNATE PROTOCOL 2

Alternate protocol title:

High volume metabolite extraction using acetone

Introductory paragraph:

Sometimes it is necessary to purify out a secondary metabolite for further chemical characterization, study, or standard use. To obtain larger quantities of the metabolite for purification, it is important to scale up the steps involved in SM extraction.

Additional Materials:

Acetone (e.g., Fisher, cat. no. A18–4)

Spatula

Blender

4L beaker

Stir bar

Magnetic stirrer (e.g., Thermo Scientific Cimarec+, cat. no. S88857108)

50 mL Glass serological pipette (e.g., Thomas-Scientific, cat. no. 7543N78)

Rotary evaporator (e.g., Buchi R-100, article no. 11100V1001)

Protocol steps with step annotations:

Inoculation

• 1Inoculate 5 μL of your strain’s spore suspension (10³ spores/mL) via point inoculation in the center of the GMM solid medium. Use 10 plates per genotype.

  Can alter the concentration of spores used, and the method of plating. See Basic Protocol 3 for more details.

• 2Grow the plates until the sample has colonized most of the plate. This is usually around 6 days if growing A. fumigatus at 37°C in the dark

  You should have a total of 10 plates per strain being used.

Extraction

• 3Once fully grown, use a spatula to place all of the agar plates + fungi into a blender.
• 4Add 250 ml of sterile ddH₂O and blend roughly until homogenized. Usually only 15–30 seconds.

  If using 10 plates and adding 250 mL of sterile water, the total volume should be around 500 mL. Scale down/up the amount of water added if using less or more plates.

• 5Move macerate into 4L beaker.
• 6Add twice the volume of acetone. Stir vigorously with a magnetic stir bar for 15 min.

  Ex. If the plates + ddH₂O is 500 mL total, add 500 mL of acetone.

• 7Add twice the volume of chloroform. Stir vigorously for another 15 min.

  Ex. If the total volume is 1 L, add 1 L of chloroform.

• 8Once 15 min has passed, slow the sir bar down to allow layer separation to form. Stir bar can be removed once separation is finished.
• 9Using a 50 mL glass pipette remove the chloroform layer (bottom layer). Evaporate in a rotary evaporator.

  Stop short of removing the entire layer to avoid grabbing any unwanted aqueous compounds.

• 10Redissolve in appropriate solvent and move into pre-weighed glass vial.
• 11If the total final weight is needed, re-evaporate in the glass vial and get weight.
• 12Store the dried extract in −20°C or −80°C freezers.
• 13When ready for use, re-suspend in solvent to desired mg/ml concentration.

ALTERNATE PROTOCOL 3

Alternate protocol title:

Extracting secondary metabolites from Aspergillus spp in liquid medium

Introductory paragraph:

This protocol was adapted from Yin et al., 2013 which was used to extract hexadehydroastechrome from A. fumigatus. When using an organic solvent that is miscible with water (i.e methanol, acetone), the culture should be frozen and lyophilized prior to extraction.

Additional Materials:

GMM liquid medium or equivalent

Dry Ice

Acetone (e.g., Fisher, cat. no. A18–4)

Methanol (e.g., Grainger, item no. 53AZ55)

Ethyl Acetate (e.g., Fisher, cat. no. E195–4)

2 L Erlenmeyer flask with baffled base (e.g., Corning, cat. no. CLS431281–6EA)

Glass lyophilizer flasks (e.g., Wilmad-LabGlass, cat. no. 14007–774)

Lyophilizer (e.g., Milrock Technology MD53, cat. no. MD3053)

Stir bar

Magnetic stirrer (e.g., Thermo Scientific Cimarec+, cat. no. S88857108)

Cotton or vacuum filtration

Protocol steps with step annotations:

Inoculation

• 1While in a biosafety cabinet, add 1 mL of a 10⁶ spores/mL spore suspension into 1 L of GMM in a 2 L Erlenmeyer flask.

  To ensure greater agitation of the culture while mixing, it is best to use Erlenmeyer flasks with baffled bases. This leads more consistent growth times between experiments.

• 2Grow the A. fumigatus culture at 25°C while shaking at 250 rpm for three days.

  Grow time can be reduced by using a temperature closer to 37°C. Ideal temperatures will vary if using a different species.

Extraction

• 3Place the fungal cultures (medium and fungal tissue) into glass lyophilizer flasks and freeze in a dry ice acetone bath.

  If wanting to extract from the fungal tissue and supernatant independently, they must be separated before lyophilization. Either pour the culture through miracloth, or centrifuge the sample at 1900 RCF for 15 min to separate or pellet the fungal tissue. The rest of the extraction protocol will be the same.

• 4Remove the lyophilized residues and add 500 mL of 10% methanol in ethyl acetate. Stir vigorously for 3.5 hours.
• 5Filter the extracts with vacuum filtration or by pouring over cotton.
• 6Put the samples into pre-weighed 20 mL glass vials and evaporate in a fume hood or with a rotary evaporator.
• 7Obtain the weight on the dried samples to calculate the mg yield.
• 8Store the dried extracts in −20°C or −80°C freezers.
• 9When ready for use, re-suspend in solvent to desired mg/ml concentration.

SUPPORT PROTOCOL 2

Support protocol title:

Creating an overlay culture

Introductory paragraph:

The following is an alternative method for plating fungal spores. The result will be a lawn of growth that grows faster than using point inoculation, which consequently speeds up experiments. However, the difference in growth conditions can change the secondary metabolite profile during the extraction.

Additional Materials:

Motorized pipet filler (e.g., Thermo Scientific S1 Pipet Fillers, cat. no. 14–387-165)

25 mL Disposable serological pipet (e.g., Dot Scientific, cat. no. 457225)

Sterile spatula or equivalent

Protocol steps with step annotations:

Option 1: Creating an overlay culture with a sporulating fungus

• 1Create two concentrations of GMM medium with agar. Typically, 0.7% agar is used for the top agar layer, and 1.5% for the bottom layer.
• 2Pour out the bottom layer onto petri dishes. If adding an antimicrobial selective agent use a motorized pipette filler to ensure the same volume is added to every plates.
• 3Calculate how many spores (total) you would like to add to every plate. Typically, somewhere between 5–10 mL of top agar is added to each plate.
• 4Cool the top agar until it is not hot to touch but is still liquid. Add the correct amount of spore suspension and plate with a motorized pipette filler.
  Ex. You want exactly 10⁶ spores to be added to each plate. You are going to add exactly 5 mL of top agar, and your spore suspension has a concentration of 10⁸ spores/mL. There are 10 plates in total. Calculations:

  $$C\mathrm{*}V=C\mathrm{*}V$$

  $${10}^{8}spores\times V\left(mL\right)={10}^{6}spores\mathrm{*}5mL$$

  $$V=0.05mLor\frac{50\mu Lsporesuspension}{5mLoftopagar}or\frac{10\mu Lsporesuspension}{mLoftopagar}$$

  $$10plates\times 5mLagarperplate+5mLforerror=55mLtotaloftopagar$$

  $$55mLtopagar\times \frac{10\mu Lsporesuspension}{mLoftopagar}=add550\mu Lofsporesuspensionto55mLoftopagar$$
• 5Cool down the top agar to ~45°C where it is cool to touch but not solid.

  If the agar is too hot, it can damage the spores. If it is too cool, it will start to solidify early.

• 6Quickly add the correct amount of spore suspension, swirl to mix, and plate on top of the solidified bottom agar with a motorized pipette filler.

Option 2: Creating an overlay culture with a non-sporulating fungus

• 7Prepare 1.5–2% agar GMM plates.
• 8Grow up small mycelial plugs in liquid shake for 24–48 hours in GMM liquid medium.

  If your fungus has a selectable marker it is recommended to use it. This protocol can be difficult to maintain sterility.

• 9Taking care to ensure sterility, grind up the fungal balls into slurry using the sterile spatula.

  Use a different sterile spatula between samples.

• 10Spread a portion of the slurry over the GMM plates to form an overlay culture.

  You only need to spread enough to lightly cover the top of the plates. You do not want there to be a large layer of liquid medium. One ‘GMM slurry’ will be enough to plate many GMM plates.

BASIC PROTOCOL 4

Basic protocol title:

Extracting DNA from filamentous fungi

Introductory paragraph:

This protocol will detail the method for extracting genomic DNA from Aspergillus spp, however it is routinely used in our lab on Penicillium spp and Alternaria spp. It will work with other filamentous fungi after minor tweaking of the growth conditions. Extracting genomic DNA can be useful for PCR amplification of gene targets, sequencing, and designing constructs for homologous or heterologous expression. From start to finish, the protocol typically takes 3–5 days with the bulk of that time being the growth and lyophilizing steps.

CAUTION: Phenol is one of the most dangerous compounds commonly found in biology labs. It can cause burns and is absorbed through the skin. Small amounts can be fatal. Always wear proper PPE and only handle in a chemical fume hood. If some does touch your skin, absorb it with PEG or glycerol before washing with water as water alone will simply spread the phenol.

Materials:

GMM + 5 g/L of Yeast Extract (liquid medium; see recipe in Reagents and Solutions)

Sporulating culture of fungus or spore suspension

LETS buffer (see recipe in Reagents and Solutions)

Phenol : CHCl3 : isoamyl alcohol (25:24:1) (e.g., Fisher, cat. no. BP1752I-100)

95% Ethanol (V/V) (e.g., Decon Laboratories, cat. no. 64–17-5)

70% Ethanol (V/V) (e.g., Decon Laboratories, cat. no. 64–17-5)

RNase A (e.g., Qiagen, mat. no. 1007885)

10 mM Tris buffer (pH 8) (see recipe in Reagents and Solutions)

Agarose (e.g., VWR, cat. no. 0710–500G)

1X TAE Buffer (see recipe in Reagents and Solutions)

Ethidium bromide (e.g., Fisher, cat. no. BP102–1; or equivalent)

Gel loading dye (e.g, New England Bio, cat. no. B7024S)

Petri Dishes 100 × 15 mm (e.g., VWR, cat. no. 25384–302)

Sterile toothpicks or equivalent

Incubation Chamber or room (e.g., MaxQ 4000, cat. no. SHKE4000)

Spatula

Brown paper towels or equivalent (e.g., Retain, cat. no. 21930)

1.5 mL microcentrifuge tube (e.g., VWR, cat. no. 111564)

Primer snap cap tubes (e.g., VWR, cat. no. 490003–692)

Gel electrophoresis machine (e.g., Fisher, cat. no. FB-SB-710)

Gel imager (e.g., Bio-rad, cat. no. 1708265)

Glass lyophilizer flasks (e.g., Wilmad-LabGlass, cat. no. 14007–774)

Lyophilizer (e.g., Milrock Technology MD53, cat. no. MD3053)

Chemical fume hood (e.g., Hamilton Pioneer Fume Hood or equivalent)

Tabletop minicentrifuge (e.g., Eppendorf Centrifuge 5424, cat. no. 05–400-002)

Protocol steps with step annotations:

Inoculation

• 1Pour out 20 mL of GMM + 5 g/L of Yeast Extract onto a petri dish while still molten.

  If needed for your strain, make sure appropriate supplements have been added to the medium. If using 60 × 15 mm petri dishes, add 10 mL of the medium.

• 2Using a toothpick, grab some Aspergillus conidia that have been grown up on solid medium and swirl into the petri dish.

  It is also possible to use a spore suspension for this inoculation. Add a small amount into the medium and swirl to mix.

• 3Incubate at 37°C for 16–24 hrs. or until there is a mycelial mat on the petri dish that has not begun conidiation.

  It is essential that the culture has not begun conidiation! It becomes very difficult to successfully extract DNA if spores are visible. Additionally, the mycelial mat does not need to cover the entire plate but needs to be large enough to easily move around (taking up at least ~1/4 of the petri dish). If the fungus is not grown enough, check every 30 min until the mycelial mat is sufficiently large.

Preparing the sample for DNA extraction

• 4Using a spatula, scoop out the mycelium and place onto the lid of the petri dish. Ball up the mycelium with the spatula and squeeze out as much liquid as possible.

  Clean the spatula with 70% EtOH between samples to prevent cross contamination.

• 5Take the mycelial ball and transfer to a paper towel. Fold the paper towel and press down on the mycelium to squeeze out the remaining liquid. Move to dry section of the paper towel and repeat until dry.

  Typically, the mycelium will be dried after 3–5 squeezes on the paper towel.

• 6Place the dried mycelium into a labeled 1.5 mL microcentrifuge tube and poke a hole into the top of the lid.
• 7Drop the mycelium containing tube into liquid nitrogen
• 8Put the 1.5 mL tube into a glass lyophilizer vial when all of the samples are ready. Lyophilize the samples overnight or until completely dry.

  To get the glass vial cold, pour a little bit of the liquid nitrogen into the vial while adding the samples. Let it sit for 1 min to evaporate. If there is excess liquid nitrogen, pour it off before adding it to the lyophilizer.

• 9Pour the lyophilized extract into a different 1.5 mL tube and break it into a fine powder with a clean toothpick.

  The sample shouldn’t be more than 100 μL in volume. Pour out excess powder.

  If working with many samples: To prevent air moisture-induced aggregation of the DNA through conformational changes (Sharma and Klibanov, 2007), immediately add the LETS buffer after breaking up the sample into a fine powder. Then move onto the next sample.

DNA extraction

• 10Add 700 μL of the LETS buffer and mix by swirling with the pipette tip. Once partially suspended, fully suspend by inverting the tube 10 times. Leave on the bench for 5 min.
• 11Move into a chemical fume hood. Transfer the supernatant into a new tube and add 700 μL of Phenol:CHCl3:isoamyl alcohol (25:24:1). Shake vigorously 30–40 times to thoroughly mix. Let the sample sit on the bench for 5 min.

  Whenever opening or working with the samples from this point on, you must be in the fume hood. The vapor of phenol can be lethal if breathed in for 30 min.

• 12Spin the samples for 7 min at 9500 x g and 4°C, or until a pellet has formed.
• 13Transfer the supernatant into a new labeled 1.5 mL tube and add an equal volume of Phenol:CHCl3:isoamyl (25:24:1). Centrifuge the tube for 7 min at 9500 x g and 4°C.
• 14Again, move the supernatant into a new labeled 1.5 mL tube and add 1 mL of 95% EtOH.

  If needed, this can be a good stopping point. The sample can be left in a −20°C overnight after adding the 95% EtOH. Anecdotally, our lab has observed that doing so might help with the quality of the extracted DNA, but this added step isn’t necessary.

• 15Shake the sample 10 times and centrifuge the tube for 7 min at 9500 x g and 4°C to pellet the DNA.
• 16Carefully decant the supernatant without losing the pellet.
• 17Wash the pellet by adding 70% EtOH but do not resuspend the pellet.

  This is another good stopping point. The DNA can be stored after adding the 70% for up to 1 year in −20°C.

• 18Spin for 2 min at 9500 x g at room temperature.
• 19Once again, discard the supernatant and remove the excess liquid with a pipette (be careful not to lose the DNA pellet). Let it dry at room temperature for 5–15 min.
• 20Add 20 μg of RNase (2 μL from 10mg/mL stock) to 40 μL of 10 mM Tris buffer (pH 8) and use to resuspend the pellet.

  If the pellet is very large, you can add 60–100 μL to decrease the concentration.

• 21Incubate the samples into a 65°C water bath for 30 min. Tap the tubes to mix halfway through.

  The incubation will denature any DNAse contamination while allowing for RNA degradation.

• 22Store in −20°C.
• 23Check the quality of the genomic DNA with a gel.

  It is common to see a large band very high up on the gel and a second band that is >10kb.

BASIC PROTOCOL 5

Basic protocol title:

Creation of DNA construct with double joint PCR

Introductory paragraph:

Prior to performing a transformation, a DNA construct must be created that will be homologously recombined into the genome. For construct creation, double joint PCR is a quick and cheap method that can be done with common molecular biology equipment (Yu et al., 2004). When deleting a gene, the DNA construct will contain a selectable marker and flanking regions identical to those around the gene of interest. When overexpressing a gene, the DNA construct will contain the gene, a selectable marker, an inducible or constitutive promoter directly in front of the gene of interest’s start codon, and the flanking regions. Several reviews detail these strategies (He et al., 2017; Li et al., 2017) and the reader is also referred to recent advances using CRISPR-Cas9 (van Rhijn et al., 2020; Lim et al., 2021).

Use of an auxotrophic background strain is common via the deletion of argB or lysB, which are required for arginine and lysine synthesis, respectively (Xue et al., 2004). Auxotrophic background strains exist for many of the commonly studied filamentous fungi. The main antifungal selective agents used in literature are hygromycin, phleomycin, and pyrithiamine.

When creating strains where multiple rounds of gene deletion/overexpression might be needed, it is recommended to use a self-excising marker. Our laboratory has found great success with the β-Rec/six Site-Specific Recombination System containing the hygromycin resistance gene hygromycin B phosphotransferase (hph) as published in (Hartmann et al., 2010).

The general rules of thumb for good primer design applies for all primer construction steps. Ideally, you want the melting temperature to be around 58–60°C. Having a GC clamp on the 5’ and 3’ ends of the primers helps improve the promoter binding specificity. Avoid repetitive elements and large runs of single nucleotides. See the internet resources section at the bottom of the paper for a linked resource that summarizes other do’s and don’ts of primer design. The following protocol will show the steps for creating gene knockout constructs, however the same general PCR reactions will be universally applicable for other constructs.

Materials:

Oligos (Primer sets)

PfuUltra II Fusion HS DNA Polymerase and 10x Buffer (e.g., Agilent, cat. no. 600672)

Agarose (e.g., VWR, cat. no. 0710–500G)

1X TAE Buffer (see recipe in Reagents and Solutions)

Gel loading dye (e.g, New England Bio, cat. no. B7024S)

Ethidium bromide (e.g., Fisher, cat. no. BP102–1; or equivalent)

Gel purification kit (Qiagen, cat. no. 28706 ;or equivalent)

Expand Long Template PCR System (Cat. No. 11 681 842 001)

Primer snap cap tubes (e.g., VWR, cat. no. 490003–692)

PCR machine (e.g., GeneAmp, cat. no. 4339386; or equivalent)

Gel electrophoresis machine (e.g., Fisher, cat. no. FB-SB-710)

Gel imager (e.g., Bio-rad, cat. no. 1708265)

Protocol steps with step annotations:

Primer Design

• 1. Create a marker gene primer set that will amplify the selectable maker. The primers should be 25–30 bp long and be on either end of the marker gene (blue arrows; Figure 3).

• 2. Create six primers (include one nested 5’ forward and one nested 3’ reverse primer) that are about 22 base pairs long. One primer set should be upstream the open reading frame (ORF) of the target gene, and the other primer set downstream of it. Make one forward and one reverse anywhere between 1.5–2kb away from ORF, and the other set with the nested primer around 1kb away from ORF (outer black and green arrows respectively; Figure 3).

• 2b. OPTIONAL: Create open reading frame primers for screening (red arrows; Figure 3).

• 3. Create the overhang primers for 5’ reverse and 3’ forward primers (inner black arrows; Figure 3). Add 25–30 bp that aligns with the selectable marker on the 5’ or 3’ end. Total length will be 52–60 bp.

Figure 3:

Design for double joint PCR. Arrows represent primers.

Double joint PCR

• 4. PCR Reaction 1: Using the Fusion enzyme and buffer, obtain the flanking regions and amplify the selectable marker.

  Run blue and black reactions on Figure 4a.

• 5. Run the flanks on a gel to verify successful PCR before moving on, they are needed for step 6. Purify out the fragments with a Qiagen Gel Purification kit.

• 6. PCR Reaction 2: Once again, using the High-Fidelity Phusion enzyme and buffer, mix the amplicons in a ratio of 1:3:1 of the 5’-flanking : Selectable Marker : 3’-flanking regions. Run ten cycles, ensuring the annealing temperature is held long enough for the entire construct. (Figure 4b).

  Exact hold times of annealing step will vary depending on enzyme used. General rule of thumb is 1 min per kb of your construct.

  This reaction creates the full DNA construct using the overhang regions as primers for each polymerase reaction.

• 7. PCR Reaction 3: Take 2–4 ml from step 6 to amplify the entire DNA construct using the nested primers. Use the Expand Long Template PCR System. The total volume should be 100 μL (Figure 4c).

  The Expand Long Template PCR System does not have surfactants. This is very important as surfactants can cause protoplast rupturing during the transformation. If using an alternative polymerase, ensure that no surfactant is included in the buffer.

• 8. Verify the PCR amplification worked by running on a gel with 2 μL of loading dye.

  One can optionally purify the DNA construct after this step instead of purifying it during the transformation with a G-50 column.

Figure 4:

The three rounds of PCR when doing a double joint PCR reaction. (A) PCR reaction 1 will amplify the three fragments. (B) PCR reaction 2 uses the overhang region to serve as the primer for the DNA polymerase, joining together the three fragments. (C) PCR reaction 3 amplifies the DNA construct that was created in step 2.

ALTERNATE PROTOCOL 4

Alternate protocol title:

Creating the DNA construct with yeast recombineering

Introductory paragraph:

Double join PCR is a cheap and relatively easy technique for synthesizing DNA constructs. However, it starts to become less effective when the DNA construct is very large (>10 kb). This is often the case when transferring entire gene clusters into a fungus for homologous expression. When such is the case, you can transform yeast competent cells with overlapping DNA fragments to have the yeast “stitch” them together. While most fungi favor non-homologous end joining over homologous recombination to resolve DNA damage, budding yeast such as S. cerevisiae efficiently use homologous recombination as their primary pathway (Gardner and Jaspersen, 2014; Zhang et al., 2011). This protocol requires the use of a Yeast and E. coli shuttle vector. Any equivalent to the ones we have will work fine for this method.

NOTE: One does not need to create frozen yeast competent cells every time the protocol is run. As written, the protocol yields 50 tubes of competent cells, enough for 50 transformations. Make the necessary adjustments based on your requirements.

Additional Materials:

Yeast competent cells (e.g., Thermo MaV203 Competent Yeast Cells, cat. no. 11281011

2x YPAD Media (see recipe in Reagents and Solutions)

ddH₂O

Sterile frozen competent cell solution: 5 % glycerol (V/V), 10% DMSO (V/V) (e.g., Fisher, cat. no. 327255000; Sigma-Aldrich, cat.no. D8418)

50% PEG 4000 or 6000 (W/V) (e.g., Calbiochem, cat. no. 528877)

LiAc 1.0 M (pH 7.5) (e.g. Fisher, cat. no. AC268640010)

Single-stranded carrier DNA (e.g., Sigma Salmon sperm DNA, cat. no. D-1626)

DNA fragments (generated by completing up to step 5 of Basic Protocol 5)

Appropriate selective media + agar for transformed yeast cells

Digested pE-YA plasmid vector (Pahirulzaman et al., 2012), or equivalent

Miniprep Plasmid Purification kit (e.g., Bio-Rad, cat. no. 732–6100l; or equivalent)

E. coli shuttle vector (e.g., Thermo DH5α Competent Cells, cat. no. 18265017)

Ice

LB Liquid Medium (see recipe in Reagents and Solutions)

LB Solid Medium w/selectable marker added

Incubation Shaker (e.g., MaxQ 4000, cat. no. SHKE4000 or equivalent)

500mL baffled base Erlenmeyer flask (e.g., Corning, cat. no. CLS431401)

OD reader (Thermo SPECTRONIC or equivalent) or Hemocytometer (e.g., Bright-Line, SKU Z359629–1EA)

Centrifuge (e.g., Thermo Sorvall ST 40R, cat. no. 75–004-525 or equivalent)

50 mL Falcon conical tube (e.g., Corning, cat. no. 14–432-22)

1.5 mL microcentrifuge tube (e.g., VWR, cat. no. 111564)

−20°C and −80°C freezer space

Tabletop minicentrifuge (e.g., Eppendorf Centrifuge 5424, cat. no. 05–400-002)

Sterile spatula

Water bath (e.g., Thermo, cat. no. TSGP02)

Protocol steps with step annotations:

OPTIONAL: Creating Frozen Yeast Competent Cells

• 1Inoculate the yeast strain into 25–50 mL of YPAD Media. Incubate overnight, 30°C @200 rpm.

  Most yeast shuttle vectors will work. If YPAD doesn’t make sense for your strain, use SD with the appropriate supplements.

• 2Prepare the 500 mL baffled base Erlenmeyer flask by filling it with 250 mL of 2x YPAD.

  Leave capped at room temp. Alternatively you can prepare this after getting the OD reading. This will be used for step 5.

• 3Incubate the culture overnight, 30°C, 200 rpm.
• 4Calculate the volume of culture that would contain 1.25 × 10⁹ cells, and move into its own tube/vial. Centrifuge at 3,000 x g for 5 min or until pelleted.

  One can obtain a concentration using a hemacytometer (see “Counting the spores using a hemocytometer” under Basic Protocol 2) or by getting and OD600 reading of the culture with 1 × 10⁷ cells/ml set to an OD reading of 1.

• 5Pour off the supernatant and resuspend the cells with fresh media from the pre-filled 500 mL Erlenmeyer flask containing 250 mL of YPAD. Once suspended, pour the cells into the 500 mL flask.

  If you had 1.25 × 10⁹ cells pelleted and resuspended in 250 mL the final concentration should be 5 × 10⁶ cells/mL.

• 6Incubate the flask at 30°C, 200 rpm, until the density has reached 2 × 10⁷ cells/mL.

  This allows the cells enough time to undergo two divisions, entering early log phase.

• 7When ready, centrifuge the cells at 3,000 x g for 5 min using sterile 50 mL falcon tubes.
• 8Pour off the YPAD supernatant and resuspend in 25 mL of ddH₂O. Transfer to a single falcon tube.
• 9Wash the cells by spinning them down at 3,000 x g for 5 min. Pour the supernatant out.
• 10Resuspend the cells with 25 mL of dd H₂O. Repeat step 9 to do a second ddH₂O wash.
• 11Resuspend the pellet in sterile frozen competent cell (FCC) solution to a concentration of 2.0 × 10⁹ cells/mL.
• 12Dispense 50 μL of the solution into 1.5 mL microcentrifuge tubes.

  This should mean there are 1 × 10⁸ cells in each tube.

• 13Slow freeze the cells in a −20°C freezer. After 3 hours, move the cells into a −80°C freezer for long term storage.

Transforming competent yeast

• 14Thaw as many frozen cells as you need.
• 15Centrifuge at 13,000 x g for 2 min and remove the supernatant.
• 16Add directly to the cell pellet as listed. 250 μL of 50% PEG, 36 μL of LiAc 1.0 M (pH 7.5), 50 μL of 2 mg/mL⁻¹ single-stranded carrier DNA.
• 17Create the DNA master mix. Add 250 ng of the digested vector, and 500 ng of each PCR product. Bring up the final volume to 14 μL with ddH₂O.
• 18Vortex until fully resuspended.
• 19Incubate at 42°C for 45 min.

  It can help to mix periodically by gently inverting the tubes.

• 20Centrifuge the 1.5 mL tubes at 13,000 x g for 30 secs and remove the supernatant.
• 21Resuspend the pellet in 1.0 mL ddH₂O and gently resuspend using the pipette tip.

  Avoid vortexing vigorously.

• 22Plate out 200–500 μL onto the appropriate selective media. The goal is to get several hundred transformants.
• 23Incubate at 30°C for 3–5 days.

Plasmid purification from yeast

• 24Using a sterile spatula, scrape the colonies into a 5 mL culture.

  Use a liquid form of the appropriate selective media that was made in step 22.

• 25Incubate at 30°C, 200 rpm overnight.
• 26Split the 5 mL culture in half and centrifuge each half in a 1.5 mL microcentrifuge tube.

  Spinning at max for 20 seconds is usually sufficient to pellet the cells. Do this twice to get the full 2.5 mL volume in the 1.5 mL Microcentrifuge.

• 27Follow the miniprep Plasmid Purification kit to purify the plasmids from the competent yeast cells.

Transforming into competent E. coli

• 28Thaw out competent E. coli cells on ice.

  You will need 100 μL of cells per tube.

• 29Add 5–10 ng of purified plasmid into labeled sterile microcentrifuge tubes.
• 30Once the cells are thawed, add 100 μL to each Microcentrifuge tube. Flick to mix and place on ice for 30 min.
• 31Remove from ice and heat shock the cells for 2 min by placing them in a 37°C water bath.
• 32Add 900 μL of liquid LB and incubate in the 37°C water bath for 30 min.
• 33Plate out two dilutions of the transformation mixture by spreading 10 μL and 100 μL onto LB + Selective Agent. Let them sit for upside down for 5 min before turning over.
• 34Incubate at 37°C overnight or until the colonies are at the desired size.
• 35Pick the colonies and screen for the desired vector via colony PCR.

  There are other screening techniques that would be sufficient here. Restriction digests, sequencing, etc.

• 36Once confirmed, purify the plasmid with the miniprep Plasmid Purification kit.

  It is recommended to make a glycerol stock of this E. coli strain once the isolate is PCR confirmed. This will prevent you from having to redo this protocol if you need more of the DNA construct in the future.

BASIC PROTOCOL 6

Basic protocol title:

Transformation of Aspergillus spp

Introductory paragraph:

Transformation of filamentous fungi allows for overexpression and deletion of genes or entire gene clusters. This protocol describes how to transform filamentous fungi with a DNA construct via homologous recombination.

After transformation, DNA must be extracted from all transformants for confirmation of successful DNA construct insertion (see Basic Protocol 4). Verification of the transformation typically involves PCR screening, Southern blot analysis, or sequencing. (Wang et al., 2021a; Southern, 2006; Cristina Stroe et al., 2020).

The negative control (most important) should typically be a sample that does not receive the DNA construct, with the positive control being a few μg of DNA known to transform well. The negative control is essential to check for contamination that grows on the selectable GMM. The positive control allows you to determine if the cells were able to transform at all. Failure of the positive control to yield transformants is a sign there is something wrong with your methodology or reagents.

Materials:

Plates containing grown fungus

Tween Water (0.1% Tween) (see recipe in Reagents and Solutions)

Liquid Minimal Medium (LMM) (see recipe in Reagents and Solutions)

Sterile ddH₂O

Lysing Enzymes (e.g., Sigma Lysing Enzymes from Trichoderma, cat. no. L1412–25G)

Yatalase (e.g., TaKaRa, product num. SD5214)

Osmotic Medium (see recipe in Reagents and Solutions)

Trapping Buffer (see recipe in Reagents and Solutions)

Ice

STC Buffer (see recipe in Reagents and Solutions)

DNA Construct to transform (see Basic Protocol 5)

10 mM tris buffer pH8 (see recipe in Reagents and Solutions)

PEG solution

GMM bottom and top agar (with selection marker) (see recipe in Reagents and Solutions)

Motorized pipet filler (e.g., Thermo Scientific S1 Pipet Fillers, cat. no. 14–387-165)

Hemocytometer (e.g., Bright-Line, SKU Z359629–1EA)

Optical microscope (e.g., Nikon Eclipse E200)

Incubation Shaker (e.g., MaxQ 4000, cat. no. SHKE4000 or equivalent)

Centrifuge (e.g., Thermo Sorvall ST 40R, cat. no. 75–004-525 or equivalent)

250 mL centrifugation bottles (e.g., Nalgene, cat. no. 05–579-20)

50 mL centrifugation bottles (e.g., Nalgene, cat. no. 3119–0050PK)

0.45 μM filter (e.g., Grainger, item no. 12K961)

Sterile 250 mL Erlenmeyer flask

Sterile 125 mL flask

Sterile spatulas

50 mL Falcon conical tube (e.g., Corning, cat. no. 14–432-22)

Motorized pipet filler (e.g., Thermo Scientific S1 Pipet Fillers, cat. no. 14–387-165)

25 mL Disposable serological pipet (e.g., Dot Scientific, cat. no. 457225)

15 mL Centrifuge Tube (e.g., Fisher, cat. no. 05–539-4)

1.5 mL microcentrifuge tube (e.g., VWR, cat. no. 111564)

Tabletop minicentrifuge (e.g., Eppendorf Centrifuge 5424, cat. no. 05–400-002)

G-50 column + matrix if not included (e.g., Cytiva G-50 Columns, cat. no. 27533001)

Protocol steps with step annotations:

Inoculation

• 1Harvest spores from at least four plates per desired transformant background and determine spore concentration with a hemocytometer.

  For more details on determining spore concentration see “Counting the spores using a hemocytometer” under Basic Protocol 2

• 2Inoculate 500 mL of sterile LMM (add supplements if appropriate) with 10⁹ spores and shake at 27°C, 250 rpm for approximately 10–12h if yeast extract (YE) was added. 12–15 hours without YE in the LMM (assuming you are using A. fumigatus). A proper culture will have mostly young germlings in small aggregates (see Figure 5).

  This is the trickiest part of the transformation as the exact ideal timing can vary between experiments, strains, minor condition changes, etc. Young germlings are larger than spores but will not have mycelial growth. If you check and there are some young germlings, but also a fair number of spores, leave the culture to grow and check every 30 min until there is an abundance of aggregated germlings.

  Ex. When running a transformation with P. expansum, we found that incubating an initial culture of 250 mL with 10⁹ spores in 250mL of LMM + YE for 10 hrs. at 25°C, 280 rpm to be ideal. This will be unique for each species!

  If the culture has overgrown and the germlings have begun growing hyphae, do not continue with the rest of the experiment. It will be impossible to create protoplasts. Simply start over with inoculating an LMM culture with the spore suspension.

Figure 5:

Example images of microscopic slides checking for germlings in P. expansum. Circled in green are germlings. Circled in red is a clumping of mycelia. You want a majority of the cells being germlings, with a few ungerminated spores. If you are seeing mycelial growth clumping together the culture has grown too long.

Making protoplasts

• 3Transfer the culture into sterile 250 mL centrifugation bottles and spin at 11500 x g for 2 min at 20°C.
• 4Decant the supernatant, taking care to not lose any germlings, and resuspend in 50 mL of ddH₂O.
• 5Transfer to the sterile 50 mL centrifugation bottle and spin down at 25000 x g for 1 min in 20°C. Decant the supernatant.
• 6Repeat the wash steps one more time (steps 4 and 5).
• 7Mix 90 mg of Lysing Enzymes, 60 mg of Yatalase, and 30 mL of Osmotic Medium. Filter through a 0.45 μM syringe filter into a sterile 125 mL Erlenmeyer.

  OPTIONAL: Prepare two Erlenmeyer flasks to test two different concentrations of germlings during step 8.

• 8Move a tip full of the germlings into the sterile 125 mL Erlenmeyer with a sterile spatula (see Figure 6B).

  Add anywhere from the amount shown in Figure 6B to 2x that amount (roughly stay within the drawn yellow box if adding more). If inoculating two flasks, it is recommended to use slightly different amounts in each one (see Figure 6C). This gives more leeway to at least one working. Ideal time for protoplasting will vary between the two flasks.

• 9Shake at 100 rpm (max 120) for ~4 hours at the same temperature as in step 2. Check periodically starting at 2-hour timepoint under a microscope to verify that the protoplasts are forming. Remove from shaker if there is an abundance of protoplasts in the culture.

  This is another step that can vary wildly depending on the species/strain. It also is very sensitive to the number of germlings added in step 7. As an example, A. fumigatus tends to take longer than A. flavus or P. expansum. Check frequently if running for the first time, and carefully document when protoplasts formed from your background strain. This will give you better estimates for how long this step will take if using similar background strains in the future.

• 10Pour the cells directly into a sterile 50 mL Falcon tube. Pipette up and down several times with a 25 mL serological pipet. Avoid pipetting vigorously but provide enough force to mechanically break up protoplast clumps and debris.
• 11Hold the tube at an angle and very slowly pipette 4 mL of room temperature trapping buffer with a P1000 (Figure 7A).

  If dispensing slowly enough two layers should start to form with the protoplasts being trapped in the middle. Holding the tube at an angle helps to dispense the liquid on the wall of the tube.

• 12Centrifuge at 1300 x g for 15 min at 20°C.
• 13Using a P1000, grab the protoplasts from the interface (Figure 7C) and transfer to a sterile 15 mL centrifuge tube.

  If you have a total of 3 mL or less keep in a single centrifuge tube. Otherwise, split the protoplasts into two separate tubes.

• 14Add STC buffer to the maximum volume and invert 8–10 times. Let it sit on ice for 2–3 min.

  The osmotic medium contains MgS0₄ which makes the protoplasts float. To properly dilute it, the STC needs to be at least 2.5x volume of the osmotic medium. Diluting the MgS0₄ allows vacuoles to leave the cells, making them sink.

• 15OPTIONAL: If there appears to be a lot of debris (partially digested protoplasts and spores) centrifuge at 200 x g (1,000 rpm TX-750 rotor) for 1 min. at 20°C. Incubate on ice for 5 min and transfer the supernatant to another 15 mL centrifuge tube.

  The mixture will appear darker if there are lots of spores and partially digested protoplasts. The pellet is the debris, the protoplasts will be left in the supernatant. DO NOT centrifuge faster or the pellet will be debris and protoplasts.

• 16Centrifuge at 7800 x g for 7 min at 15°C to collect the protoplasts. Decant and resuspend by tapping in 1 mL STC buffer. Move into 1.5 mL tube.
• 17Spin at max for 15 seconds on Tabletop minicentrifuge. Resuspend in at least 200 μL of STC per construct being transformed.

  I.e., If you have two constructs, you need 4×100μL = 400μL of STC. Add 50 for pipetting error (450 μL total).

  Recommended: Count the concentration on a hemocytometer. Save the dilution to calculate the total viable protoplasts. For each construct that is being transformed into the cells you want to have ~100μL of 10⁸ protoplasts per mL.

  If the concentration of the protoplasts is less than 5*10⁶ it is unlikely that the transformation will work.

Figure 6:

(A) Image of protoplasts under a microscope at 400x. A protoplast will appear larger than a spore, and the vacuole inside of the cell will be visible. Clumping is normal. The sample is ready when the vast majority of germlings are protoplasts under a microscope. (B) Example of the proper amount of germlings one should add during step 8. Any amount within the yellow box would be acceptable. Avoid over adding or you will have difficulty making the protoplasts. (C) It is common practice in our lab to create two flasks during step 8. Mix more germlings into one and less into the other, this should be visible as the more flask will have a darker color.

Figure 7:

Images of the trapping buffer steps 11 & 12. (A) Angling the 50 mL conical so the trapping buffer slowly runs along the wall. (B) Mixture before centrifugation. Top layer: Trapping buffer. Bottom layer: Culture. (C) Mixture after centrifugation. Protoplasts will be trapped in the interface as labeled on the image.

(optional) Purifying the DNA construct with G-50 columns

Not needed if the DNA construct has already been purified.

• 18Fill a G-50 column with G-50 matrix and spin at 600 x g for 2 min. Decant the flowthrough and place into a clean tube.
• 19Transfer the construct to the G-50 column. Centrifuge the column at 600 x g for 2 min. Save the flowthrough.

Transformation

• 20Add 20–25 μL of the G-50 purified construct and complete to 50 μL with 10 mM tris buffer pH8. Add 50 μL of STC.

  Total volume should be 100 μL.

• 21Add 100 μL of the protoplast solution, tap to mix, and incubate on ice for 50 min.
• 22Add 1.25 mL of the PEG solution. Mix gently by turning the tube and rotating it. Incubate at RT for 15–20 min.

  Do not let it sit for longer than 20 min. While the exact mechanism of PEG is only hypothesized, it is thought to help bring the DNA construct closer to the membrane by altering the water structure around plasma membranes (O’Connor, 2013). Leaving the solution sitting for too long can be harmful to the protoplasts.

• 23Add 5 mL of the STC buffer and mix by inversion. Place the mix on ice when not using.

  (optional) Can place protoplasts in 4°C overnight to increase their efficiency.

• 24Add and swirl 6 mL of the protoplast solution into cooled GMM top agar.

  This GMM top and bottom must contain your selectable marker. You need at least six plates per transformation.

  (Alternative method) Can also sandwich 500μL – 1mL protoplast suspension between the GMM bottom and top agar. First, plate the protoplast suspension and incubate at RT overnight right side up to dry. Then add top agar.

• 25Gently pour or use a pipettor to spread the GMM top agar.
• 26Incubate at 37°C for 2 to 3 days until small colonies have formed (A. fumigatus).

  Some mutations will impact the fungus’s ability to grow and thus it may take longer.

• 27Transfer the small isolates to their own GMM plate (that contains the correct selectable marker) via point inoculation and incubate until conidiation occurs.
• 28Store confirmed transformants in glycerol stocks (see Basic Protocol 1). Recommended to save 2–3 replicates.

BASIC PROTOCOL 7

Basic protocol title:

Co-culturing fungi and bacteria for extraction of secondary metabolites

Introductory paragraph:

Fungal secondary metabolite profiles change when fungi are subjected to different conditions, one being the presence of another organism. There is growing interest in understanding the implications of fungal-bacterial interactions and activation of biosynthetic gene clusters is an important aspect to be explored. In the search for novel SMs, co-culturing is useful for inducing expression of metabolites that otherwise are not expressed. This protocol is adapted from (Cristina Stroe et al., 2020) and describes methods used to co-culture fungi and bacteria for the production and extraction of secondary metabolites produced exclusively under co-culture conditions. The medium used for strain activation and co-culturing can be modified for different strains and depending on the goal of the experiment. SMs produced under co-culturing conditions on one medium will not necessarily be produced on an alternate medium. The reader is also referred to specific methods of A. fumigatus/bacteria co-culture (Zheng et al., 2015).

Materials:

Plate of bacterial strain

BHI liquid medium (see recipe in Reagents and Solutions) or other rich medium

Sterilized 33% glycerol (V/V) (e.g., Fisher, cat. no. 327255000)

BHI agar (see recipe in Reagents and Solutions) or other rich medium

CPG liquid medium (see recipe in Reagents and Solutions) or other rich medium

1x PBS (e.g., Corning, Ref. 21–040-CV)

GMM liquid medium (see recipe in Reagents and Solutions) or other minimal medium

Spore suspension of fungal strain (see Basic protocol 2)

Round bottom polystyrene tubes (e.g., Falcon, cat. no. 9217F80)

Incubation Shaker (e.g., MaxQ 4000, cat. no. SHKE4000 or equivalent)

Cryogenic tube (e.g., VWR, cat. no. 89004–320)

1.5 mL microcentrifuge tube (e.g., VWR, cat. no. 111564)

Tabletop minicentrifuge (e.g., Eppendorf Centrifuge 5424, cat. no. 05–400-002)

OD reader (Thermo SPECTRONIC or equivalent) or Hemocytometer (e.g., Bright-Line, SKU Z359629–1EA)

Miracloth (Merck Millipore, cat. no. 475855)

Protocol steps with step annotations:

Creation of bacterial glycerol stocks

• 1With a plate of the bacterial strain, inoculate a single colony into a 16 mL snap cap culture tube with a 7 mL of rich medium.

  For human bacterial isolates BHI is typically used, for other bacteria CPG or another rich medium that supports growth will work fine. (Cristina Stroe et al., 2020) uses TSB for growing Streptomyces.

• 2Incubate the culture at appropriate growth temperature (30–37°C) overnight.
• 3Vortex the grown culture until homogenous and inoculate 1mL of the bacterial culture into a cryotube with 1 mL of 33% glycerol.
• 4Vortex the cryotube to ensure a homogenous mixture.
• 5Store at −80°C.

Preparation of bacteria

• 6Streak out the bacteria from a freezer stock onto a plate of BHI agar for isolation of a single colony.
• 7Incubate plate at appropriate temperature (30–37°C) for 24–48 h or until colonies form.
• 8From the plate, inoculate a single colony into 3 mL of liquid CPG medium.

  For strains such as Streptomyces spp. which are spore-forming, spores can be collected and used to inoculate a culture instead of the colony method.

• 9Incubate culture at appropriate growth temperature (30–37°C) overnight.

  This culture should be started the day before setting up a co-culture experiment. Specific timing varies by strain. To quantify the cells most accurately by OD₆₀₀ the cultures should not be overgrown. Usually around 16 h (no more than 20 h) works well, but some strains cannot go much past 16 h.

OPTIONAL: If inoculating bacteria into a medium that differs from what it was cultured in overnight (e.g., BHI to GMM), perform the following washing steps

• 10Transfer 3, 1 mL aliquots of the overnight culture into 1.5 mL Microcentrifuge tubes.
• 11Centrifuge at 1500 x g for 5 minutes.
• 12Pipette off the supernatant.
• 13Add 1 mL of 1x PBS to each tube.
• 14Centrifuge at 1500 x g for 5 minutes.
• 15Pipette off the supernatant.
• 16Repeat steps 8–10 for a second wash.
• 17Concentrate cells in 1x PBS as desired based on growth and number of cells needed.
• 18Take OD₆₀₀ reading to quantify bacterial cells.

  For some strains there are published papers with estimates for OD₆₀₀ reading to cell # conversions. If there is not one available and you do not know the conversion for your strain it is a good idea to create your own OD₆₀₀ to CFU calibration curve to determine this before proceeding.

Option 1: Co-culturing in liquid medium (see Support Protocol 3 if co-culturing on solid medium)

• 19Inoculate 10⁸ A. fumigatus spores into GMM liquid medium.
• 20Incubate at 37C for ~16 h.

  Growth temperature should be optimized based on strain being used.

• 21Use Miracloth to separate the grown mycelia from GMM medium.
• 22Place collected mycelia into fresh GMM liquid media.
• 23Add bacteria from the overnight pre-culture into GMM flask with grown mycelia.

  The paper that this protocol is adapted from uses 1/20 of an overnight Streptomyces culture for inoculation. Typically for other interaction studies ratios of 1:100 bacterial to fungal cells work well due to the difference in growth rate. Regardless of the inoculum size, most bacteria will grow rapidly in 12–24 h.

  The medium may need to be modified depending on what will support growth of the bacterium being used. Some are adaptable and able to grow in minimal media while others cannot or will take a long time to grow.

• 24Incubate at 37°C for 12 h.
• 25Extract the metabolites for chemical analysis.

SUPPORT PROTOCOL 3

Support protocol title:

Co-culturing on solid medium

Protocol steps with step annotations:

1. Follow Basic Protocol 7 up to step 18.

2. Inoculate 10³ spores of A. fumigatus onto one side of a petri plate of GMM agar in a 10 μL spot Incubate at 37C for ~16 hrs.

   Growth temperature should be optimized based on strain being used.

3. 10 mm away from the fungal spot, inoculate bacteria diluted to an OD of 0.1 in a 10 μL spot.

4. Incubate at 37°C.

5. Follow Basic protocol 3 for extracting metabolites from solid medium.

REAGENTS AND SOLUTIONS:

Liquid vs Solid Medium General Tips

• Only add agar if making solid plates

• Fresher plates are always recommended for experiments

• When pouring plates for SM experiments, it is recommended to use motorized pipettes for consistency. This is not necessary for activating strains.

• Plates should be put into sleeve and stored at 4°C for no longer than two months or until dry

BHI (1L)

• Autoclaved; plates: 4°C, liquid: RT

• ddH₂O

• 37 g (3.7% w/v) Brain Heart Infusion broth (e.g., Millipore Sigma, Ref. 53283)

• Bring to 1L with ddH₂O

• 16 g Agar

• Autoclave

CPG (1L)

• Autoclaved; plates 4°C, liquid: RT

• ddH₂O

• 1 g Casein Acid Hydrolysate (e.g., Millipore Sigma, Cat. No. 91079–40-2)

• 1 g Yeast Extract (e.g., Millipore Sigma, Cat. No. 8013–01-2)

• 10 g Peptone (e.g., Fisher, Cat. No. 211677)

• 10 g Glucose (dextrose) (e.g., Fisher, cat. no. CAS 50–99-7)

• 16 g Agar

• Bring to 1L with ddH₂O

• Autoclave

Czapek-Dox Broth (1 L)

• Autoclaved; Plates: 4°C, Liquid: RT

• ddH₂O

• 30 g (3% w/v) of sucrose (e.g., Fisher, cat. no. 15503022)

• 3 g (0.3 % w/v) of sodium nitrate

• 1 g (0.1% w/v) of dipotassium phosphate (e.g., Sigma-Aldrich, cat. no. 340448)

• 0.5 g (0.05% w/v) of MgSO4 (e.g., Sigma-Aldrich, cat. no. M7506–500G)

• 0.5 g (0.05% w/v) of Potassium chloride (e.g., Sigma-Aldrich, cat. no. P3911)

• 10 mg (0.001% w/v) of Ferrous sulphate (e.g., Sigma-Aldrich, cat. no. 1270355)

• pH mixture to 7.3

• Add distilled water to bring total volume to 1L

• Autoclave

• Add in the order listed, heat if needed to help dissolve the compounds

Glucose Minimal Media (1 L)

Sometimes referred to as Aspergillus Minimal Media (AMM) but the recipe may vary slightly depending on the variation that was made. Xylose Minimal Media (XMM) is identical but uses xylose in replacement of glucose.

• Autoclaved; Plates: 4°C, Liquid: RT

• ddH₂O

• 50 mL of 20x nitrate salts (see nitrate salts recipe)

• 1 mL of 1000x Trace elements (see trace elements recipe)

• 10 g (1% w/v) of glucose (dextrose) (e.g., Fisher, cat. no. CAS 50–99-7)

  • Substitute for xylose if making Xylose Minimal Media

• Any supplements needed for auxotrophic mutants

• For the LMM used in transformations, add 0.5 g (0.05% w/v) Yeast extract (e.g., VWR, cat. no. 97064–368)

• pH mixture to 6.5 with NaOH

• 1M to 2M NaOH typically works best

• Add distilled water to bring total volume to 1L

• Optional: 16 g of agar (1.6% w/v)

• Autoclave

• After autoclaving: add any selective agents

• Liquid medium can be stored at room temp in clear bottle until signs of contamination

Liquid Minimal Medium used in transformation (500 mL)

• Autoclaved; stored at room temperature

• ddH₂O

• 5 g ( Glucose (dextrose) (e.g., Fisher, cat. no. CAS 50–99-7)

• 25 mL 20x Nitrate salts (see recipe)

• 500 uL Trace elements (see recipe)

• Optional 0.5 g (0.05% w/v) Yeast extract (e.g., VWR, cat. no. 97064–368)

• Supplements

• Adjust pH to 6.5

• Bring to 1 L with ddH₂O

• Autoclave

• After autoclaving: add any selective agents

• Store at room temperature with foil over the lid

Malt Extract Agar (1 L)

• Autoclaved; stored at 4°C

• ddH₂O

• 20 g (2% w/v) of Malt Extract

• 20 g (2% w/v) of glucose

• 1 g (0.1% w/v) of peptone

• 10 mg (0.001% w/v) of ZnSO₄. 7H₂O

• 5 mg (0.0005% w/v) of CuSO4. 5H20

• pH to 4.7

• Optional: 16 g of agar (1.6% w/v)

• Autoclave

• It can be difficult to fully dissolve the compounds. Bringing the solution to boil for 1 min on a hot plate while stirring can help. Alternatively, dissolve the malt extract in 500 mL and the rest of the compounds in 500 mL. Autoclave separately and mix.

Mycelium Wash (1 L)

• Autoclaved; stored at 4°C

• ddH₂O

• 147.9 g (14.7% w/v) MgS0₄ (0.6 M) (e.g., Sigma-Aldrich, cat. no. M7506–500G)

• Bring to 1 L with ddH₂O

• Autoclave

• Store at 4°C

Nitrate Salts for Mediums: 20x (1 L)

• Autoclaved; stored at room temperature

• ddH₂O

• 120 g (12% w/v) sodium nitrate (e.g., Fisher, cat. no. S343–500)

• 10.4 g (1.04 % w/v) KCl (e.g., Fisher, cat. no. BP366–500)

• 10.4 g (1.04% w/v) MgSO₄ (e.g., Sigma-Aldrich, cat. no. M7506–500G)

• 30.4 g (3.04% w/v) KH₂PO₄ (e.g., Fisher, cat. no. BP363–1)

• Bring to 1L with ddH₂O

• Store at room temperature

Oatmeal Media (1 L)

• Autoclaved; stored at room temperature

• ddH₂O

• 16 g (1.6% w/v) of old-fashioned oatmeal

• Any supplements needed for auxotrophic mutants

• Homogenize the mixture in a blender for 5 min

• Bring to 1 L with ddH₂O

• Add 16g (1.6% w/v) of Agar

• Autoclave

• Store at room temperature with foil over the lid

Osmotic medium (500 mL)

• Filter sterilized, stored at room temperature

• ddH₂O

• 147.9 g MgSO₄ (1.2 M) (e.g., Sigma-Aldrich, cat. no. M7506–500G)

• 10mM Sodium Phosphate (e.g., Sigma-Aldrich, cat. no. 342483)

• Bring to 1 L with ddH₂O

• Filter sterilize

• Store at 4°C

Potato Dextrose Media (1 L)

• Autoclaved; stored at room temperature

• ddH₂O

• 24 g (2.4% w/v) of Potato dextrose broth (e.g., BD, ref. 213400)

• Bring to 1 L with ddH₂O

• Optional 16 g (1.6% w/v) Agar

• Autoclave

• Store at room temperature

• If making solid PDM, store at 4°C

PEG solution ( 1 L)

• Autoclaved; stored at room temperature

• 60 % PEG 4000 or 6000 (W/V) (e.g., Calbiochem, cat. no. 528877)

• 2.35 g (0.234 % w/v) CaCl₂ (50 mM) (e.g., Dot Scientific inc., cat. no. DSC20010–1000)

• 50 mM Tris-HCI, pH 7.5 (see recipe)

• Bring to 1 L with ddH₂O

• Autoclave

• Store at room temperature

Stabilized Minimal Medium (1 L)

• Autoclaved; stored at room temperature

• ddH₂O

• 10 g (1% w/v) Glucose (dextrose) (e.g., Fisher, cat. no. CAS 50–99-7)

• 50 mL 20x Nitrate salts (see recipe)

• 1 mL Trace Elements (see recipe)

• 218.6 g Sorbitol (1.2 M) (e.g., Sigma, cat. no. S6021–1KG)

• Add any supplements if needed

• For SMM that is used before the transformation add:

• 1 g (0.1 % w/v) Yeast extract (e.g., VWR, cat. no. 97064–368)

• Adjust pH to 6.5

• Add 16 g (1.6% w/v) agar to bottom agar

• Add 7.5 g (0.75% w/v) agar to top agar

• Bring to 1L with ddH₂O

• Autoclave

• Store at room temperature

STC buffer (1 L)

• Autoclaved; stored at 4°C

• ddH₂O

• 218.6 g Sorbitol (1.2 M) (e.g., Sigma, cat. no. S6021–1KG)

• 0.47 g CaC12 (10mM)

• 10 mM Tris-HCI, pH 7.5 (can be made using 10 mL of 1 M Tris)

• Bring to 1L with ddH₂O

• Autoclave

TAE Buffer: 50x (1 L)

• Stored at room temperature

• ddH₂O

• 100 mL 0.5M EDTA or 18.61 g (1.86% w/v) Disodium EDTA

• 242 g (24.2% w/v) Tris

• 57.1 mL (5.71% v/v) of glacial acetic acid

• Bring to 1L with ddH₂O

TAE: 1x (10 L)

• Stored at room temperature

• 200 mL 50x TAE

• 9.8 L ddH₂O

Trace Elements (100 mL)

• Store at room temperature

• ddH₂O

• 2.20 g (0.22% w/v) ZnSO₄. 7H₂O (e.g., Fisher, cat. no. Z68–500)

• 1.10 g (0.11% w/v) H₃BO₃ (e.g., Fisher, cat. no. A74–500)

• 0.50 g (0.05% w/v) MnCl₂. 4H₂O (e.g., Fisher, cat. no. BP214–500)

• 0.16 g (0.016% w/v) FeSO₄. 7H₂O (e.g., Sigma-Aldrich, cat. no. 026–003-01–4)

• 0.16 g (0.016% w/v) CoCl₂. 6H₂O (e.g., Fisher, cat. no. AC423570050)

• 0.16 g (0.016% w/v) CuSO₄. 5H₂O (e.g., Fisher, cat. no. 18–601-914)

• 0.11 g (0.011% w/v) (NH₄)₆Mo₇O₂₄. 4H₂O (e.g., Sigma-Aldrich, cat. no. 12054–85-2)

• 5.00 g (0.5% w/v) Ethylenediamine tetraacetic acid, disodium salt dihydrate (e.g., Fisher, cat. no. S311–3)

• Dissolve each in this exact order, making sure each is dissolved. Bring up to 100 mL.

• May need to add KOH to help fully dissolve everything.

Trapping Buffer (1 L)

• Autoclaved; Stored at 4°C

• ddH₂O

• 109.3 g Sorbitol (0.6 M) (e.g., Sigma, cat. no. S6021–1KG)

• 0.1 M Tris-HCl, pH 7.0 (see recipe)

• Bring to 1L with ddH₂O

• Autoclave

• Store at 4°C

1 M Tris-HCl (pH 7.5) (1 L)

• Stored at room temperature

• ddH₂O

• 122 g TRIS (1 M) (e.g., VWR, cat. no. 0497–5KG)

• pH with concentrated HCl

• Bring to 1L with ddH₂O

• Store at room temperature

• If making at an alternative pH or molarity adjust as necessary

Tween Water (1 L)

• Autoclaved; stored at room temperature

• ddH₂O

• 0.01% (v/v) of TWEEN® 80 (e.g., MP Biomedicals, cat. no. 103170)

• Bring to 1L with ddH₂O

• Autoclave

• Store at room temperature

• To make the preparation easier, make a stock solution of 10% TWEEN® 80 (v/v) with ddH2O and add 1mL to 1000 mL of ddH₂O when more Tween Water is needed

YPAD: 2x (1 L)

• Autoclaved; stored at room temperature

• ddH₂O

• 20 g (2% w/v) Yeast extract (e.g., VWR, cat. no. 97064–368)

• 40 g (4% w/v) Peptone (e.g., BD, ref. 211677)

• 40 g (4% w/v) Glucose (dextrose) (e.g., Fisher, cat. no. CAS 50–99-7)

• 80 mg (0.008% w/v) Adenine hemisulfate (e.g., Sigma-Aldrich, cat. no. A9126)

• Bring to 1L with ddH₂O

• Autoclave

• Store at room temperature

• If making solid YPAD add agar and store at 4°C

COMMENTARY:

Background Information:

Chemists were at the forefront of early research into fungal SMs, with a strong emphasis being on function and characterization (Raistrick, 1950). In recent decades, with the advent of next generation sequencing technologies and the subsequent bioinformatics boom, significant efforts have been able to link metabolites to specific biosynthetic gene clusters (BGC), with high rates of success in A. fumigatus (Raffa and Keller, 2019; Wang et al., 2021b). The rise of genome mining software such as SMURF and ANTISMASH have streamlined the process for identifying putative BGCs in plants, bacteria, and fungi (Khaldi et al., 2010; Blin et al., 2019). However, there exists a major bottleneck in SM research. Most BGCs are silent in normal laboratory conditions as they are tightly controlled by global and cluster-specific regulators (Keller, 2019).

Throughout the years, the fungal SM research community has developed two broad approaches to “turning on” silent BGCs. The first strategy, which broadly involves altering the physical growth conditions, has been coined as OSMAC (one strain, many compounds) (Schiewe and Zeeck, 1999; Bode et al., 2002). This typically involves altering environmental growth conditions such as media, growth temperature, light, and pH (Frisvad, 2012; Bills et al., 2008; Bayram et al., 2008). For an excellent literature review on the numerous ways this approach has been leveraged, see (Pan et al., 2019). The OSMAC strategy has become less effective in recent years due to the high rediscovery rate of known SMs. Co-culturing and use of nontraditional species are potential exceptions to this as they have been historically understudied (Krause et al., 2018; Cristina Stroe et al., 2020; Nützmann et al., 2011). With the decreasing cost of sequencing fungi, a complementary approach involving both chemical and genomics (metabologenomics) and use of heterologous expression systems can place BGCs into gene cluster families to reduce rediscovery and provide a more rational means for new compound breakthroughs (Robey et al., 2020).

The second strategy involves mining fungal genomes for BGCs and utilizing genetic engineering to induce specific clusters. This allows for heterologous expression of entire clusters in model systems such as A. nidulans, or homologous editing of global/local transcription factors that might activate silent clusters (Oakley et al., 1987; Tilburn et al., 1995; Nielsen et al., 2013; Zhang et al., 2020; Raffa et al., 2021). Early transformation technologies were pioneered in the model filamentous fungus Aspergillus nidulans. Today, A. nidulans still serves as the golden standard for heterologously expressed BGCs (Caesar et al., 2020).

There are three major disadvantages to this technology. The developed genetic tools in filamentous fungi have been mostly limited to select Ascomycota species that are culturable and capable of conidiation in laboratory conditions. This includes many of the basic protocols detailed in this publication. Additionally, the difficulty of the methods themselves hinders labs that are not familiar with the techniques from successfully implementing them; something we hope in part to address with this publication. Lastly, traditional transformation methods in filamentous fungi rely on the homologous recombination (HR) pathway to replace the endogenous segment with the introduced DNA construct. With some notable exceptions (i.e., the budding yeast subphylum), most species in the fungal kingdom favor non-homologous end joining (NHEJ) over HR, resulting in very low frequencies of gene replacement. This has led to the increased use of ΔkuA and ΔkuB strains (exact gene names may vary between species), which when mutated forces the fungus to only utilize HR. Ku70 and Ku80 are two conserved core subunits in the heterodimeric Ku protein complex which mediates the NHEJ pathway. Mutation of either Ku70 or Ku80 proteins can increase the frequency of gene replacement 10–30-fold depending on the species (Krappmann, 2007).

Within the last six years, there has been an exciting push towards creating CRISPR-Cas9 systems that can genetically engineer filamentous fungi (Nødvig et al., 2015; Song et al., 2019), including A. fumigatus (van Rhijn et al., 2020). As of 2019, CRISPR-Cas9 genome editing systems had been developed in over 40 filamentous fungi and oomycetes as reviewed in (Schuster and Kahmann, 2019). One of the recent CRISPR breakthroughs for the fungal natural products field came from the development of CRISPR-mediated transcriptional activation (CRISPRa). By using a nuclease deactivated CRISPR/Cas complex, they were able to repurpose it as an artificial transcription factor capable of modulating gene expression. This allows for the activation of multiple genes via multiple guide RNAs, and was utilized by the original authors to activate a silent NRPS cluster in A. nidulans (Roux et al., 2020). There is no doubt CRISPR based technologies will begin playing a larger role in the future of the fungal natural products field. This will be vital for reducing the time it takes to activate silent clusters, in addition to enabling the manipulation of nontraditional organisms.

Critical Parameters:

(Basic Protocol 3) Whenever running a secondary metabolism extraction, the utmost care must be taken with the growth conditions. As an example, if one follows a previous publication perfectly but decides to grow the strain at room temperature instead of 25°C as was listed in the publication, there is a chance their SM won’t be produced in the same quantity. Take detailed laboratory notes, and carefully plan out your experiments before conducting them.

(Basic Protocol 4) During the DNA extraction, the critical step is incubating until there is a large mycelial mat on the dish. This can be tricky to get right, as in our lab’s experience, the optimal time to grow a strain may vary from experiment to experiment. If it appears that some of your strains are ready for DNA extraction, but other petri dishes need to be grown more, place the ready plates in a 4°C refrigerator to slow down growth. Remember, if a plate has already made spores, you must start this step over for that sample.

(Basic Protocol 6) There are two tricky steps in the transformation protocol that require observations under a microscope before moving on. This can be daunting to those running the protocol for the first time, as they are unsure of what constitutes as ‘perfect’ vs ‘under/overgrown’. When creating germlings, you should see spores that have small bumps protruding out from them. Long hyphal branches are what you want to avoid. When creating the protoplasts, look for a ball inside of a ball under the microscope (vacuole inside the cell). Protoplasts will appear larger than spores, making them easier to distinguish. It is impossible to have 100% protoplasts, but having a majority is what you are looking for.

Understanding Results:

Extracting Secondary Metabolites

To verify that your extract worked, analytical chemistry methods need to be employed. Thin layer chromatography and ultraviolet based methods are relatively fast and great if you know what compound you are looking for. When a higher resolution of data is needed, mass spectrometry, crystallography, and nuclear magnetic resonance can be excellent tools. While mass spec (i.e., LCMS) can be run on crude samples, your compound of interest must be purified before running on NMR or crystallography. As stated earlier (Sticher, 2008; Bucar et al., 2013; Raja et al., 2017) are great reviews with more details on compound identification.

Verifying the transformation was successful

After you have completed the transformation protocol and have small colonies growing on the selective media, transfer the colonies to their own isolate plates. This is best if done before conidiation has occurred to ensure you are transferring only from one colony. You should transfer a minimum of 20 isolates, but more is better. Extract the genomic DNA from the isolates for PCR verification with screening primers. For the isolates that are confirmed with PCR, double-check that the insertion recombined into the correct location using Southern blotting or DNA sequencing. It is recommended to save several replicates of successful transformations as glycerol stocks for future use.

Time Considerations:

Making and activating glycerol stocks can be done in several hours and is a very routine process. The only necessary preparation is ensuring you have made the proper medium for plating out the fungus. Similarly, extracting metabolites from fungal cultures is a relatively routine and simple task. The most challenging step is determining which organic solvent is best for your compound of interest. Depending on how long the culture is sonicated or mixed, the process can take anywhere from a single hour to overnight.

Genetic transformations are more time consuming. From start to finish, if everything works well, the process can be completed in full within 3–4 weeks. The transformation itself takes ~2 days. Verification of successful transformants around 1 week. The rest of the time is taken up waiting for the fungus to grow.

Overall, in the best-case scenario, where your SM of interest is expressed in common growth conditions, one can obtain a crude sample within 2–3 weeks. Most of that time is taken up by allowing the fungus to grow. In the worst-case scenario, you may never get your BGC to be turned on despite months (if not years if you aren’t careful) of effort. The OSMAC approach can quickly become overwhelming with the pairwise combinations of conditions that can be changed. Months can be spent trying various medias, temperatures, plating methods etc. When trying to genetically activate BGC expression, internal transcription factors may not always regulate the BGC adjacent to it (Wang et al., 2021a). Additionally, attempts to heterologously express entire clusters in model organisms are not always fruitful.

It is important to bear this in mind. Knowing when your time is better suited pursing alternative avenues is crucial to researching natural products. There are numerous cases of researchers pursuing a specific BGC but never succeeding in turning on its transcription despite months of work. SMs are highly attuned compounds that improve the fitness of the organism it evolves in. There can be significant variation even within the same species (Drott et al., 2020). It goes without saying that the natural products community only understands a small fraction of the complex regulatory networks that control their underlying expression.

Table 2.

Troubleshooting Guide for Common Issues in Fungal Secondary Metabolism Lab Work

Problem	Possible Cause	Solution
My metabolite of interest is not showing up during chemical analysis.	The gene cluster that produces the metabolite is silent in the growth conditions tested.	1. Use the OSMAC strategy and change the growth variables. 2. Make sure you are using the proper organic solvent during the extraction. If unsure what to use, ethyl acetate and chloroform are the best for picking up the widest diversity of polar and non-polar compounds. 3. If the BGC has an internal transcription factor, transform the strain to overexpress it. 4. You might need to utilize heterologous expression to activate the silent gene cluster (see Basic Protocols 4-6).
After running the transformation, none of the replicates tested via PCR, southern, or sequencing showed the correct insertion.	The rate of gene recombination in some strains can be as low as 2% and thus can be very rare (Zhang et al., 2011).	1. You may not have tested enough replicates. When using non ΔKu70/Ku80 backgrounds, it is recommended to test a minimum of 50 strains. It is common for a vast majority to be false positives even when grown on selective medium. Test more of the isolates from the transformation. 2. Consider using a ΔKu70/Ku80 background if available for your strain. Make sure the pleotropic impacts of doing so won’t hinder any conclusions being drawn from the studies. 3. Sometimes the DNA construct itself needs to be remade, perhaps with different primer sets and overhang regions.
The metabolite extraction looks very cloudy.	Agarose or fungal tissue is inside of the sample and needs to be removed before running on HPLC/LCMS-MS etc.	Filter the solution multiple times if needed. Alternatively, you can centrifuge the sample and move over the supernatant to pellet the insoluble materials. It’s important to have a very clean sample to prevent clogging up the instrumentation used in chemical analysis.
My strain did not grow properly or sporulate on GMM.	The medium is not optimal for your species/strain.	1. Many species will never sporulate in laboratory conditions. While this can make molecular biology techniques that rely on spores incredible difficult if not impossible to conduct, metabolic extractions from the mycelium still might yield interesting results. 2. Ensure that you have added the necessary supplements if relevant. Additionally, look to the literature to see what other groups commonly grow your strains/species in. While commonly used for a wide array of fungi, GMM can be substituted for an alternative media in all protocols detailed.
I am having frequent contamination issues.	Not taking appropriate measures to ensure sterility.	It is incredibly easy to have accidental contamination in fungal laboratories due to the increased number of spores being present in the air. Ensure you are following these sterile technique rules of thumb: 1. Anything that touches a live culture should only be opened under sterile biosafety cabinets. 2. Use filter tips when doing any work that involves live cultures. 3. Avoid using a Bunsen burner for sterile steps. While commonly used in non-sporulating yeast and bacterial labs, it often will not properly prevent contamination in filamentous fungal labs. 4. In our experience, some researchers have a natural skin microbiota that makes them more prone to causing contamination during their experiments (despite taking other precautions). Consider double gloving if worried this may be the case for yourself. 5. The only way to ensure a biosafety cabinet is sterile between strains/replicates in an experiment is to turn on a UV light for 15 min between samples. While this is not always necessary, it is highly recommended when creating or using glycerol stocks. Especially if said strain has no selective maker.
My point inoculations aren’t growing as a single circle in the center of the plate.	You are moving the plate before the spore suspension can dry or are pipetting the liquid out too forcefully.	To ensure the tightest possible point inoculation, pipette the spore suspension very slowly and avoid pushing to the second stop. Allow the spore suspension to completely dry before moving. If in a biosafety cabinet, this can be quickly done by leaving the lid off the plate for 10–20 min. Otherwise, let the plate sit at room temp for several hours until dry before moving.
Fungus or bacterium not growing well.	Growth of one is overpowering the other.	Try to modify the ratio of the initial inoculum to use more cells of the one that is not growing well.
Bacterium not growing in minimal medium.	Missing essential nutrients.	The minimal media sufficient to support fungal growth will not always be effective for bacteria. Try supplementing the medium listed here with any missing components that are required for your bacterium to grow.