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. Author manuscript; available in PMC: 2023 Jan 25.
Published in final edited form as: Methods Mol Biol. 2022;2489:41–52. doi: 10.1007/978-1-0716-2273-5_3

Investigating Fungal Biosynthetic Pathways Using Heterologous Gene Expression: Aspergillus nidulans as a Heterologous Host

Danielle A Yee 1, Yi Tang 1,2,*
PMCID: PMC9873597  NIHMSID: NIHMS1867179  PMID: 35524044

Abstract

Fungal natural products encompass an important source of pharmaceutically relevant molecules. Heterologous expression of biosynthetic pathways in chassis strains enables the discovery of new secondary metabolites and characterization of pathway enzymes. In our laboratory, biosynthetic genes in a clustered pathway have been refactored in engineered heterologous hosts such as Aspergillus nidulans. Here we describe the assembly of heterologous expression vectors, transformation into A. nidulans, and detection of new compounds in the transformant strains.

Keywords: Aspergillus nidulans, heterologous expression

1. Introduction

Secondary metabolites from fungi and their derivatives comprise a broad class of natural products with rich structural diversity and powerful biological activity.[1] Recent genome sequencing efforts have revealed many natural product gene clusters in fungi are not expressed under standard laboratory culturing conditions.[2] To access silent gene clusters, pathway genes can be reconstituted in well-established heterologous hosts such as Aspergillus nidulans and Aspergillus oryzae.[3–8] Use of these platforms has allowed for the discovery of countless natural products and characterization of their biosynthetic pathways.[9–10] Here we describe an episomal heterologous expression system in A. nidulans A1145, which can accommodate up to 12 genes on three plasmids.[11] For a cleaner background during compound detection, the modified strain A. nidulans A1145ΔSTΔEM can be used, in which endogenous production of sterigmatocystin [12] and emericellamide [13] has been abolished.[14] The general workflow for this system is as follows: first, heterologous expression plasmids for A. nidulans are assembled through yeast homologous recombination. Following this, the plasmids are transformed into A. nidulans protoplasts. Individual transformant colonies are then assayed for compound production. RT-PCR (reverse transcription-polymerase chain reaction) can be performed to verify genes are successfully expressed in the transformant strains.

2. Materials

2.1. Strains:

  1. A. nidulans A1145[11] (genotype: pyrG89; pyroA4; nkuA::argB; riboB2) or A. nidulans A1145ΔSTΔEM[14]

  2. S. cerevisiae JHY651[15] or other yeast strain auxotrophic for uracil

  3. E. coli TOP10 (Invitrogen), DH10b (Thermo Scientific), etc.

  4. Original fungal host strain with target gene cluster (if genomic DNA needs to be acquired)

2.2. Plasmids:

  1. pYTR (see Note 1)

  2. pYTP (see Note 2)

  3. pYTU (see Note 3)

2.3. Reagents for cloning:

Many of the commercial kits are optional. If following other protocols, additional materials are required.

  1. Restriction enzymes PacI, NotI, SwaI, BamHI, PshAI

  2. Agarose gel for DNA electrophoresis

  3. Gel DNA Recovery Kit

  4. Quick-DNA Fungal/Bacterial Microprep Kit (Zymo Inc. USA)

  5. Primers to amplify genes and promoters

  6. Proof-reading DNA polymerase

  7. Solid YPEG medium: 5 g bacto-peptone, 2.5 g yeast extract, 7.5 mL glycerol, 5 g agar, 250 mL ddH2O. Autoclave and let solution cool to around 55 °C. Add 10 mL of 100% ethanol. Mix thoroughly and pour plates.

  8. Liquid YPD medium: 5 g bacto-peptone, 2.5 g yeast extract, 5 g dextrose, 250 mL ddH2O. Autoclave and store at room temperature.

  9. Frozen-EZ Yeast Transformation II Kit (Zymo Inc. USA)

  10. Solid SDCAA(-U) medium: 5 g dextrose, 5 g agar, 200 mL ddH2O. Autoclave. Prepare supplements: 1.25 g casamino acids, 1.7 g yeast nitrogen base without amino acids, 10 mg adenine, 10 mg tryptophan, 50 mL ddH2O. Filter sterilize. Add supplements to melted agar, mix thoroughly, and pour plates.

  11. Zymoprep™ Yeast Plasmid Miniprep I (Zymo Inc. USA)

  12. Isopropanol

  13. 1000X carbenicillin (50 mg/mL): Dissolve 0.5 g of carbenicillin in 10 mL ddH2O. Filter sterilize. Prepare 1 mL aliquots and store at −20 °C.

  14. 1000X ampicillin (100 mg/mL): Dissolve 1 g of ampicillin in 10 mL ddH2O. Filter sterilize. Prepare 1 mL aliquots and store at −20 °C.

  15. Solid LB medium: 6.25 g LB powder, 5 g agar, 250 mL ddH2O. Autoclave. To prepare LB plates with carbenicillin, let melted agar cool to around 55 °C, then add 250 μL of 1000X carbenicillin. Mix thoroughly and pour plates.

  16. Liquid LB medium: 6.25 g LB powder, 250 mL ddH2O. Autoclave and store at room temperature. Aliquot and add ampicillin as needed.

  17. Plasmid Miniprep Kit

  18. Yeast RNA Isolation Kit

  19. SuperScript III First-Strand Synthesis System (Invitrogen)

  20. Oligo-dT primer

  21. GoTaq Master Mix (Promega)

2.4. Media and solutions for A. nidulans transformation and culturing:

  1. 100X uracil (500 mM): 2.24 g, 40 mL of ddH2O. Add 10N NaOH until fully dissolved. Filter sterilize.

  2. 100X uridine (1 M): 9.76 g, 40 mL of ddH2O. Filter sterilize.

  3. 1000X pyridoxine (0.5 mg/mL): 20 mg, 40 mL of ddH2O. Filter sterilize.

  4. 1000X riboflavin (0.125 mg/mL): 5 mg, 40 mL of ddH2O. Filter sterilize.

  5. 20X nitrate salts: 120 g NaNO3, 10.4 g KCl, 10.4 g MgSO4•7H2O, 30.4 g KH2PO4. Add ddH2O. up to 1 liter, stir until dissolved, and store at room temperature.

  6. Trace elements : 2.20 g ZnSO4•7H2O, 1.10 g H3BO3, 0.50 g MnCl2•4H2O,0.16 g FeSO4•7H2O, 0.16 g CoCl2•5H2O, 0.16 g CuSO4•5H2O, 0.11 g (NH4)6Mo7O24 •4H2O. Add each to 80 mL of ddH2O in the order shown. Add more ddH2O to bring the total volume to 100 mL. Adjust pH to 6.5 with 1 N KOH.

  7. Solid CD-sorbitol medium (1 L): 10 g glucose, 50 mL 20X nitrate salts, 1 mL trace elements, 218.6 g sorbitol (1.2 M), 20 g agar. Autoclave then pour plates as needed. Add the appropriate supplements to individual empty petri dishes before pouring in the agar media. If not making plates immediately, solid media can be stored at room temperature and re-melted in the microwave.

  8. Solid CD medium (1 L): Prepare as described above for solid CD-sorbitol medium, omitting the sorbitol.

  9. Liquid CD medium (1 L): 10 g glucose, 50 mL 20x nitrate salts, 1 mL trace elements. Autoclave. Aliquot as needed and add the appropriate supplements.

  10. Liquid CD-ST medium (1 L): 20 g starch, 20 g peptone (acidic digest) or casamino acids (acidic digest), 50 mL 20x nitrate salts, 1 mL trace elements. Add starch to 100 mL of ddH2O and mix with a stir bar for 10 min. Add boiling ddH2O to bring the total volume to 950 mL. Add the remaining ingredients. Continue to mix until everything is dissolved. Autoclave. Aliquot as needed and add the appropriate supplements.

  11. Solid CD-ST medium (1 L): Prepare as described above for liquid CD-ST medium. Add 20 g of agar after everything is dissolved. Autoclave then prepare plates with the appropriate supplements.

  12. Osmotic medium (500 mL): 147.9 g MgSO4 (1.2 M), 10mM sodium phosphate buffer (NaPB) (can be made from a 2M NaPB stock: 90.9 g Na2HPO4 and 163.4 g NaH2PO4 or 187.9 g NaH2PO4per liter, pH 6.5). Adjust pH to 5.8 with 1M Na2HPO4 (about 25 mL). Filter sterilize and store at 4°C. Tip: start with 450 mL of water, then the total volume will end up to be about 500 mL.

  13. Trapping Buffer (1 L): 109.3 g sorbitol (0.6 M), 0.1 M Tris-HCI, pH 7.0 (can be made using 100 mL 1 M Tris). Autoclave and store at 4°C.

  14. STC buffer (1 L): 218.6 g sorbitol (1.2 M), 1.47 g CaC12 (10mM), 10 mM Tris-HCI, pH 7.5 (can be made using 10 mL 1 M Tris). Autoclave and store at 4°C.

  15. PEG solution (100 mL): 60% PEG 4000 (BDH), 50 mM CaCl2, 50 mM Tris-HCI, pH 7.5 (can be made using 50 mL 1 M Tris). Autoclave and store at room temperature.

  16. Lysing enzyme, yatalase, 0.2 μm syringe filters

2.5. Other materials for A. nidulans transformation,culturing, and analysis:

  1. Two sterile 125 mL flasks

  2. Sterile 30 mL Corex tube or sterile 50 mL Falcon tube

  3. Sterile cell strainer (Fisher, Cat No. 22363547, optional)

  4. Solvents e.g. ethyl acetate, acetone, methanol

3. Methods

3.1. Cloning of plasmids for A. nidulans expression:

The plasmids for heterologous expression in A. nidulans A1145 are denoted as pYTU, pYTP, and pYTR[5]. Their features include auxotrophic markers for uracil (pyrG), pyridoxine (pyroA), and riboflavin (riboB), respectively, as well as the AMA1 origin of replication for Aspergillus (Fig. 1). For cloning purposes, these vectors also contain the uracil auxotrophic marker (URA3) and 2-μm origin for S. cerevisiae, and the ampicillin resistance marker (ampR) and ColE1 origin for E. coli. pYTU contains the starch inducible promoter PglaA, pYTP contains the starch inducible promoter PamyB, and pYTR contains the constitutive promoter PgpdA from Aspergillus niger. Additional commonly used promoters include the constitutive PgpdA promoters from Penicillium oxalicum (PPOgpdA) and Penicillium expansum (PPEgpdA), as well as the constitutive PcoxA promoter from A. niger. Typically, one to four genes expressed under different promoters can be inserted into each plasmid.

Figure 1.

Figure 1.

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Vectors for heterologous expression in A. nidulans: pYTU, pYTP, and pYTR.

  1. If using the original promoters included in the pYT vectors, digest pYTU with the restriction enzymes PacI/NotI and pYTP and pYTR with PacI/SwaI or BamHI/SwaI following the manufacturer’s instructions. If using different promoters, digest pYTU with PshAI/NotI, or PshAI/PacI, and pYTP and pYTR with NotI/PacI, NotI/BamHI, or NotI/SwaI (see Note 1). Run digestion reactions on an agarose gel and recover DNA using a commercial kit such as Zymoclean™ Gel DNA Recovery Kit.

  2. To obtain template DNA for amplification of target genes, culture the original fungal host on solid or liquid media such as potato dextrose agar or broth. Isolate the strain’s genomic DNA using a commercial kit such as Quick-DNA Fungal/Bacterial Microprep Kit, phenol-chloroform extraction,[16] or microwave total genomic DNA extraction.[17]

  3. Amplify the genes of interest and their native terminators (300-500 bp downstream from the stop codon) by PCR using the genomic DNA of the original host as the template. A high-fidelity polymerase such as Q5® High-Fidelity DNA Polymerase (NEB), AccuPrime Pfx DNA Polymerase (Invitrogen), or Phusion (NEB) should be used. If replacing the original promoters or if more than one gene is to be inserted into each plasmid, amplify the promoters to be used by PCR. Use overhang primers to introduce 25-40 bp of homology between adjacent fragments. Run PCR products on an agarose gel and recover DNA using a commercial kit such as Zymoclean™ Gel DNA Recovery Kit.

  4. Co-transform the overlapping DNA fragments and their corresponding digested vectors into S. cerevisiae JHY651[3] or other yeast strain auxotrophic for uracil to assemble the expression plasmids in vivo by yeast homologous recombination. Before preparing competent cells, yeast strains should be grown on YPEG agar plates and cultured in YPD liquid media. Yeast competent cells can be prepared using the PEG-lithium acetate method [18] or a commercial kit such as Frozen-EZ Yeast Transformation II Kit (Zymo Inc. USA).Transformants should be plated on SDCAA(-U) agar plates. Yeast colonies will appear after 1-3 days of incubation at 28°C.

  5. Extract the assembled plasmids from yeast using a commercial kit such as Zymoprep™ Yeast Plasmid Miniprep I (Zymo Inc. USA), and transform into E. coli TOP10, DH10b, etc. by electroporation to isolate single clones. Plate cells on LB agar plates with 50 μg/mL of carbenicillin. E. coli colonies will appear after 12-16 hours of incubation at 37 °C.

  6. Culture single E. coli colonies in 3-5 mL of LB medium with 100 μg/mL of ampicillin for 12-16 hours, shaking at 250 rpm, 37°C. Extract the plasmids from the overnight cultures using a commercial kit such as Zyppy™ Plasmid Miniprep Kit (Zymo Inc. USA) or QIAprep Spin Miniprep Kit (Qiagen). Confirm correct assembly by performing digestion checks with restriction enzymes followed by DNA sequencing.

3.2. Procedure for transformation of plasmids into A. nidulans:

3.2.1. Germlings:

  1. Grow A. nidulans A1145[11] or A1145ΔSTΔEM[14] on CD agar supplemented with uracil, uridine, pyridoxine, and riboflavin for 3-5 days at 37°C. If using cells from a frozen protoplast stock, streak the strain on CD-sorbitol agar with the appropriate supplements.

  2. When green spores appear, inoculate 25 mL of liquid CD medium containing the appropriate supplements in a sterile 125-mL flask with fresh spores (2 x 106 spores/mL, about 3 cm2 of spores) using a sterile cotton-tipped applicator and shake at 28°C, 250 rpm for 16-20 hours. A proper culture should have an abundance of young germlings in small aggregates.

3.2.2. Digestion:

  1. Harvest the culture by centrifugation at 4300 x g for 20 min at 20°C.

  2. Remove the supernatant and resuspend the pellet in 10 mL of osmotic medium. Spin down by centrifugation at 4300 x g for 20 min at 20°C.

  3. Dissolve 30 mg of lysing enzymes from Trichoderma harzianum and 20 mg of Yatalase in 10 mL of osmotic medium and sterilize with a 0.2 μm syringe filter.

  4. Remove the supernatant from the centrifuged cells and resuspend the pellet in the sterilized lysing enzyme mixture. Transfer directly into a sterile 125-mL flask.

  5. Digest cells by shaking at 80 rpm for 4-6 hours at 28°C. Protoplasts are thin-walled and about 2 times as large as spores.

3.2.3. Harvesting cells:

  1. Pour cells directly into an autoclaved 30 mL Corex tube and gently overlay with 10 mL of trapping buffer. Centrifuge in Beckmann equivalent rotor at 5200 x g for 20 min at 4°C (see Note 5).

  2. After centrifugation, protoplasts will accumulate in the cloudy layer at the interface of the two buffers. Remove the protoplasts from the interface with a pipet and transfer them to a sterile 15 mL falcon tube.

  3. Add 2 volumes of STC buffer and centrifuge at 4300 x g for 20 min at 4°C (see Note 6).

  4. Decant the supernatant. Resuspend the protoplasts in STC buffer at a concentration of 108- 109 (usually 1 mL) with minimal pipetting, which can damage the protoplasts. Aliquot 100 μL of protoplasts each in sterile 1.5 mL centrifuge tubes. If desired, store aliquots for future use at −80°C (see Note 7).

3.2.4. Transformation:

  1. For each transformation, add 3 μL of each plasmid to 100 μL of protoplasts, and incubate on ice for one hour. As a negative control, include one transformation with the appropriate empty pYT vectors. Plasmid stock DNA concentrations should be at least 100 ng/μL (see Note 8).

  2. Add 600 μL of PEG solution to each tube. Mix gently by turning the tube on its side and rotating it. Incubate at room temperature for 20 min.

  3. Using a pipet, drop the PEG mixture on CD-Sorbitol agar plates with the appropriate supplements if transforming only one or two plasmids. Using a spreader is not necessary and can damage the protoplasts. Incubate at 37 °C right side up to let dry. Colonies will appear after 2-4 days.

3.3. Procedure for production of compounds and biotransformation

3.3.1. Production of compounds

  1. For expression of genes under starch inducible promoters, transformants must be cultured in CD-ST media. For expression of genes under constitutive promoters, CD or CD-ST media may be used (see Note 9). For liquid cultures, inoculate the spores from selected transformants in 10 mL of liquid media with appropriate supplements using a sterile cotton-tipped applicator. Shake cultures at 250 rpm at 28°C. For solid cultures, use a sterile cotton-tipped applicator to streak selected transformants onto CD or CD-ST agar plates. Incubate at 28°C.

  2. Check cultures for compound production every other day, beginning after two days of growth. For liquid cultures, use a pipet with autoclaved cut tips to transfer 500 μL of culture to a 1.5 mL microcentrifuge tube (see Note 10). Centrifuge at maximum speed for 5 min. Transfer the supernatant to a new microcentrifuge tube. Add 500 μL ethyl acetate to the supernatant and vortex for 1 min. Add 500 μL acetone or methanol (see Note 11) to the cell pellet and vortex for 15 min. For solid cultures, cut out a 1 cm2 piece of agar and transfer to a microcentrifuge tube with 500 μL of acetone or methanol and vortex for 15 min.

  3. Centrifuge the samples at max speed for 5 min. Transfer the organic layers to new microcentrifuge tubes and dry in a speed vacuum.

  4. Resuspend the dried extracts in 100 μL of methanol. Centrifuge at maximum speed for 5 min. and transfer 50 μL of the extract to a LC-MS vial. Inject 20 μL of extract on the LC-MS for compound detection.

3.3.2. Biotransformation

  1. Use the same culturing conditions as described previously for compound production. Substrates for the biotransformation assay can be added at the beginning of culturing or after the cells have grown for a few days. Add the substrate to the culturing medium to a final concentration of 200-500 μM. If adding substrates at the beginning of a solid culture, allow melted agar to cool before adding the substrate. If feeding to a solid culture after cells have grown, the substrate can be layered on top of the plate.

  2. To check cultures for biotransformation products, follow the same procedure for metabolite analysis as described in steps 2-4 above.

3.4. Procedure for RT-PCR (reverse transcription-polymerase chain reaction) to verify gene expression

  1. Use the same culturing conditions as described previously for compound production and biotransformation. Cells can be harvested for RNA extraction when compounds are expected to be produced.

  2. Extract RNA from the cells using RiboPure™ Yeast RNA Isolation Kit (Ambion) following the manufacturer’s instructions. Digest residual genomic DNA in the RNA extracts with DNase I (provided in the kit) at 37 °C for 4-6 hours. Follow the manufacturer’s instructions to inactivate the DNase.

  3. Use SuperScript III First-Strand Synthesis System (Invitrogen) for cDNA synthesis with oligo-dT primers following directions from the user manual.

  4. Using the synthesized cDNA as the template, set up PCR reactions to amplify fragments of the genes for heterologous expression using GoTaq Master Mix (Promega) following the manufacturer’s instructions. To check for gene expression, it is not necessary to amplify the entire open reading frames of the genes. If possible, design primers flanking the introns in the target genes so the smaller cDNA product can be separated from possible bands from genomic DNA contamination. As a positive control, include a PCR reaction to amplify a region of actA (actin gene for A. nidulans) flanking its introns to assess the quality of the cDNA. Run the PCR reactions on an agarose gel. Bands with the correct cDNA product size will indicate the genes are expressed (see Note 12).

Acknowledgements

Research in related areas is supported by NIH 1R35GM118056 to YT.

4. Notes

1.

Selection is by riboB, final concentration 0.125 μg/mL riboflavin.

2.

Selection is by pyroA, final concentration 0.5 μg/mL pyridoxine HCl.

3.

Selection is by pyrG, final concentration 10 mM uridine and 5 mM uracil.

4.

If using SwaI, perform the digestions with each enzyme separately. First, digest with only SwaI using NEB buffer 3.1 at 25 °C, then column purify the reaction with a commercial kit such as DNA Clean & Concentrator (Zymo Inc. USA). Next, digest the recovered DNA with the second restriction enzyme using NEB Cutsmart® buffer at 37°C.

5.

Alternatively, pour cells into a sterile 50 mL Falcon tube and gently overlay with 10 mL of trapping buffer. Centrifuge at 4300 xg for 30 min at 4°C.

6.

As another alternative to steps 1-3, pour cells through a sterile cell strainer (Fisher, Cat No. 22363547) into a sterile 50 mL Falcon tube. Add an equal volume of STC buffer and gently invert the tube to mix. Centrifuge at 4300 x g for 20 min at 4°C. Decant the supernatant and gently resuspend the pellet in 10 mL of STC buffer with minimal pipetting to wash the cells. Centrifuge at 4300 xg for 20 min at 4°C.

7.

Fresh protoplasts have the highest transformation efficiency. Frozen protoplasts can be used up to three weeks after storage but will rapidly lose competency.

8.

It is optimal to add less than 10 μL of DNA solution to maintain the concentration of the STC buffer components. If plasmid DNA concentrations are low, plasmid stocks eluted in water can be lyophilized and resuspended in a smaller volume of STC buffer to increase DNA concentration.

9.

Strains grow faster and to a higher cell density in CD-ST, which is a richer medium compared to CD. However, the background metabolite profile is generally cleaner in CD compared to CD-ST.

10.

Uncut pipet tips may be used in early stages of culturing. It is necessary to use cut tips once the mycelium has grown too thick to be aspirated by an uncut pipet tip.

11.

Depending on their solubility, some compounds may only be detected by extracting with certain solvents but not others. Therefore, it may be advantageous to test different solvents for extraction if target compounds cannot be detected.

12.

To confirm genes are properly spliced, the entire open reading frames of the genes can be amplified by PCR using a high-fidelity polymerase. The resulting PCR products can be purified and sent for DNA sequencing.

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