Efficient generation of uridine/uracil auxotrophic mutants in homokaryotic Cordyceps militaris strains for constructing a food-grade expression platform

Abstract

Cordyceps militaris is a well-known medicinal mushroom widely exploited in traditional medicine and nutraceuticals. In this study, we aim to establish a new platform for improving the production of beneficial ingredients in this fungus. We successfully generated uridine/uracil auxotrophic mutants (ΔpyrG) in five homokaryotic C. militaris strains. The efficiency of the pyrG deletion by homologous recombination reached 100% in all the C. militaris strains. Genetic transformation of the C. militaris ΔpyrG strains mediated by Agrobacterium tumefaciens using the native pyrG auxotrophic marker resulted in high transformation yields of 109-810 transformants per 10⁵ conidia. Additionally, the pyrG marker from Aspergillus oryzae was also functional for the genetic transformation of C. militaris ΔpyrG. We further showed that the gpd1 and tef1 genes were strongly expressed during the mycelial growth of C. militaris, and their promoter sequences were integrated into binary vectors for enhancing recombinant expression. With the constructed platform, the strong heterologous expression of the DsRed protein was proven, and the genomic integration of the endogenous CmFE gene encoding a serine protease under the regulation of the tef1 promoter significantly increased the activity of this enzyme in C. militaris. Our work provides a promising platform for food-grade recombinant expression in C. militaris.

1. Introduction

The genus Cordyceps Fr. comprises more than 400 species, of which Cordyceps militaris (L.) Fr. is one of the most well-known species used in traditional medicine and nutraceuticals. This edible fungus produces numerous bioactive constituents, including cordycepin, adenosine, pentostatin, ergothioneine, carotenoids, polysaccharides, and digestive enzymes that are beneficial to human health (Chen et al., 2022; Wang et al., 2022b; Zheng et al., 2011a). Cordyceps militaris is a heterothallic fungus that requires a combination of two homokaryotic strains carrying the opposite mating-type locus (MAT1-1 or MAT1-2) for sexual reproduction via fruiting body formation (Zheng et al., 2011a). However, recent studies also indicated that C. militaris can exist as heterokaryotic strains harboring both MAT1-1 and MAT1-2 in the genome, or as homokaryotic strains carrying only MAT1-1 or MAT1-2. A heterokaryotic strain (MAT1-1/MAT1-2) of C. militaris can be separated into two homokaryotic strains by single monokaryotic ascospore isolation from fruiting bodies (Lee et al., 2017; Lu et al., 2016; Vu et al., 2023). Notably, some homokaryotic C. militaris strains are able to form fruiting bodies and produce high contents of cordycepin, a bioactive substance with antimicrobial and anticancer activities (Vu et al., 2023, 2024; Zheng et al., 2011a). Both the types of homokaryotic strains have been fully sequenced for the whole genome, and the available genome database of the homokaryotic C. militaris Cm01 strain (MAT1-1) facilitates genetic approaches in this fungus (Kramer & Nodwell, 2017; Zheng et al., 2011a).

Currently, different strategies for genetic engineering and genome editing have been developed in filamentous fungi and medicinal mushrooms to promote the production of beneficial ingredients and bioactive compounds (Li et al., 2017; Michielse et al., 2005; Shen et al., 2023; Tarafder et al., 2024). In fungi, genetic manipulation usually requires one of the most common transformation methods, including the polyethylene glycol (PEG)-mediated protoplast transformation and Agrobacterium tumefaciens-mediated transformation (ATMT) (Li et al., 2017; Michielse et al., 2005; Wang et al., 2024; Zheng et al., 2011b). Compared to the PEG-mediated protoplast transformation, the ATMT can use directly fungal spores as the starting material for genetic transformation instead of fragile protoplasts that require a costly and time-consuming procedure for the preparation. Genomic integration of T-DNA via ATMT is also a special feature for the stable maintenance of target expression constructs. Additionally, ATMT can be employed to generate libraries of T-DNA insertional mutants for discovering novel genes in a fungus (Binh et al., 2021; Li et al., 2017; Zheng et al., 2011b). Although a few recent attempts on establishing an ATMT system using auxotrophic markers have been initially successful in C. militaris (Wang et al., 2022a; Yan et al., 2024), the majority of genetic manipulation approaches via ATMT in this fungus still mainly use antibiotic resistance markers (Lou et al., 2021; Wang et al., 2017, 2021a, 2021b; Wāng et al., 2020; Xia et al., 2017; Zheng et al., 2011b). The employment of antibiotic resistance markers for genetic manipulation may raise concerns about adverse effects or safety for downstream applications (Arnau et al., 2020; Son & Park, 2020; Vandermeulen et al., 2011). Therefore, the development of a reliable and efficient ATMT-based platform with suitable binary vectors harboring nutritional markers to support the food-grade production of beneficial ingredients in C. militaris is necessary.

In previous studies, we demonstrated that the ATMT using the pyrG gene as a nutritional selection marker is very efficient in filamentous fungi (Binh et al., 2021; Nguyen et al., 2017; Tran et al., 2023). In the present work, we deleted the pyrG gene in C. militaris to generate uridine/uracil auxotrophic mutants and constructed an efficient ATMT system for genetic manipulation in this medicinal mushroom. The constructed ATMT system employed only the pyrG auxotrophic marker for genetic transformation instead of drug-resistant markers. The uridine/uracil auxotrophic ATMT efficiency could reach 109−810 transformants per 10⁵ fungal spores depending on the tested C. militaris strains. Binary vectors carrying the pyrG selectable marker and an expression cassette under the regulation of the native gpd1 promoter or tef1 promoter were also constructed for genetic manipulation in the uridine/uracil auxotrophic C. militaris strains. With the established platform, the heterologous expression of the DsRed fluorescent reporter gene and the overexpression of the endogenous CmFE gene encoding a serine protease were successfully implemented in this fungus.

2. Materials and methods

2.1. Microbial strains

Five homokaryotic C. militaris strains (Supplementary Fig. S1), including H8 (MAT1-1), N19 (MAT1-1), G4 (MAT1-1), G2 (MAT1-2), and N9787 (MAT1-2), were employed for constructing uridine/uracil auxotrophic mutants. Escherichia coli DH5α was used for DNA cloning and plasmid propagation. Agrobacterium tumefaciens (Ag. tumefaciens) AGL1 was employed for the genetic transformation of uridine/uracil auxotrophic C. militaris strains. All the microbial strains were provided by the Genomics Unit, National Key Laboratory of Enzyme and Protein Technology, University of Science, Vietnam National University, Hanoi.

2.2. Media for microbial cultivation and genetic transformation

Luria-Bertani medium (10 g peptone, 5 g yeast extract, 5 g NaCl, in 1 L distilled water) was used for bacterial cultivation.

Potato dextrose agar (PDA) medium (20 g glucose, the infusion broth from 200 g peeled fresh potato, 16 g agar, in 1 L distilled water) and Czapek-Dox agar (CDA) medium (30 g sucrose, 3 g NaNO₃, 1 g KH₂PO₄, 0.5 g KCl, 0.5 g MgSO₄.7H₂O, 0.01 g FeSO₄.7H₂O, 0.005 g CuSO₄.5H₂O, 0.001 g ZnSO₄.7H₂O, 16 g agar, in 1 L distilled water, pH 5.5) were employed for fungal cultivation.

CDA supplemented with 0.1% (w/v) uridine and 0.1% (w/v) uracil (CDA+Uri+Ura) was used for genetic transformation, and CDA+Uri+Ura supplemented with 0.2% (w/v) 5-fluoroorotic acid (5-FOA) (CDA+Uri+Ura+5-FOA) was recruited for screening 5-FOA resistant transformants.

Induction medium (IM) medium (40 mL 1M MES solution, 400 mL 2.5x MM salt solution, 0.9 g glucose, 5 mL glycerol, 200 μM acetosyringone, 16 g agar, in 1 L distilled water), and IM supplemented with different concentrations of uridine and/or uracil were employed for the co-cultivation process of the ATMT protocol. For liquid IM, 1.8 g glucose was used. The 1M MES solution (pH 5.3) was prepared by dissolving 195.2 g MES (2-(N-morpholino)ethanesulfonic acid) in 1 L distilled water. The 2.5x MM salt solution (3.625 g KH₂PO₄, 5.125 g K₂HPO₄, 0.375 g NaCl, 1.25 g MgSO₄.7H₂O, 0.165 g CaCl₂.2H₂O, 0.0062 g FeSO₄.7H₂O, 1.25 g (NH₄)₂SO₄, in 1 L distilled water) was autoclaved and separately stored at room temperature.

2.3. Spore suspension preparation

The wild-type homokaryotic C. militaris strains (Supplementary Fig. S1) were cultivated on PDA, while uridine/uracil auxotrophic C. militaris strains were grown on CDA+Uri+Ura. Culture plates were incubated at 25 °C in the dark for 3 d to promote fungal mycelial growth, and consequently under an illumination cycle of 12 h light and 12 h dark with a light intensity of 500−700 lux for 5 d to promote sporulation. Sterile distilled water was added to the agar surface of each culture plate, and conidia (asexual spores) were separated from fungal mycelia with a sterile glass spreader. The fluid containing spores was filtered through Miracloth (Calbiochem, Darmstadt, Germany), and the filtrate was collected in a sterile Falcon conical tube. After centrifugation at 3743 ×g for 10 min, the spore pellet was carefully washed three times with sterile distilled water to remove maximally trace elements and medium nutrients. Finally, the spore pellet was resuspended in sterile distilled water to obtain a spore suspension. The spore suspension was adjusted to the concentration of 10⁶ or 10⁷ spores/mL, and stored at 4 °C for the next experiments.

2.4. Genomic DNA and cDNA preparation

One milliliter of a spore suspension (10⁶ spores/mL) was inoculated in an Erlenmeyer flask containing 50 mL of potato dextrose broth (PDB) medium or Czapek-Dox broth (CDB) medium. The flask was shaken at 200 rpm, 25 °C for 3 d. Fungal mycelial biomass was harvested by filtering the culture through Miracloth. Fungal biomass was pulverized in liquid nitrogen to obtain fine powder for genomic DNA extraction. For PCR screening, genomic DNA extraction was conducted as previously described (Nguyen et al., 2016). For real-time PCR analysis, genomic DNA and total RNA samples were extracted from the fungal biomass powder using PureHelix™ Genomic DNA Prep Kit (NanoHelix, Daejeon, South Korea) and PureHelix™ Total RNA Purification Kit (NanoHelix, Daejeon, South Korea) following the manufacturer’s instructions, respectively. The cDNA was prepared from the total RNA using SensiFAST™ cDNA Synthesis Kit (Meridian Bioscience, Ohio, USA) following the protocol described in the manufacturer’s manual.

2.5. PCR amplification and real-time PCR analysis

Phusion high-fidelity DNA polymerase (Thermo Scientific, Waltham, USA) was employed to ensure accurate PCR amplifications for DNA cloning, while GoTaq Green Master Mix (Promega, Madison, USA) was used for PCR screening. A procedure for PCR amplification comprised 94 ºC (5 min); 35 cycles of 94 ºC (30 s), 58−60 ºC (30 s), 72 ºC (1 min); and 72 ºC (10 min). Primers used for PCR amplifications are listed in Supplementary Table S1.

For gene copy number determination by real-time PCR (qPCR), 50 ng/µL genomic DNA was used, while 100 ng/µL cDNA was employed for evaluating gene expression in C. militaris. Rotor-Gene Q (Qiagen, Hilden, Germany), SensiFAST^TM SYBR® No-ROX Kit (Meridian Bioscience, Ohio, USA), and specific primers (Supplementary Table S1) were employed for qPCR amplifications. A procedure of qPCR amplification comprised 50 °C (2 min), 95 °C (10 min), 40 cycles of 95 °C (15 s), and 60 °C (30 s). The copy number of the CmFE gene in recombinant strains was compared to the wild-type homokaryotic C. militaris G2, which carries only a single CmFE. The 2^−∆Ct formula was employed to calculate the gene copy number, where ΔCt=Ct of the strain of interest - Ct of the reference strain (Schmittgen & Livak, 2008).

2.6. Construction of a binary vector for the pyrG gene deletion in Cordyceps militaris

The pyrG cassette of 2.610 kb that comprises the 5’ flanking region of 779 bp, the pyrG open reading frame (ORF) of 1104 bp, and the 3’ flanking region of 727 bp was amplified from genomic DNA of C. militaris G2 by PCR using the primer pair CmpyrG-F/CmpyrG-R (Supplementary Table S1). The PCR product was digested with EcoRI and HindIII, and purified with MEGAquick-spin^TM Plus Total Fragment DNA Purification Kit (iNtRON Biotechnology, Gyeonggi-do, South Korea). The digested DNA product was ligated to the pKO2 binary vector (Thai et al., 2024), which was also treated with EcoRI and HindIII using T4 DNA ligase to generate a recombinant vector (namely pCmpyrG). The pCmpyrG binary vector was further treated with StuI to remove a short fragment of 378 bp, and self-ligated by T4 DNA ligase to obtain a new construct for the pyrG gene deletion. The obtained plasmid, pCmG, was used to delete pyrG in C. militaris by homologous recombination.

2.7. Deletion of the pyrG gene in homokaryotic Cordyceps militaris strains

For the deletion of the pyrG gene, the binary vector pCmG was transformed into Ag. tumefaciens AGL1 by electroporation. The transformed bacterial cells were pre-induced in the liquid IM containing 200 µM acetosyringone (AS) at 200 rpm, 28 °C for 6 h (OD₆₀₀ = 0.6−0.8). A volume of 100 µL of the pre-induced bacterial culture was mixed with 100 µL of a fungal spore suspension (10⁶ spores/mL) in a 1.5 mL tube. The mixture was spread on the 90-mm filter paper membrane (Sartorius, Göttingen, Germany) and placed on the IM agar plate. The plate was incubated at 22 °C in darkness for 60 h. The filter paper was then transferred to the selective medium CDA+Uri+Ura+5-FOA with cefotaxime (300 µg/mL). The plate was additionally incubated for 7−10 d. Fungal transformants appearing on the plate were collected as potential uridine/uracil auxotrophic mutants (∆pyrG). The 5-FOA resistant transformants were simultaneously cultivated on different media, including CDA, CDA+Uri+Ura, and CDA+Uri+Ura+5-FOA at 25 °C for 5−7 d. The transformants purified by single spore isolation were grown in CDB+Uri+Ura for genomic DNA extraction. The pyrG deletion in these transformants was confirmed by PCR using two specific primer pairs, CmpyrG-F/CmpyrG-R and CmpyrG-ORF-F/CmpyrG-ORF-R (Supplementary Table S1).

2.8. Construction of binary vectors for boosting recombinant expression in Cordyceps militaris

2.8.1. pEX3.1A carrying the Aspergillus oryzae pyrG auxotrophic marker (AopyrG) and the DsRed gene under the regulation of the Cordyceps militaris gpd1 promoter

The 5’ upstream sequence of the gpd1 gene encoding the glyceraldehyde 3-phosphate dehydrogenase (GenBank: XM_006669697) extracted from the genome database of C. militaris Cm01 (https://mycocosm.jgi.doe.gov/Cormi1/Cormi1.home.htmL) under the locus name CCM_04549m.01 was used to design the specific primer pair Cmgpd1-F/Cmgpd1-R (Supplementary Table S1). The gpd1 promoter sequence was amplified from the genomic DNA of C. militaris G2 by PCR using Phusion high-fidelity DNA polymerase. The PCR product was digested with SpeI and PmlI, and purified for the ligation to the pEX2B binary vector (Nguyen et al., 2017) to replace the amyB promoter at the respective restriction sites. The constructed binary vector named pEX3.1A was confirmed by PCR and by the digestion with EcoRI and XhoI.

2.8.2. pEX3.1B carrying the AopyrG auxotrophic marker and the DsRed gene under the regulation of the Cordyceps militaris tef1 promoter

The 5’ upstream sequence of the tef1 gene encoding the elongation factor 1-alpha (GenBank: XM_006665968) was extracted from the C. militaris Cm01 genome database (https://mycocosm.jgi.doe.gov/Cormi1/Cormi1.home.htmL) under the scaffold name scaffold_00001:2566913-2567433. The tef1 promoter sequence was amplified from the genome of C. militaris G2 using the primer pair Cmtef1-F/Cmtef1-R (Supplementary Table S1). The PCR product was purified and treated with SpeI and PmlI. The digested product was ligated into the pEX2 binary vector (Nguyen et al., 2016) at the respective restriction sites to replace the Aspergillus nidulans gpdA promoter. The recombinant plasmid (namely pEX3.1B) was verified by the digestion with EcoRI.

2.8.3. pEX3.2A carrying the CmpyrG auxotrophic marker and the DsRed gene under the regulation of the Cordyceps militaris gpd1 promoter

The C. militaris pyrG (CmpyrG) cassette, including the 5’ upstream sequence (promoter) of 980 bp, the open reading frame (ORF) of 1104 bp, and the 3’ downstream sequence (terminator) of 250 bp, was amplified from genomic DNA of C. militaris G2 using the primer pair CmpyrG-F1/CmpyrG-R1 (Supplementary Table S1). The PCR product was treated with EcoRI and SpeI. The digested product was purified and ligated to the respective restriction sites in pEX3.1A to replace the AopyrG marker. The recombinant binary vector (namely pEX3.2A) carrying the CmpyrG auxotrophic marker was verified by the digestion with XhoI.

2.8.4. pEX3.2B carrying the CmpyrG auxotrophic marker and the DsRed gene under the regulation of the Cordyceps militaris tef1 promoter

The CmpyrG marker was isolated from pEX3.2A by EcoRI and SpeI. The purified fragment was ligated to the respective restriction sites in pEX3.1B to replace the AopyrG marker. The recombinant plasmid (namely pEX3.2B) carrying the CmpyrG auxotrophic marker and the DsRed expression cassette under the regulation of the tef1 promoter was verified by the digestion with EcoRI.

2.8.5. pEX3.2B-CmFE carrying the CmFE gene under the regulation of the Cordyceps militaris tef1 promoter

The endogenous CmFE gene sequence (GenBank: MK032688) encoding a serine protease (Katrolia et al., 2020) was amplified from the genomic DNA of C. militaris G2 using Phusion high-fidelity DNA polymerase and the primer pair CmFE-ORF-F/CmFE-ORF-R (Supplementary Table S1). PCR product was digested with PmlI and BamHI, and purified for the ligation to pEX3.2B at the respective restriction sites. The obtained plasmid pEX3.2B-CmFE was used for overexpressing the CmFE gene in C. militaris G2∆pyrG.

2.9. Evaluating the influence of key factors on the ATMT efficiency in the uridine/uracil auxotrophic Cordyceps militaris strains

The uridine/uracil auxotrophic C. militaris G2 strain and the pCmpyrG binary vector containing T-DNA with only the CmpyrG auxotrophic marker were employed for evaluating the influence of some key factors on the efficiency of ATMT. These factors included co-cultivation temperatures (20, 22, 25, 28 °C), uridine/uracil concentrations (0.01% uridine, 0.01% uracil, 0.01% uridine + 0.01% uracil, 0.02% uridine, 0.02% uracil, 0.02% uridine + 0.02% uracil) added to IM, time intervals for co-cultivation (24, 48, 60, 72 h), and spore concentrations (10⁴, 10⁵, 10⁶, 10⁷ spores/mL). The ATMT procedure was conducted as described above, and the CDA medium was used for selecting prototrophic transformants. Data were expressed as mean ± standard deviation. Statistical analyses were conducted with GraphPad Prism 8 (GraphPad Software, San Diego, USA) using one-way ANOVA with Multiple comparisons and Tukey’s test. Significant differences were considered with p < 0.05.

2.10. Examining fungal transformants expressing the DsRed reporter gene

The binary vectors (pEX3.1A, pEX3.1B, pEX3.2A, pEX3.2B) carrying the pyrG auxotrophic marker (AopyrG from A. oryzae or CmpyrG from C. militaris) were recruited for genetic transformation of the uridine/uracil auxotrophic C. militaris strains via the ATMT method. Fungal transformants were transferred to the CDA plates. The plates were incubated at 25 °C for 5 d. For examining the DsRed expression, some transformants were selected for cultivating on sterile microscopic slides containing PDA. The slides were maintained in sterile Petri plates containing some moistened filter paper pieces at 25 °C for 5 d. The DsRed expression in the fungal transformants was detected under an Axioplan fluorescence microscope system (Carl Zeiss, Göttingen, Germany).

2.11. Assay of protease activity

One milliliter of a spore suspension (10⁶ spores/mL) from the wild-type C. militaris G2 strain or the CmFE-overexpressing strains was inoculated to a 250 mL flask containing 50 mL of the CDB medium. The flasks were incubated in an orbital shaker at 150 rpm, 25 °C for 7 d. Fungal cultures were filtered through Miracloth to remove mycelial biomass. Consequently, filtrates were centrifuged at 13362 ×g, 4 °C for 10 min, and supernatants as crude enzyme solutions were used for protease activity assay. Protease activity was determined by using Azocasein (Megazyme, Bray, Ireland) following the manufacturer’s instructions. The reaction was incubated at 37 °C for 3 h. The absorbance of the supernatant solutions was measured against the reaction blank at 440 nm. Experiments were performed in triplicate. Statistical analysis was carried out with GraphPad Prism 8 using one-way ANOVA with Multiple comparisons and Tukey’s test. Significant differences were considered with p < 0.05.

3. Results

3.1. Identification of a pyrG/URA3 orthologous gene in the Cordyceps militaris genome

We used a deduced PyrG protein sequence (AO090011000868) from Aspergillus oryzae RIB40 as a query to search for its orthologs in the genome of the C. militaris Cm01 strain (https://mycocosm.jgi.doe.gov/Cormi1/Cormi1.home.htmL). The C. militaris Cm01 strain was identified to be a homokaryotic strain, which carries only the mating-type locus MAT1-1 in the genome (Zheng et al., 2011a). The result showed that there is only a single pyrG orthologous gene (CmpyrG) present in the C. militaris Cm01 genome with the locus name CCM_05256m.01. The same sequence of C. militaris pyrG was also detected in the GenBank database under different accession numbers, including EGX91099, XP_006670464, and XM_006670401. This gene has a length of 1104 bp encoding a putative polypeptide of 367 amino acids. Comparative analysis revealed that the PyrG protein sequence of C. militaris exhibits high homology to its orthologs from the entomopathogenic fungus Beauveria bassiana and mycoparasitic Trichoderma species with amino acid identities of 78.14-92.33%. However, C. militaris PyrG displayed low homology of 47.19-49.44% when compared to its orthologs from saprophytic Aspergillus and Penicillium species, and only 45.8% to Ura3 of the yeast Saccharomyces cerevisiae (Fig. 1A). Phylogenetic analysis confirmed that the C. militaris PyrG protein is more closely related to the PyrG orthologs from the Beauveria and Trichoderma species rather than those from the Aspergillus and Penicillium species (Fig. 1B).

Fig. 1 - Comparative analysis of the Cordyceps militaris PyrG with other fungal PyrG/Ura3 proteins. A: The amino acid (aa) identity between the C. militaris PyrG and other PyrG/Ura3 homologs by analyzing with Clustal Omega (Sievers & Higgins, 2018). B: The phylogenetic tree was constructed with MEGA X (Kumar et al., 2018) using the Neighbor-Joining method with 1000 bootstrap replicates. The deduced PyrG/Ura3 protein sequences were extracted from the GenBank database. Genetic scale bar, statistical reliability (%) at nodes of the tree branches, and accession numbers are indicated.

3.2. Generation of uridine/uracil auxotrophic mutants by the pyrG deletion is effective in homokaryotic Cordyceps militaris strains

In this study, we generated a binary vector harboring a T-DNA construct with the integration of the pyrG gene deletion cassette (Supplementary Fig. S2). This cassette was transformed into five wild-type homokaryotic C. militaris strains (Supplementary Fig. S1) via the ATMT method. To confirm the uridine/uracil auxotrophy, the 5-FOA resistant transformants from the transformation plates were examined for their growth on different media (CDA, CDA+Uri+Ura, and CDA+Uri+Ura+5-FOA). The results showed that all the tested transformants (100%) were uridine/uracil auxotrophic and truly resistant to 5-FOA (Fig. 2A, Supplementary Fig. S3A). To confirm whether the 5-FOA-resistant transformants were the pyrG deletion mutants (∆pyrG), the genomic DNA samples of these transformants were prepared for PCR analysis with two specific primer pairs CmpyrG-F/CmpyrG-R and CmpyrG-ORF-F/CmpyrG-ORF-R (Supplementary Table S1). In principle, if homologous recombination occurs between the pyrG deletion construct and the native pyrG locus, a 378 bp fragment of the pyrG open reading frame (ORF) will be deleted from the genome of C. militaris (Fig. 2B). Our results showed that with the primer pair CmpyrG-F/CmpyrG-R, a DNA band of 2.610 kb was amplified in the wild-type C. militaris strain, while a shorter band of 2.232 kb was present in all the tested mutants. Furthermore, using the primer pair CmpyrG-ORF-F/CmpyrG-ORF-R to amplify the internal portion of the pyrG ORF, a DNA band of 0.611 kb appeared in the wild-type C. militaris strains, but no bands were obtained for the pyrG deletion mutants. These results revealed that the 378 bp fragment of the pyrG ORF was successfully deleted from the genomes of the tested mutants (Fig. 2C, Supplementary Fig. S3B). Taken together, all the 5-FOA resistant transformants (100%) generated in the homokaryotic C. militaris strains were proven as the pyrG deletion mutants.

Fig. 2 - Deletion of the pyrG gene in the homokaryotic Cordyceps militaris. A: Evaluating the growth of the uridine/uracil auxotrophic mutants on different culture media. Three representative mutants (M1, M2, M3) were selected for the illustration. B: The scheme for the pyG deletion (∆pyrG) by homologous recombination in C. militaris. C: Confirmation of the ∆pyrG mutants by PCR using two specific primer pairs (Supplementary Table S1). PCR products were analyzed on agarose gels. M stands for the 1 kb DNA ladder. Genomic DNA from the wild-type (WT) strain was used for the reference control, and sterile distilled water was employed for the negative control (-).

3.3. Genetic transformation of the uridine/uracil auxotrophic Cordyceps militaris is highly efficient with the ATMT method

We used the pCmpyrG binary vector (Supplementary Fig. S2A) with the T-DNA construct carrying only the native pyrG selectable marker from C. militaris for the genetic transformation of the uridine/uracil auxotrophic mutant strains. The first experiments of genetic transformation in C. militaris G2 ∆pyrG were based on our previously published ATMT procedures for the filamentous fungi A. oryzae and Penicillium rubens (Nguyen et al., 2016; Tran et al., 2023) with co-cultivation temperatures of 20−28 °C, the time of 60 h for co-cultivation and the uridine/uracil supplementation of 0.02%. The results showed impressively a high transformation yield of 453 ± 21 transformants per 10⁵ conidia when the co-cultivation was performed at 25 °C. The data also indicated the feasibility of the genetic transformation of the uridine/uracil auxotrophic C. militaris at the lower and higher temperatures (20, 22, 28 °C) with the yields of 140−174 transformants per 10⁵ conidia (Fig. 3A). We then chose the co-cultivation temperature of 25 °C to examine effects of other factors, including the time intervals for co-cultivation (24, 48, 60, 72 h), uridine/uracil supplementations (0.01−0.02% of uridine, uracil, or in combination), and spore concentrations (10³−10⁶ conidia/plate) on the ATMT efficiency in C. militaris ∆pyrG. Our results revealed that the supplementation of a combination of 0.01% uridine and 0.01% uracil to IM for the co-cultivation at 25 °C for 60 h significantly increased the ATMT efficiency up to 783 ± 77 transformants per 10⁵ conidia (Fig. 3B). Examinations of changes in other time intervals for co-cultivation (24, 48, 72 h) and spore concentrations (10³, 10⁴, 10⁶ conidia/plate) did not show improvements in transformation efficiency (Fig. 3C, D). The optimal procedure for the ATMT method in the uridine/uracil auxotrophic C. militaris is summarized in Supplementary Fig. S4.

Fig. 3 - Examination of key factors for genetic transformation of the uridine/uracil auxotrophic Cordyceps militaris by ATMT. A: Different temperatures for co-cultivation. B: Supplementations of different concentrations of uridine, uracil, or a combination of uridine (Uri) and uracil (Ura) to IM. C: Time intervals for co-cultivation. D: Different spore concentrations of the uridine/uracil auxotrophic C. militaris. Data were calculated from three replicates and expressed as mean ± standard deviation. Different lowercase letters above columns indicate statistically significant differences (p < 0.05).

We further evaluated the optimal ATMT procedure in four other uridine/uracil auxotrophic C. militaris strains, including H8∆pyrG, N19∆pyrG, G4∆pyrG, and N9787∆pyrG. The results indicated this ATMT procedure was also effective in these uridine/uracil auxotrophic strains of C. militaris with high transformation yields of 109-810 transformants per 10⁵ conidia (Table 1). Our data reveal that the uridine/uracil auxotrophy-based ATMT efficacy varied significantly among the tested C. militaris strains and appeared to be independent of the mating types of these strains.

Table 1. The ATMT efficiency in the uridine/uracil auxotrophic Cordyceps militaris strains

Uridine/uracil auxotrophic C. militaris strains	Mating type	Transformation efficiency (transformants per 105 conidia)
H8∆pyrG	MAT1-1	109 ± 35
N19∆pyrG	MAT1-1	189 ± 47
G4∆pyrG	MAT1-1	810 ± 102
G2∆pyrG	MAT1-2	783 ± 77
N9787∆pyrG	MAT1-2	392 ± 55

3.4. Successful construction of binary vectors harboring the native gpd1 and tef1 promoters for enhancing heterologous expression in Cordyceps militaris

We first examined the expression of four common genes, including tub1 (tubulin beta chain, GenBank: XM_006669203), tef1 (elongation factor 1-alpha, GenBank: XM_006665968), gpd1 (glyceraldehyde-3-phosphate dehydrogenase, GenBank: XM_006669697) and rpb1 (polymerase II large subunit, GenBank: KC242729), in the homokaryotic C. militaris G2 during mycelial growth under liquid shaking cultivation. These genes are well-known as reference genes for real-time PCR analysis in filamentous fungi and were previously evaluated for their expression in C. militaris under different developmental stages of fruiting body formation on solid culture media (Flatschacher et al., 2023; Lian et al., 2014). The calculation for the expression levels of all four genes was based on the threshold cycle (Ct) values of real-time PCR analysis. The results showed that the Ct values for tub1, tef1, gpd1, and rpb1 corresponded to 16.97, 14.59, 15.88, and 21.66, respectively. Our data also revealed that during the mycelial growth of C. militaris under shaking cultivation, tef1 and gpd1 were strongly expressed with the Ct values ranging from 14.59 to 15.88 (Fig. 4A).

Fig. 4 - The construction of binary vectors carrying the native gpd1 and tef1 promoters for enhancing recombinant expression in Cordyceps militaris. A: The distribution of cycle threshold (Ct) values by real-time PCR analysis for the tested genes in C. militaris under liquid shaking cultivation. The experiment was conducted with three independent replicates for each gene, and the mean values are presented as the line crossing the box. B: Four binary vectors (pEX3.1A, pEX3.1B, pEX3.2A, and pEX3.2B) were constructed for recombinant expression in C. militaris. The T-DNA of these vectors contains the pyrG auxotrophic marker from A. oryzae or C. militaris, and the DsRed expression cassette under the regulation of the gpd1 promoter (Pgpd1) or the tef1 promoter (Ptef1) from C. militaris. Single sites of the restriction enzymes were indicated. LB (left border) and RB (right border) display the T-DNA constructs of the binary vectors.

The 5’ upstream sequences of gpd1 (963 bp) and tef1 (904 bp) were amplified from the genome of the wild-type C. militaris G2 by PCR using the primer pairs Cmgpd1-F/Cmgpd1-R and Cmtef1-F/Cmtef1-R, respectively (Supplementary Table S1). The native gpd1 promoter sequence was used to replace the amyB promoter in the pEX2B binary vector (Nguyen et al., 2017) to generate pEX3.1A, while the native tef1 promoter sequence was employed for the replacement of the gpdA promoter in the pEX2 binary vector to obtain pEX3.1B. Further, we substituted the A. oryzae pyrG auxotrophic marker (AopyrG marker) in pEX3.1A and pEX3.1B with the native C. militaris pyrG auxotrophic marker (CmpyrG marker) to generate pEX3.2A and pEX3.2B. The native pyrG marker including the promoter sequence of 980 bp, the open reading frame of 1104 bp, and the terminator of 250 bp was amplified by PCR using the primer pair CmpyrG-F1/CmpyrG-F1 (Supplementary Table S1). Especially, the DsRed reporter gene in the newly constructed binary vectors is changeable and can be replaced with a gene of interest via the available restriction enzyme sites (Fig. 4B).

Using the established ATMT procedure in the uridine/uracil auxotrophic C. militaris, we demonstrated that all these binary vectors are functional to recover the wild-type prototrophic phenotype for the C. militaris ∆pyrG strains via the add-back event of the pyrG marker (Fig. 5A; Supplementary Fig. S5A). To confirm the prototrophic growth recovery in the transformants, we selected randomly 80 transformants generated from two independent uridine/uracil auxotrophic C. militaris strains. The results showed that all the selected transformants could grow prototrophically on the minimal CDA medium without a uridine/uracil supplementation (Supplementary Fig. S6A, B). Furthermore, the successful integrations of the T-DNA carrying the DsRed expression cassette into the genomes of the selected transformants were confirmed by PCR using the DsRed-specific primer pair (Supplementary Figs. S5B, S6C). To examine the expression of the DsRed gene, we grew some selected transformants directly on microscopic slides containing the CDA medium. After the incubation of 5 d at 25 °C, the culture slides were examined under a fluorescence microscope with a filter for detecting the DsRed signal. The results showed that all the tested transformants from different uridine/uracil auxotrophic C. militaris strains displayed obvious signals for DsRed. In some cases, the strong DsRed signal could be clearly observed in the whole fungal mycelia and the conidiophores with conidia (Fig. 5B, C; Supplementary Fig. S5B, C). Taken together, we conclude that the native gpd1 and tef1 promoters represent strong constitutive promoters for enhancing gene expression in C. militaris. Notably, the pyrG selectable marker from A. oryzae with a low homology of amino acids (less than 50%) compared to the C. militaris pyrG is still functional for the genetic transformation in the uridine/uracil auxotrophic C. militaris.

Fig. 5 - Genetic transformation of the uridine/uracil auxotrophic Cordyceps militaris strains using the ATMT method. A: The binary vectors pEX3.1B and pEX3.2B were evaluated for their functionality via genetic transformation of two different uridine/uracil auxotrophic C. militaris strains (H8∆pyrG, G2∆pyrG). Selected transformants were transferred to CDA and CDA+Uri+Ura for the verification of prototrophic growth. B: The C. militaris H8 strain expressing the DsRed reporter gene. C: The C. militaris G2 strain expressing the DsRed reporter gene. These strains were examined for the DsRed signal under fluorescence microscopy using the slide culture method.

3.5. The enhanced expression of the CmFE gene encoding a serine protease in Cordyceps militaris using the constructed ATMT system

We constructed the binary vector pEX3.2B-CmFE carrying the C. militaris CmFE gene under the regulation of the native tef1 promoter to validate the established ATMT platform for overexpressing this endogenous target gene. The CmFE gene encoding a serine protease with fibrinolytic activity was previously reported in C. militaris (Katrolia et al., 2020). The CmFE overexpression cassette from pEX3.2B-CmFE was integrated into the genome of C. militaris G2∆pyrG using the optimal ATMT procedure. Two independent CmFE overexpression strains (OE11, OE18) were selected and examined for their prototrophic growth ability. The results showed that these strains could normally grow on the CDA medium like the wild-type C. militaris G2 strain, whereas the uridine/uracil auxotrophic G2∆pyrG was unable to grow on this minimal medium. PCR analysis using a combined primer pair Cmtef1-F/CmFE-ORF-R that specifically amplified the CmFE overexpression cassette (Supplementary Table S1) confirmed the successful integration of the respective T-DNA construct into the genomes of these selected strains (Fig. 6A). The results from real-time PCR quantification estimated that overexpression strains OE11 and OE18 possess 2 or 3 copies of CmFE in the genome, respectively in comparison to the wild-type strain with only a single copy of this gene (Fig. 6B). These overexpression strains were grown in the CDB medium for protease activity assay. The data showed that both the CmFE-overexpressing strains exhibited better protease activity (91 U/mL for OE11, and 95 U/mL for OE18) compared to the wild-type G2 strain (46 U/mL). However, strain OE18 possessing the genomic integration of the estimated 3 copies for CmFE showed similar protease activity compared to strain OE11 with the estimated 2 copies for this gene in the genome (Fig. 6C). The reason for this situation still needs to be clarified in the future with more CmFE-overexpressing strains and additional experiments for expression level quantifications.

Fig. 6 - The overexpression of the endogenous CmFE gene in Cordyceps militaris using the newly constructed ATMT system. A: The pEX3.2B-CmFE binary vector was used for the overexpression of the CmFE gene. Two selected transgenic strains were examined for prototrophic growth on CDA. The presence of the CmFE-overexpression cassette in the genomes of these strains was confirmed by the PCR amplification with the specific primer pair Cmtef1-F/CmFE-ORF-R. M stands for the 1 kb DNA ladder. Genomic DNA from the uridine/uracil auxotrophic C. militaris G2∆pyrG strain and the plasmid pEX3.2B-CmFE were used as DNA templates for the negative control (-) and the positive control (+), respectively. B: Estimation of copy number of the CmFE gene based on the cycle threshold (Ct) values determined by real-time PCR analysis in two CmFE-overexpressing strains (OE11, OE18) compared to the wild-type (WT) C. militaris G2 strain. C: Determination of protease activity in the CmFE-overexpressing strains compared to the wild-type strain. The experiments were performed in triplicate and data are presented as mean ± deviation. The asterisks (****) indicate a statistically significant difference with p < 0.0001, ns stands for “not significant” with p > 0.05.

4. Discussion

Currently, genetic transformation methods in C. militaris still mainly use dominant resistance markers, including antibiotic resistance genes and herbicide resistance genes for selecting transformants (Lou et al., 2021; Wang et al., 2017, 2021a, 2021b; Wāng et al., 2020; Xia et al., 2017; Zheng et al., 2011b). The employment of dominant resistance markers may pose risks of spreading resistance genes and does not meet safety standards in food-grade production (Arnau et al., 2020; Vandermeulen et al., 2011). Uridine and uracil are required for the growth and development of fungi, and the genes responsible for their biosynthesis are designated as URA3 in yeasts and pyrG in filamentous fungi (Nguyen et al., 2017; Pronk, 2002). The pyrG gene encoding orotidine 5'-monophosphate decarboxylase is required for the uridine/uracil biosynthesis in fungi, and disruption of this gene leads to uridine/uracil auxotrophy. Orotidine 5'-monophosphate decarboxylase can also convert 5-FOA to 5-fluorouracil which is toxic to wild-type fungal cells. As a result, pyrG-deficient fungal mutants are unable to grow on the minimal medium, and become resistant to 5-FOA (Binh et al., 2021; Ko et al., 2008; Nguyen et al., 2017; Tran et al., 2023).

Our previous work proved that the deletion of pyrG in the filamentous fungus A. oryzae by using the ATMT method resulted in the uridine/uracil auxotrophic transformants with the ability of 5-FOA resistance. The results from PCR analysis indicated that all of the 5-FOA resistant A. oryzae transformants (100%) existed as the pyrG deletion mutants (Nguyen et al., 2017). However, using the same strategy, the efficiency of the pyrG deletion in the penicillin-producing fungus P. rubens was very low at only 1%, and the majority of the 5-FOA resistant transformants appeared as ectopic strains for unknown reasons (Tran et al., 2023). In this study, we indicated that the generation of uridine/uracil auxotrophic mutants by the deletion of the pyrG gene using ATMT is very effective in homokaryotic C. militaris strains. All the 5-FOA resistant transformants (100%) appearing on the selective medium were proven as the pyrG deletion mutants. (Fig. 2; Supplementary Fig. S3). Therefore, the construction of uridine/uracil auxotrophic strains as recipients for genetic transformation in the medicinal mushroom C. militaris becomes simple and time-saving.

ATMT has been demonstrated to be highly efficient for genetic transformation in filamentous fungi. However, the efficacy of this transformation method is usually influenced by some factors such as temperatures and time intervals for the co-cultivation step, spore concentrations, acetosyringone concentrations, uridine/uracil supplementations (Binh et al., 2021; de Groot et al., 1998; Michielse et al., 2005; Vu et al., 2018). In a recent study conducted by Li et al. (2022), the authors generated a uridine/uracil auxotrophic C. militaris mutant by deleting the pyrG gene and reported a relatively low ATMT efficiency in this mutant strain with only 75 ± 35 transformants per 10⁶ conidia. In our present study, we have provided full research for establishing a highly efficient ATMT system with suitable binary vectors based on the uridine/uracil auxotrophic mechanism. The constructed ATMT system was evaluated in five different uridine/uracil auxotrophic C. militaris strains. We used the optimal acetosyringone concentration of 200 µM as previously published for ATMT in C. militaris and other filamentous fungi (de Groot et al., 1998; Zheng et al., 2011b), and examined changes in some other factors. We found that the best conditions for the ATMT method in the uridine/uracil auxotrophic C. militaris should include the uridine/uracil supplementation of 0.01% to IM, the spore concentration of 10⁵ conidia per plate, and the co-cultivation at 25 °C for 60 h. Under these conditions, the uridine/uracil auxotrophy-based ATMT efficiency in our study could reach 109−810 transformants per 10⁵ conidia depending on each fungal strain (Fig. 3; Table 1). These results are comparable to the yields of 30−600 transformants per 10⁵ conidia obtained from the hygromycin resistance-based ATMT in the C. militaris JM4 strain (Zheng et al., 2011b). The major limitation of the hygromycin resistance-based ATMT system is that it requires hygromycin as a costly antibiotic selection agent and the respective hygromycin resistance gene for genetic transformation events. Using antibiotic resistance genes as selection markers in genetic manipulation is not acceptable for the production of commercial bioproducts (Arnau et al., 2020; Vandermeulen et al., 2011). In comparison to other uridine/uracil auxotrophy-based ATMT systems established in some industrially relevant filamentous fungi, the current study provides a similar ATMT system, but with a superior transformation efficiency of up to 810 transformants per 10⁵ conidia, which is much higher than those in A. oryzae (106 transformants/10⁵ conidia), P. rubens (175 transformants/10⁵ conidia) and P. lilacinum (287 transformants/10⁵ conidia) (Binh et al., 2021; Nguyen et al., 2017; Tran et al., 2023).

In fungi, gene expression engineering strategies always require a broad spectrum of strong promoters, especially native promoters (Mojzita et al., 2019). The constitutive promoters gpd1/gpdA and tef1 have been widely exploited for enhancing recombinant expression in yeasts and filamentous fungi (Adnan et al., 2022; Kitamoto et al., 1998; Nevoigt et al., 2006; Umemura et al., 2020). By using quantitative real-time PCR, we indicated that two common reference genes gpd1 and tef1 were strongly expressed during the mycelial growth of C. militaris under liquid shaking cultivation. The promoter sequences of these genes were then integrated into the expression cassette of the binary vectors of our ATMT system for activating the expression of target genes. We showed that these native promoters were able to boost the heterologous expression of the DsRed reporter gene in C. militaris (Figs. 4, 5; Supplementary Fig. S5). Fibrinolytic enzymes are important agents for treating blood-clotting disorders. A serine protease encoded by the endogenous CmFE gene in C. militaris was reported to possess fibrinolytic activity (Katrolia et al., 2020). Using the ATMT system constructed in this study, we showed that the genomic integration of approximately 2−3 copies of the CmFE gene under the regulation of the native tef1 promoter significantly increased the protease activity in C. militaris (Fig. 6). A similar result was also reported in Aspergillus awamori, where the integration of 4 copies of a Fusarium solani cutinase expression cassette into the genome of A. awamori by ATMT led to the enhanced cutinase activity (Gouka et al., 1999).

Conclusively, the uridine/uracil auxotrophy-based ATMT system established in our work represents a safe and efficient platform for food-grade recombinant expression in the industrially important mushroom C. militaris.

Disclosure

The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.

Acknowledgments

This study was funded by Vietnam National University, Hanoi under grant number KLEPT.20.03. We thank Dr. Tran Thi Thanh Huyen, VNU University of Science for her assistance in some experiments.

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