Establishment of an Efficient Polyethylene Glycol (PEG)-Mediated Transformation System in Pleurotus eryngii var. ferulae Using Comprehensive Optimization and Multiple Endogenous Promoters

Pleurotus eryngii var. ferulae, a fungus of the genus Pleurotus, efficiently degrades lignin, especially during co-cultivation with other fungi. However, low transformation efficiency and heterologous gene expression restrict systematic studies of the molecular mechanisms and metabolic control of natural products in this mushroom. In this study, the homologous resistance marker carboxin (cbx) was used to establish a polyethylene glycol-mediated transformation (PMT) system in P. eryngii var. ferulae. Optimization of the transformation process greatly improved the number of positive transformants. In particular, we optimized: (i) protoplast preparation and regeneration; (ii) screening methods; and (iii) transformation-promoting factors. The optimized transformation efficiency reached 72.7 CFU/μg, which is higher than the average level of Pleurotus sp. (10–40 CFU/μg). Moreover, three endogenous promoters (Ppfgpd1, Ppfgpd2, and Ppfsar1) were screened and evaluated for different transcription initiation characteristics. A controllable overexpression system was established using these three promoters that satisfied various heterologous gene expression requirements, such as strong or weak, varied, or stable expression levels. This study lays the foundation for recombinant protein expression in P. eryngii var. ferulae and provides a method to investigate the underlying molecular mechanisms and secondary metabolic pathway modifications.


Introduction
Pleurotus eryngii var. ferulae, a member of the Pleurotus species, is a tetrapolar heterothallic edible mushroom [1]. The wild-type strain is mainly distributed in Europe, the Middle East, North Africa, along the Mediterranean coast, and China [2,3]. P. eryngii var. ferulae is edible and has several medicinal properties, including anti-tumor, antioxidant, anti-obesity, and immunoregulatory properties [4][5][6][7]. Moreover, P. eryngii var. ferulae secretes large amounts of lignin-degrading enzymes (laccase, manganese peroxidase, and lignin peroxidase) for industrial use [8]. These enzymes can be used to degrade lignin, provide bioenergy, decolor dyes, and treat sewage [9]. Our previous studies confirmed that co-cultivating P. eryngii var. ferulae with certain fungi can increase the transcription of multiple laccase isoenzymes [10,11]. However, the lack of an efficient genetic transformation system restricts further research on the regulatory mechanisms and prevents metabolic engineering.
The cell wall of mushroom mycelium is mainly composed of glucan and chitin. These substances form thick, strong regions that block the entry of genetic materials [17,21]. Hence, preparing high-quality protoplasts is the most critical step for genetic transformation. Additions of carrier DNA and functional substances during transformation play an auxiliary role in transformation efficiency. Strong promoters facilitate the high-level transcription and expression of heterologous genes in fungi. Homologous promoters are conducive to RNA polymerase and transcription factor recognition, reduce methylation, and improve transcription initiation efficiency [22]. The transcription initiation efficiencies of the 35S promoter (P CaMV35S ) from the cauliflower mosaic virus, the glyceraldehyde-3-phosphate dehydrogenase promoter (P gpd ), and the small GTPase promoter (P ras ) from L. edodes are all far lower than those of homologous promoters in G. lucidum [23]. Our previous studies found that pfgpd1, pfgpd2, pfsar1 (small COPII coat GTPase gene), and pfras all maintain high transcription levels under diverse culture conditions [24]. Intracellular expression of enhanced green fluorescent protein (EGFP) can be used to verify the transcription initiation efficiency of these promoters and further clarify promoter selection for a new strain genetic transformation system. This study describes a genetic transformation system for P. eryngii var. ferulae. We improved the transformation efficiency and positive transformant rate by optimizing the three core steps in the transformation process: protoplast preparation and regeneration, PEG-mediated transformation, and the effect of different added substances. Further, egfp was used as a reporter gene to compare the transcriptional initiation efficiency of four endogenous promoters in P. eryngii var. ferulae. This work provides an important foundation for research on regulatory mechanisms and metabolic engineering using P. eryngii var. ferulae.

Plasmid Construction
According to the test of fungal sensitivity, cbx resistance was used to screen positive transformants. Referring to a previous study [25], cbx is a 1,4-oxathiin derivative, which blocks the growth of basidiomycetes by inhibiting the activity of the succinate dehydrogenase B subunit (SDHB). The mutant SDHB with an amino acid substitution (His239 to Leu) confers resistance to cbx. Sdhb was amplified using genomic DNA as a template using the primers listed in Table S1. The complete pfsdhb sequence (1905 bp) was obtained using gene walking technology. This sequence consists of a promoter, the Sdhb open reading frame (ORF), and a terminator. His (CAC) at position 239 of SDHB was replaced with Leu (CTC) via site-directed mutagenesis. The mutant Pfsdhb fragment was inserted into the pMD19T simple vector backbone (Takara) to generate the pKAB plasmid ( Figure S1).

PEG-Mediated Protoplast Transformation
One hundred microliters of STC buffer containing 10 8 mL −1 protoplasts were gently mixed with 10 mg plasmid, functional substances (salmon sperm deoxyribonucleic acid (SS-DNA), lambda DNA (λDNA), spermidine, or heparin), and 150 µL PTC buffer (40% PEG4000, 50 mM CaCl 2 , and 10 mM Tris-HCl pH 7.5). The mixture was incubated on ice for 10 min. Then, 1 mL PTC buffer was added, and the mixture was incubated for an additional 30 min at 28 • C. The protoplasts were recovered by centrifugation (4 • C, 5 min at 4000 g) and resuspended in 1 mL CYM regeneration medium. Then, the protoplasts were statically cultured at 25 • C for 0-4 days (post-transformation culture without cbx resistance). The transformed protoplasts were incubated in monolayers or double layers in CYM regeneration medium with 2 mg/mL cbx for 10-14 days at 25 • C. Monolayer screening denotes that there is only one layer of the resistance medium in the plate. Double-layer screening means that the bottom medium contains no resistance medium and the upper medium contains cbx for transformant selection. Each transformant was cultured through five passages in medium containing 4 mg/mL cbx.
2.6. Transformant Verification pfsdhb was amplified using genomic DNA from the wild-type strain and transformants as templates. The molecular weight of the fragments was determined by agarose gel electrophoresis. The nucleic acid sequence and mutation site were determined by Sangon Biotech Company (Shanghai, China) [26].

Fluorescence Intensity Analysis
Egfp was inserted into the multiple cloning site of pKAB1-pKAB4 according to the above method. The transformants were transferred to CYM medium and cultured for 7 days at 25 • C while being shaken at 150 rpm. The hyphae were removed with tweezers and placed on a glass slide. A confocal laser microscope (Leica, Wetzlar, Germany) was used to compare the difference in green fluorescence intensity in the hyphae of different transformants.

RT-qPCR
The four transformants were cultured in PDB and WBM medium for 7 days. A Biospin Plant Total RNA Extraction Kit (BIOER, Hangzhou, China) was used to extract mycelial RNA from all transformants. cDNA was constructed using HiScript III RT SuperMix for qPCR containing gDNA wiper (Vazyme, Nanjing, China), as described previously [27]. The primers were designed by the primer-blast of the NCBI website (https://www.ncbi.nlm. nih.gov/tools/primer-blast/, accessed on 24 November 2021; Table S1). Sar1 was selected as the internal reference gene [28]. The target genes were quantified using the 2 −∆∆Ct method. At least three independent biological and technical replicates were performed for each sample.

Statistical Analysis
Data were presented as the mean ± standard deviation (SD). Duncan's multiple range tests (p ≤ 0.05) were used for data analysis. SPSS v.11.4 (IBM Corp., Armonk, NY, USA) was used to process data.

Resistance Gene Selection and Fungal Sensitivity
Wild-type P. eryngii var. ferulae was extremely sensitive to cbx; 2 mg/L cbx completely inhibited mycelial growth ( Figure S2a). In contrast, 200 mg/L hygB achieved the same effect ( Figure S2b), which indicates that selection with cbx is sensitive and has low cost. Plasmid pKAB containing the mutant pfsdhb gene was transformed into P. eryngii var. ferulae via PMT (Figure 1). Once pKAB was integrated into the genome, the transfected gene was stably maintained in the genome even after five passages. The pfsdhb fragment was amplified and sequenced using the transformant genome as a template. We observed two alleles at the mutation site (Figure 1), indicating that the transformant genome contained both original and mutant pfsdhb and that the heterologous gene integration method was insertion rather than replacement. The original pfsdhb gene was retained while the mutant pfsdhb from pKAB existed independently in the P. eryngii var. ferulae genome for replication and transcription. A similar phenomenon appeared for this fragment during G. lucidum transformation [29]. Thus, a genetic transformation system was established for the first time in P. eryngii var. ferulae.

Protoplast Preparation and Regeneration of P. eryngii var. ferulae
Only a few positive P. eryngii var. ferulae transformants were obtained using the initial transformation conditions. Improving the quantity and quality of P. eryngii var. ferulae protoplasts seemed the most basic solution. There are noteworthy differences in the reagents and methods used to prepare mushroom protoplasts, even for closely related species. Lysozyme in MgSO 4 buffer solution generated the largest number of P. eryngii var. ferulae protoplasts (Figure 2 and Figure S3). Mannitol and sucrose, which are typically used for P. eryngii and P. ostreatus, respectively, are not suitable for P. eryngii var. ferulae mycelia [30,31]. One reason is that lysozyme has variable substrate selectivity in different osmotic pressure stabilization buffers. Another reason is the diverse chitin and β-1,3-glucan content in different mycelial cell walls. Excessively high concentrations of stabilizer and lysozyme, reaction time, and reaction temperature all have a significant negative impact on the regeneration capability of protoplasts. Excessive enzymatic hydrolysis makes fungal cell wall repair difficult. The optimal conditions for protoplast preparation and regeneration were determined for P. eryngii var. ferulae (Figure 2). We selected 0.6 M MgSO 4 as the osmotic pressure stabilizer and mycelia were enzymatically hydrolyzed with 20 mg/mL lysozyme (final concentration) at 30 • C for 2 h. This returned 2.33 × 10 8 CFU/mL protoplasts, and the regeneration rate reached 2.93%.

Transformant Screening Methods
Screening methods were also optimized ( Figure 3). By adopting a double-layer plate method, the number of transformants obtained greatly improved from 0.8 to 5.4 CFU/µg, which is 6.75 times higher than that obtained using the monolayer plate method. This indicates that when transformants exist in protoplast form, they remain sensitive to cbx resistance. Therefore, the resistance-free medium in the lower layer ensures protoplast survival. In addition, increasing the post-transformation culture time improved the transformant yield, but an excessively long post-transformation culture time significantly increased the false positive transformant rate. Therefore, adding a 2-day resistance-free culture step after transformation yielded the highest number of transformants (11.8 CFU/µg) while ensuring a high positive rate.

Effect of Promoting Factors on Enhancing Transformation Efficiency
As the amount of SS-DNA, λDNA, and spermidine increased, the transformation efficiency of the exogenous plasmid in P. eryngii var. ferulae was higher (Figure 4a-d), with the highest efficiency using 50 µg, 70 µg, and 0.4 µmol, respectively. Heparin sodium had no effect on transformation. Moreover, these four promoting factors did not change the positive transformant rate, which remained at about 60.3%. The optimal additive was composed of 50 µg SS-DNA, 70 µg λDNA, and 0.4 µmol spermidine. This combination achieved the highest transformation efficiency (72.7 CFU/µg), which is 6.16 times higher than that before optimization. This result indicates that these promoting factors do not interfere with each other in the mixed system and that the efficiency of combined use is higher than that of each compound alone.
To ensure heterologous gene expression, the transcription initiation efficiency of the four endogenous promoters was compared using the fluorescence intensity of mycelia ( Figure 5). The hyphae of P. eryngii var. ferulae::pfgpd1-egfp and P. eryngii var. ferulae::pfsar1-egfp showed strong fluorescence intensity in each hypha. In contrast, the fluorescence of P. eryngii var. ferulae::pfgpd2-egfp was weak, while fluorescence from P. eryngii var. ferulae::pfras-egfp was not observed.
The type and number of core cis-acting elements on the four endogenous promoters (P pfgpd1 , P pfgpd2 , P pfsar1 , and P pfras ) were predicted and analyzed using PlantCARE ( Figure 5). All four promoters contain a TATA box, which defines the initial binding position of RNA polymerase II. There was a significant difference in the numbers of CAAT and GC boxes. P pfgpd1 and P pfsar1 had five and six CAAT boxes and one and two GC boxes, respectively. CAAT and GC boxes belong to the transcriptional regulatory region, which is involved in transcriptional activation [32,33]. Therefore, P pfgpd1 and P pfsar1 have strong transcription initiation efficiency. In contrast, we observed only one CAAT box and one GC box in P pfgpd2 and no GC boxes in P pfras . This is directly related to the low frequency of recruitment of RNA polymerase II in these transformants. P pfgpd1 and P pfsar1 had high transcription initiation efficiencies under different culture conditions ( Figure 6). P pfgpd1 was especially effective in CYM and WBM culture media. The transcription initiation efficiency of P pfsar1 was consistent using different culture conditions. These results indicate that P pfsar1 is a stable promoter in P. eryngii var. ferulae; egfp transcription driven by P pfgpd2 was relatively low. However, the effect was much better than that of P pfras . In general, these three important endogenous promoters (P pfgpd1 , P pfgpd2 , and P pfsar1 ) exhibit distinct characteristics for expressing heterologous genes and can satisfy a wide range of experimental needs. (a) pKAB-egfp, which does not contain any promoter, was used as the negative control; (d,g,j,m) P pfgpd1 , P pfgpd2 , P pfsar1 , and P pfras were analyzed for core cis-responsive elements and inserted into the pKAB-overexpression plasmid to express the egfp gene. In the diagrams, filled squares indicate TATA boxes, filled inverted triangles indicate CAAT boxes, and filled circles indicate GC boxes; (b,c), (e,f), (h,i), (k,l). (n,o) Phase-contrast, fluorescence, and merged images of transformant mycelium, including P. eryngii var. ferulae::pfgpd1-egfp, P. eryngii var. ferulae::pfgpd2-egfp, P. eryngii var.

Discussion
Genetic transformation is essential for investigating molecular mechanisms and facilitating mushroom breeding [12]. Previous studies showed that P. eryngii var. ferulae demonstrates excellent laccase production and dye decolorization capabilities during cocultivation [11,24,27]. However, the lack of a genetic transformation system is an obstacle that restricts further molecular studies. Transformation methods for mushrooms are complicated and differ greatly between species. For example, the transformation conditions are markedly different for the similar species P. ostreatus and P. eryngii [34,35]. Therefore, transformation in P. eryngii var. ferulae must be systematically investigated. In the present study, we optimized P. eryngii var. ferulae transformation to improve protoplast quantity and quality and to enhance transformation efficiency. We used four endogenous promoters to compare heterologous gene expression in this species.
Mutant sdhb, which confers antifungal resistance, results in a 60.3% positive transformant rate. Currently, the most frequently used antibiotic for screening mushrooms is heterologous hygB resistance [12]. However, screening with hygB has a high false-positive transformation rate. P. eryngii var. ferulae growth was completely inhibited when treated with 200 mg/L hygB, which indicates that the homologous selection marker is better than heterologous resistance. Thus, cbx should be the first choice for screening genetic transformation of edible fungi [36].
In this study, the number of positive transformants after comprehensive optimization was approximately 89.9-fold higher than that of the original transformation method. This transformation efficiency was prominent in the entire mushroom transformation system ( Table 1). High-quality protoplasts lay the foundation for successful transformation [37]. During the enzymatic hydrolysis of hyphae, lysozyme reduces protoplast viability, especially when using excessive amounts of lysozyme or extended reaction time. Suboptimal reaction conditions increase the number of protoplasts but decrease the vitality, which causes a decreased protoplast regeneration rate. Optimized transformation conditions determine the number and ratio of positive transformants. Protoplasts are relatively fragile in early growth stages and are more likely to die if they are directly cultured on plates containing antifungal compounds. Adopting the double-layer plate method and increasing the post-transformation culture time provided sufficient recovery time for the transformed protoplasts. Additionally, promoting factors can improve transformation efficiency. Both carrier DNA (SS-DNA and λDNA) reduce plasmid degradation by intracellular nucleases in P. eryngii var. ferulae. Similarly, adding 50 µg λDNA increases the transformation efficiency of P. ostreatus 50-fold [43]. Spermidine can bind to plasmids and initiate endocytosis, which improves plasmid entry into protoplasts [44]. Heparin plays an indispensable role in the genetic transformation of G. lucidum [45]. However, 4-20 µmol of heparin sodium had no effect on P. eryngii var. ferulae. A combination of SS-DNA, λDNA, and spermidine in the transformation reaction increases the P. eryngii var. ferulae transformation efficiency to reach 72.7 CFU/µg DNA.
Endogenous promoters increase the probability of plasmid integration into the genome and are more easily recognized by the fungus itself to improve transcription initiation [22,23]. This study compared the performance of four endogenous promoters in P. eryngii var. ferulae. Both P pfgpd1 and P pfsar1 have excellent transcription initiation efficiencies under diverse culture conditions. Multiple CAAT and GC boxes are distributed in the two promoters. In eukaryotes, the number of core cis-acting response elements and the distance from the transcription initiation site play a crucial role in the frequency of transcription initiation [33]. The efficiency of P pfsar1 was not affected by the carbon source, nitrogen source, or polyphenols in the medium. This explains why the sar1 gene can be used as a housekeeping gene in P. ostreatus, owing to its strong promoter stability [28]. Therefore, combined P pfsar1 and P pfgpd1 can replace heterogeneous P CaMV35S and can be used to construct a dual promoter silencing vector to improve silencing efficiency [46,47]. Only one CAAT box exists in P pfgpd2 , which leads to weak expression. Thus, these three important endogenous promoters (P pfsar1 , P pfgpd1 , and P pfgpd2 ) can be utilized to build a comprehensive exogenous protein expression system in P. eryngii var. ferulae: (i) P pfgpd1 can be used to express and obtain large amounts of exogenous proteins, especially when cultured with CYM and WBM; (ii) stable transcription initiation features of P pfsar1 can be exploited to elucidate molecular mechanisms, such as the mechanism underlying differential regulation of similar transcription factors; and (iii) P pfgpd2 can be used to express cytotoxic proteins, which can reveal the function of target genes while maintaining cell vitality.

Conclusions
The PMT system was successfully established in P. eryngii var. ferulae for the first time. High-quality protoplasts laid the foundation for this transformation. Adopting the double-layer plate method, post-transformation culture for 2 days, and the addition of 50 µg SS-DNA, 70 µg λDNA, and 0.4 µmol spermidine during the transformation process maximized transformation efficiency. Moreover, three important endogenous promoters (P pfgpd1 , P pfgpd2 , and P pfsar1 ) showed different types of transcription initiation features. A controllable overexpression system can be established using these three promoters, which can be used to select various expression levels. This work provides a method that facilitates studies of molecular mechanisms and novel secondary metabolic pathways in P. eryngii var. ferulae. This work also proposes a template to establish a genetic transformation system for wild filamentous fungi.