Efficient expression of heterologous genes by the introduction of the endogenous glyceraldehyde-3-phosphate dehydrogenase gene intron 1 in Ganoderma lucidum

Background Ganoderma lucidum, a well-known medicinal mushroom, has received wide attention as a promising cell factory for producing bioactive compounds. However, efficient expression of heterologous genes remains a major challenge in Ganoderma, hindering metabolic regulation research and molecular breeding of this species. Results We show that the presence of glyceraldehyde-3-phosphate dehydrogenase gene (gpd) intron 1 at the 5′ end of, the 3′ end of, or within the heterologous phosphinothricin-resistant gene (bar) is efficient for its expression in G. lucidum. The enhanced expression of bar is exhibited by the higher accumulation of mRNA and increased amounts of protein. Moreover, the insertion of the gpd intron 1 in the β-glucuronidase gene (gus) elevates its mRNA accumulation and enzyme activity, which facilitates the use of this reporter gene in Ganoderma. Conclusions This study has demonstrated the importance of the introduction of gpd intron 1 for the efficient expression of bar and gus in G. lucidum. The presence of the gpd intron 1 in heterologous genes increases levels of mRNA accumulation and protein expression in basidiomycete Ganoderma. The developed method may be utilized in upregulating the expression of other heterologous genes in Ganoderma. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-021-01654-8.

Molecular genetic approaches tools such as genetic transformation, disruption, and deletion of target genes have been developed in G. lucidum [12][13][14][15][16]. However, efficient expression of heterologous genes remains a main challenge in Ganoderma, although it would be valuable for metabolic regulation and molecular breeding. Successful expression of heterologous genes in basidiomycetes may depend on several factors, including an effective promoter, codon optimization, and the presence of introns [12,17,18]. Among those elements, introns have a significant effect on the expression of heterologous

Open Access
Microbial Cell Factories *Correspondence: xjuwei@163.com; jwxu@kust.edu.cn 1 Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China Full list of author information is available at the end of the article genes. In some mushroom species such as Clitopilus passeckerianus, Coprinus cinereus, and Schizophyllum commune, the addition of an intron is required for the efficient expression of the green fluorescent protein gene (gfp), the hygromycin-resistant gene, and the phleomycin-resistant gene [19][20][21].
Intron-containing G. lucidum genes represent 85.4% of predicted genes (approximately 16,113) in its 43.3-Mb genome [8], indicating the importance of introns in Ganoderma. In a previous study, we found that an extra fragment, including gpd exon 1, intron 1, and 3-bp exon 2 at the 5′ end of gfp and phosphinothricin-resistant gene (bar), was essential for their expression in G. lucudium [16]. However, the role of gpd intron 1 in regulating the expression of heterologous genes has not been yet thoroughly investigated in G. lucidum. Furthermore, how gpd intron 1 affects gene transcription, protein expression, and enzyme activity when heterologous genes are expressed in G. lucidum remains unclear.
In this study, we show that the presence of gpd intron 1 at different locations is effective in enhancing the expression of heterologous bar in G. lucidum. The efficient expression of heterologous genes is due to higher accumulation of mRNA and increased amount of protein. Moreover, the insertion of gpd intron 1 in the gus enhances its mRNA and enzyme activity, which facilitates the use of this reporter gene in Ganoderma.

Endogenous gpd intron 1 increases the expression of the heterologous bar in G. lucidum
Our previous study has shown that the insertion of the gpd fragment containing the first exon (6 bp), the first intron (67 bp), and part of the second exon (3 bp) at the 5′ end of the bar and gfp exerts a significant influence on protein expression. To investigate the effect of the endogenous gpd intron 1 on protein expression in G. lucidum, we constructed plasmids in which the heterologous codon-optimized phosphinothricin-resistance gene (opbar)-flag was regulated by the endogenous gpd promoter and the succinate dehydrogenase gene (sdh) terminator of G. lucidum (pJW-EXP-opbar-flag) (Fig. 1). Alternatively, the opbar-flag was cloned in similar plasmids that contained the gpd intron 1 directly upstream of the start codon (pJW-EXP-in-opbar-flag), 6 bp downstream of the translation start site of bar (pJW-EXP-in (M)-opbar-flag), and directly downstream of the stop codon (pJW-EXP-opbar-flag-in) (Fig. 1). These plasmids also contained a carboxin-resistance cassette, allowing selection of transformants on CYM plates with carboxin. After G. lucidum protoplasts were transformed with intron-containing and intronless opbar-flag plasmids (pJW-EXP-opbar-flag), we obtained carboxin-resistant colonies with all used plasmids. However, when these carboxin-resistance transformants were re-screened on CYM plates including phosphinothrin, no phosphinothrin-resistant colonies were obtained with plasmid pJW-EXP-opbar-flag. The phosphinotrhrin-resistance transformants were obtained with intron-containing plasmids pJW-EXP-in-opbar-flag, pJW-EXP-in (M)opbar-flag, and pJW-EXP-opbar-flag-in ( Fig. 2A, Additional file 1: Figs. S1A and S2A). PCR analysis showed that the opbar-flag has integrated into the genomes of transformants pJW-EXP-opbar-flag, transformants pJW-EXP-in-opbar-flag, pJW-EXP-opbar-in (M)-flag, and pJW-EXP-opbar-flag-in (Fig. 2B, Additional file 1: Figs. S1B and S2B). These results indicated that the introduction of the endogenous gpd intron 1 is important for efficient expression of heterologous bar in G. lucidum. Moreover, this effect was independent of the position of the gpd intron 1. Positive effects of intron on transgene expression have also been observed in other basidiomycetes such as Agaricus bisporus, C. cinereus, Phanerochaete chrysosporium, S. commune, C. passeckerianus and Trichoderma viride [20][21][22][23][24]. Enhancement of gene expression by introns may play a role in those organisms that generally possess introns in their genes [22].

Enhancement is associated with increased levels of mRNA
The transformant pJW-EXP-in-opbar-flag was chosen with the tranformant pJW-EXP-opbar-flag to study how the gpd intron 1 affects gene transcription and protein expression. The presence of opbar-flag in the genome of transformants pJW-EXP-opbar-flag and pJW-EXPin-opbar-flag was further determined by molecular hybridization. The genome of transformants pJW-EXPopbar-flag and pJW-EXP-in-opbar-flag were digested with NheI and probed with an opbar fragment (Fig. 3A, B). Southern blot analysis verified that these transformants had insertions of opbar, with single or multiple copies in their genomes. Single-copy integration of opbar-flag event was detected in the genomes of transformants pJW-EXP-opbar-flag T1, T4, and T5, and pJW-EXP-in-opbar-flag T1, T3, and T5. To minimize copy-number effect, the transcription level of opbar was analyzed in transformants pJW-EXP-opbar-flag T1 and T5, and pJW-EXP-in-opbar-flag T1 and T3 carrying a single-copy opbar by real-time qRT-PCR analysis. The results showed high transcription levels in transformants pJW-EXP-in-opbar-flag T1 and T3 bearing gpd intron 1 in opbar and low levels for transformants pJW-EXP-opbar-flag T1, and T5 containing opbar without gpd intron 1. No significant transcription difference was observed between pJW-EXP-in-opbar-flag T1 and T5. The transcription level of opbar in transformants pJW-EXP-in-opbar-flag T1 and T3 was, respectively, 12.3-and 10.1-fold higher than in transformant pJW-EXP-in-opbar-flag T1 (Fig. 3C). Our results indicated that gpd intron 1 increased the transcription level of heterologous opbar in G. lucidum. Previous studies have suggested that introns increased the level of mRNA possibly by enhancing maturation and stability of transcripts [25]. The difference in transcription level between pJW-EXP-in-opbar-flag T1 and T3 might be relevant to integration position effects [26].

The effect of the gpd intron 1 on protein yield of heterologous opbar
Proteins isolated from transformants pJW-EXP-opbarflag T1, and T5, and pJW-EXP-in-opbar-flag T1 and T3 were analyzed by western blotting (Fig. 4). A protein of ≈ 22 kDa was detectable in the transformants pJW-EXP-in-opbar-flag T1 and T3. However, it was not observed in transformants pJW-EXP-opbar-flag T1 and T5 containing opbar without the gpd intron 1. The band at 22 kDa that was recognized by flagspecific antibodies was the opbar-flag protein, as the molecular weight was similar to the predicted value. As shown in Fig. 4, the amount of opbar-flag protein also increased with the introduction of gpd intron 1. These results coincided with those of qRT-PCR analysis and phosphinothrin-resistance screening. Gpd intron 1 enhanced protein expression both at the RNA and protein levels. Opbar protein levels did not correlate with opbar mRNA levels in the intronless G. lucidum transformants, indicating that gpd intron 1 may also enhance the efficiency of translation besides increasing mRNA content by affecting transcription and stability of mRNA [27,28]. The detailed mechanism of enhancement of introns on the translation of heterologous genes requires further investigation.

Introduction of gpd intron1 enhances β-glucuronidase (GUS) expression
To confirm the positive effect of gpd intron 1 on the expression of heterologous genes, we also constructed plasmids pJW-EXP-opgus and pJW-EXP-in-opgus (Fig. 5A) and transformed these into Ganoderma protoplasts. These plasmids contain a carboxin-resistant cassette for transformation selection and the full codon-optimized gus (opgus) cassette (pJW-EXPopgus) or the intron-opgus cassette. Carboxin-resistant colonies were screened for transgenes by genome PCR (Additional file 1: Fig. S3) and subsequently were further analyzed by Southern blotting. The results showed that opgus and intro-opgus had integrated into the genome of the recipient as single copies in transformants pJW-EXP-opgus T1, T2, T3, and T4, and transformants pJW-EXP-in-opgus T1 (Fig. 5B). Real-time qRT-PCR analysis showed that the transcription level of opgus in transformant pJW-EXP-in-opgus T1 was 3.8-fold higher than that in transformants pJW-EXPopgus T1 and T2 without gpd intron 1 (Fig. 6A). The increase in transcription level relative to the intronless control ranged from 3.8-fold for opgus to 12.3-fold for opbar, which may be dependent on the cDNA sequence and the transcription level of the intronless heterologous gene [27,29]. Histochemical staining analysis was also performed to detect expression of opgus in wild-type (WT), and transformants pJW-EXP-opgus T2 and pJW-EXP-in-opgus T1. Figure 6B shows that GUS activity was observed in the mycelia of transformants pJW-EXP-opgus T2 and pJW-EXP-in-opgus T1, while no blue staining was detected in the WT mycelia. Moreover, GUS activity in transformant pJW-EXP-inopgus T1 was higher than that in transformant pJW-EXP-opgus T2. Again, our results showed that gpd intron 1 enhanced the opgus transcription and GUS enzyme activity in G. lucidum. It may thus be possible

Conclusions
This work demonstrated the importance of introducing gpd intron 1 for the efficient expression of opbar and gus in G. lucidum. The presence of gpd intron 1 in heterologous genes enhances mRNA accumulation and protein expression in basidiomycete Ganoderma. The developed method can be applicable to upregulate the expression of other heterologous genes in Ganoderma.
The opgus and in-opgus genes were PCR amplified from plasmids pUC57-opgus and pUC57-in-opgus using primers opgus F/opgus R and in-opgus F/opgus R (Additional file 1: Table S1), respectively. The obtained products were fused into plasmid pJW-EXP that was digested with NheI and SmaI using the ClonExpress MultiS One Step Cloning Kit (Vazyme, Nanjing) to generate plasmids pJW-EXP-opgus and pJW-EXP-in-opgus, respectively.

Genetic transformation of G. lucidum and selection of transformants
Transformation of G. lucidum protoplasts was performed according to our previous studies [13,33]. The suspensions of G. lucidum protoplasts and plsmids in PTC buffer (50 mM CaCl 2 , 10 mM Tris-HCl buffer (pH 7.5), and 60% w/v PEG 4000) were plated onto CYM selective plates containing 0.6 M mannitol and 2 µg/L carboxine. After 14 days of incubation at 30 °C, carboxin-resistant transformants were screened for phosphinothricinresistance protein and GUS expression. The transformants expressing opbar were screened on CYM plates with 150 mg/L phosphinothricin. The genome of these transformants was extracted for PCR amplification of the fused fragment of gpd promoter and opbar using primers gpd-id-F/opbar-id-R. The transformants expressing opgus were identified by PCR amplification of the fused fragment of gpd promoter and opgus using primers gpd-id-F/opgus-R.

Nucleic acid isolation
Ganoderma lucidum mycelia were harvested, frozen, and powdered in liquid nitrogen. Genomic DNA was isolated employing the cetyltrimethylammonium bromide (CTAB) method. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocols and treated with RNase-free DNase I before use.

Southern blots
DNA samples from G. lucidum transformants were digested with NheI and separated on a 0.7% agarose gel. The opbar (0.54 kb) and opgus (0.95 kb) fragment amplified from plasmids pJW-EXP-opbar-flag and pJW-EXP-opgus with primers bar probe F/bar probe F and gus probe F/gus probe R were used as probes. Southern blot analysis was conducted under conditions recommended for the digoxigenin (DIG) hybridization system by Mylab ™ (Beijing, China).

Quantitative real-time (qRT)-PCR analysis
Approximately 1 mg total RNA was used as template, and reverse transcription was performed using the Pri-meScript ™ RT reagent Kit (Takara, China) following the manufacturer's instructions. The transcription levels of opbar and opgus were determined by qRT-PCR as previously described [6,16]. The 18S-rRNA transcript was used as internal control to normalize relative transcription levels. For opbar and opgus, the transcription level in opbar-flag and opgus strains was set to 1.0, and the relative transcription levels in other strains were presented as fold changes relative to the reference level. The primers qRT-bar-F/qRT-bar-R, qRT-gus-F/qRT-gus-R, and qRT-18S-F/qRT-18S-R (Additional file 1: Table S1) were used for amplification of the opbar, opgus, and 18S rRNA genes, respectively.

Western blot analysis
Ganoderma lucidum mycelia were washed and homogenized in 1 mL of lysis buffer (25 mM Tris base (pH 7.4), 200 mM glycine, 5 mM EDTA, 1 mM phenylmethyl sulfonyl fluoride, and 1 mM 2-mercaptoethanol). The homogenates were centrifuged at 12,000×g for 20 min at 4 °C, then protein concentration in the supernatants was determined using the Bradford method. For western bolt analysis, 30 µg of protein was separated on a 12% SDS-polyacrylamide gel and electro-transferred to a polyvinylidene difluoride membrane in TBST buffer [10 mM Tris/HCl (pH 7.4), 100 mM NaCl, 0.1% Tween 20] for 1 h. Membranes were blocked with non-fat milk dissolved in TBSF buffer at room temperature for 2 h, followed by incubation with primary antibodies (anti-Flag, PROTEINTECH, Cat No. 20543-1-AP at 1:5000 dilution; anti-tubulin rabbit polyclonal antibody, BBI, Cat No. D110015 at 1:5000 dilution). Membranes were washed with TBSF and incubated with an HRP-conjugated goat anti-rabbit IgG (BBI, Cat No. D110058 at 1:5000 dilution). The blots were washed with TBSF buffer again, and the bands were visualized using an enhanced chemiluminescene method.

Statistical analysis
Data were generated in three independent sample measurements, and all data are presented as the mean ± standard deviation. The results were considered significant for p values < 0.05 in a two-tailed analysis.