Overexpression of the Squalene Epoxidase Gene Alone and in

Article pubs.acs.org/JAFC

Overexpression of the Squalene Epoxidase Gene Alone and in Combination with the 3‑Hydroxy-3-methylglutaryl Coenzyme A Gene Increases Ganoderic Acid Production in Ganoderma lingzhi De-Huai Zhang,†,‡ Lu-Xi Jiang,†,‡ Na Li,§ Xuya Yu,† Peng Zhao,† Tao Li,† and Jun-Wei Xu*,† †

Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, 650500, China Faculty of Science, Kunming University of Science and Technology, Kunming, 650500, China

§

S Supporting Information *

ABSTRACT: The squalene epoxidase (SE) gene from the biosynthetic pathway of ganoderic acid (GA) was cloned and overexpressed in Ganoderma lingzhi. The strain that overexpressed the SE produced approximately 2 times more GA molecules than the wild-type (WT) strain. Moreover, SE overexpression upregulated lanosterol synthase gene expression in the biosynthetic pathway. These results indicated that SE stimulates GA accumulation. Then, the SE and 3-hydroxy-3-methylglutaryl coenzyme A (HMGR) genes were simultaneously overexpressed in G. lingzhi. Compared with the individual overexpression of SE or HMGR, the combined overexpression of the two genes further enhanced individual GA production. The overexpressing strain produced maximum GA-T, GA-S, GA-Mk, and GA-Me contents of 90.4 ± 7.5, 35.9 ± 5.4, 6.2 ± 0.5, and 61.8 ± 5.8 μg/100 mg dry weight, respectively. These values were 5.9, 4.5, 2.4, and 5.8 times higher than those produced by the WT strain. This is the first example of the successful manipulation of multiple biosynthetic genes to improve GA content in G. lingzhi. KEYWORDS: ganoderma, ganoderic acid, squalene epoxidase, fermentation, genetic engineering, co-overexpression production.10,27,29 For instance, homologous overexpression of GA biosynthetic genes and the heterologous expression of the Vitreoscilla hemoglobin gene in G. lucidum significantly increased GA production.10,28 HMGR, SQS, and LS gene overexpression implicate important roles of these genes in the regulation of GA biosynthesis in G. lingzhi.12,13,29,30 Genetic manipulation of GA biosynthetic genes is crucial in elucidating the regulation of GA biosynthesis and enhancement of GA production. SE, also known as squalene monooxygenase, catalyzes the first oxygenation step in GA biosynthesis by converting squalene to 2,3-oxidosqualene.4,9,11 In suspension-cultured Panax ginseng cells, increasing SE gene transcription improved the accumulation of triterpene saponin.31 In P. ginseng, silencing the SE gene via RNA interference decreased ginsenoside production.32 In the basidiomycete Hypholoma sublateritium, SE gene overexpression increased the production of triterpene clavaric acid by 33%−97%.33 These results suggested that SE is an important enzyme in the triterpene biosynthetic pathway. The SE gene has been cloned and characterized from different fungi, such as H. sublateritium,33 Trametes versicolor (GenBank accession no. XM_008045850), and Taiwanof ungus camphoratus (GenBank accession no. KT070558). However, the SE gene has not been isolated from G. lingzhi. Moreover, whether SE gene overexpression can increase GA production remains unknown.

1. INTRODUCTION Mushrooms are excellent sources of biologically active medicinal compounds. Ganoderma linzhi is a well-known and widely used traditional Chinese medicinal mushroom.1,2 Ganoderic acids (GAs), which are C30 lanostane-type triterpenoids, are responsible for the pharmacological activities of G. lingzhi. Ganoderic acid has antitumor, antimetastasis, antiinvasion, and anti-HIV pharmacological effects.1,3,4 Recent studies have shown that different GA molecules exhibit various bioactivities. For example, ganoderic acid (GA)-S and GA-Mk induce the apoptosis of cervical carcinoma HeLa cells.5,6 GAMe and GA-T inhibit the growth and metastasis of lung cancer.7,8 GAs, like ergosterol, are synthesized from mevalonic acid through the isoprenoid pathway (Figure 1).4,9,10 In G. lingzhi, GAs are derived from a lanostane skeleton, and the skeleton undergoes oxidation, reduction, and acylation to form various individual GAs.4,11 Some key structural genes in the GA biosynthetic pathway, such as 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMGR), squalene synthase (SQS), and lanosterol synthase (LS), have been isolated and characterized in Ganoderma lucidum.9,12,13 Submerged fermentation of mushrooms is a promising approach to the mass production of secondary metabolites. Considerable efforts have been made to enhance GA production in the submerged cultivation of G. lucidum, such as the optimization of cultivation conditions,3,14−17 addition of elicitors, 18−23 and development of bioprocessing strategies.24−26 A highly productive G. lingzhi strain may allow for the sustainable and large-scale production of valuable secondary metabolites. Genetically engineering G. lucidum has been recently reported as an effective approach to increase GA © 2017 American Chemical Society

Received: Revised: Accepted: Published: 4683

February 10, 2017 April 8, 2017 May 22, 2017 May 22, 2017 DOI: 10.1021/acs.jafc.7b00629 J. Agric. Food Chem. 2017, 65, 4683−4690

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of four GAs detected from the cultured mycelia of G. lingzhi (A). The biosynthetic pathways of GAs (B).

2. MATERIALS AND METHODS

Given the complexity of the systems that regulate GA biosynthesis, previous research studies that focused on the overexpression of a single biosynthetic gene, such as SQS and LS gene, have only modestly improved GA production.12,13 The manipulation of multiple genes in the GA biosynthetic pathway is a promising approach for enhancing metabolite production.10,34−36 For example, in Ophiorrhiza pumila, the cooverexpression of geraniol-10-hydroxylase (G10T) and strictosidine synthase (STR) genes produced higher levels of camptothecin than the individual overexpression of G10H gene or STR gene. 34 In Saccharomyces cerevisiae, the coordinated overexpression of SE gene (erg1) and lanosterol 14-α demethylase gene (erg11) increased content of sterol to beyond that of the strain that overexpressed erg1 or erg11 alone.37 However, the effect of coexpression of multiple biosynthetic genes on GA production remains unknown. HMGR is known to be the major rate-limiting enzyme in the MVA pathway.38−40 The overexpression of the N-terminally truncated HMGR gene in G. lucidum caused more accumulation of precursors but did not cause significant differences in individual GA content.29 Previous studies showed that SE is an important enzyme in the postsqualene part of the sterol/ ginsenoside biosynthetic pathways.32,37,41,42 Therefore, it would be interesting to investigate whether the combined overexpression of the HMGR and SE genes would enhance GA production. In this work, the SE gene was cloned from G. lingzhi and overexpressed. Then, the effect of SE gene overexpression on GA biosynthesis was investigated in a submerged G. lingzhi culture. Moreover, GA production was analyzed in engineered G. lingzhi that coexpressed HMGR and SE genes. This work aims to advance the understanding of GA biosynthesis regulation, as well as develop a more efficient fermentation process for GA production.

2.1. Strains and Culture Conditions. Ganoderma lucidum CGMCC 5.616 from the China General Microbiological Culture Collection Center, which is conspecific with Ganoderma lingzhi,13 was cultured in accordance with the description by Xu et al.15 The details of culture mediums and conditions are shown in the Supporting Information. Plasmids were propagated in Escherichia coli DH5α. G. lingzhi protoplasts were regenerated in complete yeast medium (CYM) that contained 10 g/L maltose, 20 g/L glucose, 2 g/L yeast extract, 2 g/L tryptone, 4.6 g/L KH2PO4, 0.5 g/L MgSO4, 0.6 M mannitol, and 10 g/L agar. 2.2. Vector Construction and Genetic Transformation of G. lingzhi. The SE gene of G. lingzhi was amplified from genomic DNA using primers SE-NheI-F (5′-GCTAGCATGTGGTCCGTCTCGTACGACATCA-3′) and SE-SmaI-R VGB-SmaI-R (5′-GGGCCCTCACGGGCGGATCTCCGTCC-3′). Using a standard molecular procedure, the amplified NheI-SE gene-SmaI fragment was inserted in the NheI-SmaI sites of the pJW-EXP30 backbone to create the pJWEXP-SE plasmid. G. lingzhi protoplasts were exposed to the pJW-EXPSE plasmid in accordance with polyethylene glycol mediated transformation, as described previously.28,30 For cotransformation, 2 μg of plasmid pJW-EXP-SE and 2 μg of pJW-EXP-HMGR30 were used as transforming plasmids. Candidate transformants were individually subcultured onto CYM plates that contained 2 mg/L carboxin. The stability of the integrated DNA was assessed by subculturing transformants five times on carboxin-free CYM plates. Carboxin resistance was then tested by transferring the transformants on CYM medium that was supplemented with 2 mg/L of carboxin. Using whole-genome PCR analysis, stable transformants were screened for the integration of the fused glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter and SE (or HMGR) gene. The primers gpdSE-F (5′-CAAGGCGGTCAACAGGTAA-3′), gpd-SE-R (5′-CGTCCATAGTAGCGGCAAA-3′), gpd-HMGR-F (5′-AGTTTCTGTGGTGCTGTTGC-3′), and gpd-HMGR-R (5′-CGAGTTAGCTTCGTCCGTCT-3′) were used for PCR verification. 2.3. Nucleic Acid Extraction and cDNA Synthesis. The genomic DNA of G. lingzhi was isolated using the cetyltrimethylammonium bromide (CTAB) method. The total RNA of G. lingzhi was extracted using a TRIzol reagent (Invitrogen, Carlsbad, CA). The quality and quantity of nucleic acid were determined by agarose gel 4684

DOI: 10.1021/acs.jafc.7b00629 J. Agric. Food Chem. 2017, 65, 4683−4690

Article


Figure 2. Amino acid sequence alignment of GLSE (Ganoderma lingzhi) and SEs from other fungi: DSSE (Dichomitus squalens, XP_007365331), TVSE (Trametes versicolor, XM_008045850), TCSE (Taiwanof ungus camphoratus, KT070558), LSSE (Laetiporus sulphureus, KZT04024), and GFSE (Grifola frondosa, OBZ72334). Three conserved motifs in the SE of G. lingzhi are double underlined. electrophoresis and spectrophotometric measurements. After treatment with RNase-free DNase I (Fermentas, Canada), 1 μg of total RNA was reverse-transcribed with SuperScript RNase H First-strand synthesis kit (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. 2.4. Measurement of Gene Expression by Quantitative RealTime PCR (qRT-PCR). The transcription levels of HMGR, SQS, SE, and LS genes were measured by qRT-PCR. The qRT-PCR protocols and primers for the amplification of the HMGR, SQS, and LS genes were described previously.15,26,28,29 To amplify the SE gene, the following primer sets were used: SE-qRT-F (5′-CGGTCGAGAAGGAGGCGTTCTT-3′) and SE-qRT-R (5′-TGTCGTGGGTCCCGATCTGGTAT-3′). Transcription levels were normalized relative to that of the reference gene G. lingzhi 18S rRNA. Post qRTPCR calculations were performed with the 2−ΔΔCt method.43 2.5. Determination of Biomass and Total and Individual GA Contents. G. lingzhi mycelia were precipitated by centrifuging a sample at 10000g for 10 min. The precipitated mycelia were then washed thrice with distilled H2O and dried to a constant weight at 45 °C. Dry cell weight was measured with the gravimetric method. Total crude GAs and individual GAs were extracted and analyzed in accordance with a previously described method.15,29 The detailed analytical method is described in the Supporting Information. 2.6. Statistical Analysis. Data were the average of three independent sample measurements. Error bars indicate the standard deviation from the mean of biological triplicates. Statistical analysis was performed using Student’s t-test. The difference was considered significant when p < 0.05 in a two-tailed analysis.

with the oligonucleotides SE-NheI-F and SE-SmaI-R as primers and G. lingzhi genomic DNA. The SE gene (GenBank accession no. KY211742) is 1722 bp long and contains a 1404 bp open reading frame. The gene encodes a protein that comprises 467 amino acids, with a theoretical molecular mass of 51.7 kDa and a pI of 8.45. The SE gene includes five introns of 738, 376, 58, 175, and 57 nucleotides at positions 1−738, 802−1177, 1241− 1298, 1418−1592, and 1666−1722, respectively, from the translation initiation codon. The G. lingzhi SE shows high sequence similarity with other SE protein from fungi, such as Dichomitus squalens (82% identity), Trametes versicolor (75%), Laetiporus sulphureus (68%), Taiwanof ungus camphoratus (67%), and Grifola f rondosa (67%). SE is a flavin adenine dinucleotide (FAD) dependent monooxygenase. Protein sequence analysis revealed that the G. lingzhi SE has three strictly conserved motifs that are involved in FAD binding and squalene recognition (Figure 2).33 SE catalyzes the conversion of squalene to 2,3-oxidosqualene, which is the rate-limiting step in sterol biosynthesis in mammals and yeast.44,45 To explore the function of SE in the regulation of GA biosynthesis, the G. lingzhi SE gene was overexpressed using a pJW-EXP-SE plasmid (Figure 3A). The SE gene was coupled to the glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter from G. lingzhi in the pJW-EXP-SE plasmid. G. lingzhi protoplasts were transformed with the pJW-EXP-SE plasmid through our previously reported transformation method.10,30,46 Candidate transformants were grown on selective complete yeast medium (CYM) supplemented with 2 mg/L carboxin (Sigma, St. Louis, MO) after five rounds of subculture on carboxin-free CYM. The transformants

3. RESULTS AND DISCUSSION 3.1. Construction of the SE Gene-Overexpressing G. lingzhi Transformants. The SE gene was amplified by PCR 4685


Article


3.2. Overexpression of the SE Gene Increases GA Production. The effects of SE gene overexpression on biomass and GA production were studied in submerged culture of G. lingzhi. Table 1 shows the biomass and individual GA contents of the SE transformants SE1, SE2, and SE3 as well as the WT strain on day 9. The SE transformants accumulated higher levels of the four GAs than the WT strain. These results confirmed that the transformants consistently overexpressed the SE gene. From the information shown in Table 1, transformant SE2 was selected, along with the WT strain, to further study the time profiles of the biomass, total GA content, and individual GA accumulation. Figure 5A shows that the maximum dry cell weight (DW) of SE2 and the WT strain was 8.05 and 7.78 g/L, respectively. SE gene overexpression did not significantly affect cell growth. This result was consistent with a previous study, in which silencing the SE gene in Trichoderma harzianum transformants did not affect cell growth.41 Total GA content in SE transformants and the WT strain reached their maximum levels on day 9 and gradually declined thereafter. The maximum total GA content in the SE transformant was 2.42 mg/100 mg DW, which was 1.3-fold higher than that obtained in the WT strain (Figure 5B). GA-T, GA-S, GA-Mk, and GAMe were detected as the major individual GA components from the cultured mycelia of G. lucidum CCGMC 5.616.15 The time profiles of individual GA accumulation in SE2 and WT strain are shown in Figure 6. In both strains, GA-T and GA-Me contents reached their maximum levels on day 12 of the fermentation (Figures 6A and 6D). By contrast, GA-S and GAMk contents reached their maximum values on day 9 and decreased thereafter in the SE transformant (Figures 6B and 6C). The maximum levels of GA-T, GA-S, GA-Mk, and GA-Me in SE2 were 57.7 ± 4.2, 19.1 ± 2.1, 4.3 ± 0.2, and 33.6 ± 2.4 μg/100 mg dry weight (DW), respectively, which were 3.2, 2.4, 1.8, and 2.9 times higher than that of the WT strain. Individual GA levels in the SE transformants are comparable to those obtained in G. lingzhi that overexpressed the LS gene.13 The overexpression of the SE gene enhanced GA production in G. lingzhi, which suggests that SE is an important enzyme in GA biosynthesis. Similar observations were also reported for H. sublateritium33 and S. cerevisiae,45 in which overexpressing a SE gene increased triterpenoid clavaric acid production and βamyrin titer, respectively. In P. ginseng, silencing the SE gene reduced triterpenoid ginsenoside production.32 SE gene overexpression in G. lingzhi may increase lanosterol accumulation in cells,37 thus increasing flux toward GA biosynthesis. SE gene overexpression increased contents of total and individual GAs, indicating that SE gene expression is positively correlated with GA biosynthesis. HMGR gene overexpression increases squalene accumulation, but does not significantly change individual GA content in G. lucidum.29 This suggests that there are other important enzymes involved in regulation of GA

Figure 3. PJW-EXP-SE plasmid (A). Identification and characterization of the SE transformant (B). Amplification patterns of genomic PCR obtained with the primers gpd-SE-F and gpd-SE-R from different strains. Lane N is the negative control; lane WT is the WT strain; lane S is the SE transformant; lane P is the pJW-EXP-SE plasmid as a positive control; lane M is the DNA marker.

that retained carboxin resistance were detected via PCR, followed by sequencing of the PCR products. The presence of the fused gpd promoter and SE gene in the transformant and positive control were confirmed by genome PCR with primers gpd-SE-F and gpd-SE-R (Figure 3B). The lane of the SE transformant showed a 2.36 kb amplification band. However, no corresponding band was found in the lane of the wild-type (WT) strain. Additionally, no morphological difference was observed by light microscopy between the SE transformant and WT strains of shaking culture (data not shown). qRT-PCR results showed that the transformant produced higher SE transcript levels (at approximately 5-fold) compared with the untransformed WT strain under submerged culture conditions (Figure 4).

Figure 4. Transcriptional levels of the GA biosynthetic genes in the WT strain and the SE transformant. Expression level of the samples from the WT strain is defined as 1.0, and expression levels in the transgenic strain are displayed in relation to the reference sample. The error bars indicate the standard deviations from three independent samples. d is days, and * indicates statistical significance (p < 0.05) compared to the WT strain.

Table 1. Maximum Biomass and Individual GA Contents of the SE Transformants SE1, SE2, and SE3 and the WT Strain individual GA content (μg/100 mg DW) cell line WT SE-1 SE-2 SE-3 a

biomass (g/L) 7.86 7.93 8.01 8.77

± ± ± ±

0.34 0.21 0.27 0.31

GA-Mk 2.23 3.83 3.79 3.71

± ± ± ±

GA-T

0.21 0.06*a 0.43* 0.34*

16.23 53.81 54.45 60.19

± ± ± ±

1.21 1.34* 1.45* 1.99*

GA-S 8.01 22.72 25.45 21.79

± ± ± ±

0.98 4.12* 4.19* 5.41*

GA-Me 10.67 29.66 31.12 27.99

± ± ± ±

0.89 1.23* 1.51* 1.85*

The asterisk symbol (*) means significantly different from value for WT (p < 0.05). 4686


Article


Figure 5. Time profiles of cell growth (A) and total GA content (B) in the WT strain (blank triangles) and the SE transformant (dark triangles) under submerged culture conditions. Symbols are the same as those in Figure 4.

Figure 6. Time profiles of contents of GA-T (A), GA-S (B), GA-Mk (C), and GA-Me (D) in the WT strain (blank triangles) and the SE transformant (dark triangles) under submerged culture conditions. Symbols are the same as those in Figure 4.

previous studies have reported that increased SE gene expression contributed to triterpenoid overproduction in H. sublateritium and P. ginseng, respectively.31−33 In G. lucidum, the higher expression of biosynthetic genes, such as HMGR, SQS, and LS genes, favored GA accumulation.15,19,26,47 HMGR and SQS gene expression levels were unaffected by SE gene overexpression in G. lingzhi, and this result is in good agreement with previous studies that reported that the gene expression levels of HMGR and SQS were not influenced by genetically manipulating the SE gene in T. harzianum and P. ginseng, respectively.32,41 This observation was also correlated with previous reports for G. lingzhi, wherein the expression levels of upstream genes were not noticeably altered by the overexpression of downstream genes in the GA biosynthetic pathway.12,13 3.4. Overexpression of the SE Gene in Combination with the tHMGR Gene Further Improved GA Production. The overexpression of multiple genes in the GA biosynthetic pathway may considerably increase GA production.10,27 We investigated the effect of simultaneously overexpressing SE and HMGR genes on GA production in G. lingzhi. The pJW-EXP-

biosynthesis. Our results implicate SE as an important gene for GA biosynthesis in G. lingzhi. 3.3. The Effect of SE Gene Overexpression on GA Biosynthetic Gene expression. The above results show that homologous SE gene overexpression increases GA biosynthesis. It is unknown, however, whether other GA biosynthetic genes would respond to SE overexpression. Thus, the expression levels of the HMGR, SQS, SE, and LS genes were examined in the SE transformant and WT strain to elucidate the possible molecular mechanisms of GA hyperproduction. Samples were analyzed at 6 and 9 d in accordance with our preliminary experiments. Gene expression levels were normalized against that of the G. lingzhi 18S rRNA gene. Figure 4 shows that SE gene overexpression significantly improved the expression levels of the SE and LS genes. Maximum SE and LS transcription levels in the SE transformant were 5.3- and 2.3fold higher than those in the WT strain, respectively. The levels of SE and LS genes in the transformants were higher than those in the WT strain, although they declined from days 6 to 9. These results indicate that the increased transcript levels of SE and LS genes might enhance GA production. Similarly, 4687


Article

Journal of Agricultural and Food Chemistry HMGR30 and pJW-EXP-SE plasmids were cotransformed into G. lingzhi protoplasts. The integration of the HMGR gene and SE genes into the genome of the transformant was confirmed by genomic PCR, followed by sequencing of the amplified products. The expected products of 938 bp and 2.13 kb, which represent genes that correspond to the fusion of the gpd promoter-HMGR and gpd promoter-SE genes, respectively, were present in the H-S transformant (Figure 7). qRT-PCR

limitation that prevents the hyperproduction of individual GAs in HMGR transformants may have been partially overcome by the overexpression of genes in the postsqualene portion of the GA biosynthetic pathway. Our results indicate that, in G. lingzhi, HMGR and SE gene overexpression more effectively enhanced GA production than individual HMGR or SE gene expression. This may likely result from the combined upregulation of HMGR and SE gene channel metabolic flux from the general MVA pathway into specific triterpene GAs in G. lingzhi.29,37 This strategy can also be applied to genetically engineer complex pathways, such as the triterpenoid pathway, to effectively produce important secondary metabolites in other mushrooms. Modifying SE gene expression affects GA biosynthesis in G. lingzhi. The overexpression of SE genes upregulates LS gene expression and increases GA production in a submerged G. lingzhi culture. Moreover, the combined overexpression of SE and HMGR genes further enhances GA production. Our results indicate that SE plays an important role in GA biosynthesis. The simultaneous overexpression of SE and HMGR genes in G. lingzhi more effectively increases the production of antitumor GAs than the overexpression of an individual gene.

■

Figure 7. Amplification patterns of genomic PCR from the WT strain and the H-S transgenic strain. Lane N is the negative control; lane WT2 is the genomic PCR analysis of the WT strain with primers gpdSE-F and gpd-SE-R; lane S is the genomic PCR analysis of the H-S transgenic strain with the primers gpd-SE-F and gpd-SE-R; lane P2 is the pJW-EXP-SE plasmid as a positive control; lane WT1 is the genomic PCR analysis of the WT strain with primers gpd-HMGR-F and gpd-HMGR-R; lane H is the genomic PCR analysis of the H-S transgenic strain with the primers gpd-HMGR-F and gpd-HMGR-R; lane P is the pJW-EXP-HMGR plasmid as a positive control; lane M is the DNA marker.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00629.

■

Culture of G. lingzhi, analyses of GAs, transcriptional levels of the HMGR and SE genes, and HPLC chromatograms (PDF)

AUTHOR INFORMATION

Corresponding Author

analysis revealed that H-S transformant overexpressed the HMGR and SE genes (Supporting Information). Table 2 shows the maximum total GA and individual GA content in the WT, HMGR, SE, H-S1, H-S2, and H-S3 transgenic strains. The individual GA contents in the H-S transgenic strain were significantly higher compared with those of the WT strain, HMGR, and SE transformants. For example, the maximum GAT, GA-S, GA-Mk, and GA-Me levels in the H-S2 transgenic strain were 90.4 ± 7.5, 35.9 ± 5.4, 6.2 ± 0.5, and 61.8 ± 5.8 μg/ 100 mg DW respectively, which were 5.9-, 4.5-, 2.4-, and 5.8fold higher than those of the WT strain. The obtained individual GA levels in the H-S transformant were approximately two times higher than that reported for the SEoverexpressing and LS-overexpressing G. lucidum.13 The

*E-mail: [email protected]; [email protected]. Tel/fax: +86871-65920676. ORCID

Jun-Wei Xu: 0000-0002-0871-2171 Author Contributions ‡

D.-H.Z. and L.-X.J. contributed equally to this work.

Funding

This work was supported by the National Natural Science Foundation of China (No. 31360495 and No. 21566016). Notes

The authors declare no competing financial interest.

Table 2. Maximum Total GA and Individual GA Content in the WT, HMGR, SE, H-S1, H-S2, and H-S3 Transgenic Strains individual GA content (μg/100 mg DW) cell line WT HMGR SE H-S1 H-S2 H-S3

biomass (g/L) 7.98 8.17 8.19 8.34 8.14 8.21

± ± ± ± ± ±

0.14 0.23 0.07 0.34 0.2 0.1

total GA content (mg/100 mg DW) 1.10 2.76 2.41 3.58 3.85 3.64

± ± ± ± ± ±

0.03 0.38*b 0.21* 0.27* 0.35* 0.24*

GA-Mk 2.56 2.41 3.39 5.56 6.24 7.25

± ± ± ± ± ±

0.13 0.04 0.05* 0.68* 0.53* 0.66*

GA-T

GA-S

± ± ± ± ± ±

7.92 ± 1.12 8.11 ± 0.52 24.16 ± 3.2* 30.81 ± 2.7* 35.9 ± 4.48* 37.25 ± 4.6*

15.20 16.01 56.39 98.56 90.44 89.19

1.3 0.3 3.6* 6.6* 7.5* 5.9*

GA-Me 10.61 11.18 33.28 55.36 61.87 57.25

± ± ± ± ± ±

0.28 1.1 2.61* 5.36* 5.80* 4.6*

a

H-S, G. lingzhi strains overexpressing the HMGR and SE genes. bThe asterisk symbol (*) means significantly different from value for WT (p < 0.05). 4688


Article


■

(17) Zhao, W.; Xu, J. W.; Zhong, J. J. Enhanced production of ganoderic acids in static liquid culture of Ganoderma lucidum under nitrogen-limiting conditions. Bioresour. Technol. 2011, 102, 8185− 8190. (18) Liang, C. X.; Li, Y. B.; Xu, J. W.; Wang, J. L.; Miao, X. Y.; Tang, Y. J.; Gu, T.; Zhong, J. J. Enhanced biosynthetic gene expressions and production of ganoderic acids in static liquid culture of Ganoderma lucidum under phenobarbital induction. Appl. Microbiol. Biotechnol. 2010, 86, 1367−1374. (19) Ren, A.; Li, X. B.; Miao, Z. G.; Shi, L.; Jaing, A. L.; Zhao, M. W. Transcript and metabolite alterations increase ganoderic acid content in Ganoderma lucidum using acetic acid as an inducer. Biotechnol. Lett. 2014, 36, 2529−2536. (20) Ren, A.; Qin, L.; Shi, L.; Dong, X.; Mu, D. S.; Li, Y. X.; Zhao, M. W. Methyl jasmonate induces ganoderic acid biosynthesis in the basidiomycetous fungus Ganoderma lucidum. Bioresour. Technol. 2010, 101, 6785−6790. (21) Xu, Y. N.; Xia, X. X.; Zhong, J. J. Induced effect of Na plus on ganoderic acid biosynthesis in static liquid culture of Ganoderma lucidum via calcineurin signal transduction. Biotechnol. Bioeng. 2013, 110, 1913−1923. (22) You, B. J.; Lee, M. H.; Tien, N.; Lee, M. S.; Hsieh, H. C.; Tseng, L. H.; Chung, Y. L.; Lee, H. Z. A novel approach to enhancing ganoderic acid production by Ganoderma lucidum using apoptosis induction. PLoS One 2013, 8, e53616. (23) Zhang, J. M.; Zhong, J. J.; Geng, A. L. Improvement of ganoderic acid production by fermentation of Ganoderma lucidum with cellulase as an elicitor. Process Biochem. 2014, 49, 1580−1586. (24) Tang, Y. J.; Zhang, W.; Zhong, J. J. Performance analyses of a pH-shift and DOT-shift integrated fed-batch fermentation process for the production of ganoderic acid and Ganoderma polysaccharides by medicinal mushroom Ganoderma lucidum. Bioresour. Technol. 2009, 100, 1852−1859. (25) Zhu, L. W.; Zhong, J. J.; Tang, Y. J. Multi-fed batch culture integrated with three-stage light irradiation and multiple additions of copper ions for the hyperproduction of ganoderic acid and Ganoderma polysaccharides by the medicinal mushroom Ganoderma lucidum. Process Biochem. 2010, 45, 1904−1911. (26) Li, H. J.; Zhang, D. H.; Han, L. L.; Yu, X.; Zhao, P.; Li, T.; Zhong, J. J.; Xu, J. W. Further improvement in ganoderic acid production in static liquid culture of Ganoderma lucidum by integrating nitrogen limitation and calcium ion addition. Bioprocess Biosyst. Eng. 2016, 39, 75−80. (27) Qin, H.; Xu, J. W.; Xiao, J. H.; Tang, Y. J.; Xiao, H.; Zhong, J. J. Cell factories of higher fungi for useful metabolite production. Adv. Biochem. Eng./Biotechnol. 2015, 155, 199−235. (28) Li, H. J.; He, Y. L.; Zhang, D. H.; Yue, T. H.; Jiang, L. X.; Li, N.; Xu, J. W. Enhancement of ganoderic acid production by constitutively expressing Vitreoscilla hemoglobin gene in Ganoderma lucidum. J. Biotechnol. 2016, 227, 35−40. (29) Xu, J. W.; Xu, Y. N.; Zhong, J. J. Enhancement of ganoderic acid accumulation by overexpression of an N-terminal truncated 3-hydroxy3-methylglutaryl CoA reductase gene in the basidiomycete Ganoderma lucidum. Appl. Enviorn. Microbiol. 2012, 78, 7968−7976. (30) Yu, X.; Ji, S. L.; He, Y. L.; Ren, M. F.; Xu, J. W. Development of an expression plasmid and its use in genetic manipulation of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher basidiomycetes). Int. J. Med. Mushrooms 2014, 16, 161−168. (31) Zhao, C. L.; Cui, X. M.; Chen, Y. P.; Liang, Q. Key enzymes of triterpenoid saponin biosynthesis and the induction of their activities and gene expressions in plants. Nat. Prod. Commun. 2010, 5, 1147− 1158. (32) Han, J. Y.; In, J. G.; Kwon, Y. S.; Choi, Y. E. Regulation of ginsenoside and phytosterol biosynthesis by RNA interferences of squalene epoxidase gene in Panax ginseng. Phytochemistry 2010, 71, 36−46. (33) Godio, R. P.; Fouces, R.; Martin, J. F. A squalene epoxidase is involved in biosynthesis of both the antitumor compound clavaric acid

ABBREVIATIONS USED GA, ganoderic acid; SE, squalene epoxidase; WT, wild-type; DW, dry cell weight; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A; SQS, squalene synthase; LS, lanosterol synthase; CYM, complete yeast medium; gpd, glyceraldehyde-3-phosphate dehydrogenase gene

■

REFERENCES

(1) Bishop, K. S.; Kao, C. H. J.; Xu, Y.; Glucina, M. P.; Paterson, R. R. M.; Ferguson, L. R. From 2000 years of Ganoderma lucidum to recent developments in nutraceuticals. Phytochemistry 2015, 114, 56−65. (2) Paterson, R. R. Ganoderma − a therapeutic fungal biofactory. Phytochemistry 2006, 67, 1985−2001. (3) Upadhyay, M.; Shrivastava, B.; Jain, A.; Kidwai, M.; Kumar, S.; Gomes, J.; Goswami, D. G.; Panda, A. K.; Kuhad, R. C. Production of ganoderic acid by Ganoderma lucidum RCKB-2010 and its therapeutic potential. Ann. Microbiol. 2014, 64, 839−846. (4) Xu, J. W.; Zhao, W.; Zhong, J. J. Biotechnological production and application of ganoderic acids. Appl. Microbiol. Biotechnol. 2010, 87, 457−466. (5) Liu, R. M.; Li, Y. B.; Zhong, J. J. Anti-proliferation and induced mitochondria-mediated apoptosis of ganoderic acid Mk from Ganoderma lucidum mycelia in cervical cancer HeLa cells. Lat. Am. J. Pharm. 2012, 31, 43−50. (6) Liu, R. M.; Zhong, J. J. Ganoderic acid Mf and S induce mitochondria mediated apoptosis in human cervical carcinoma HeLa cells. Phytomedicine 2011, 18, 349−355. (7) Tang, W.; Liu, J. W.; Zhao, W. M.; Wei, D. Z.; Zhong, J. J. Ganoderic acid T from Ganoderma lucidum mycelia induces mitochondria mediated apoptosis in lung cancer cells. Life Sci. 2006, 80, 205−211. (8) Wang, G.; Zhao, J.; Liu, J. W.; Huang, Y. P.; Zhong, J. J.; Tang, W. Enhancement of IL-2 and IFN-γ expression and NK cells activity involved in the anti-tumor effect of ganoderic acid Me in vivo. Int. Immunopharmacol. 2007, 7, 864−870. (9) Shi, L.; Ren, A.; Mu, D. S.; Zhao, M. W. Current progress in the study on biosynthesis and regulation of ganoderic acids. Appl. Microbiol. Biotechnol. 2010, 88, 1243−1251. (10) Xu, J. W.; Zhong, J. J. Genetic engineering of Ganoderma lucidum for the efficient production of ganoderic acids. Bioengineered 2015, 6, 357−360. (11) Chen, S. L.; Xu, J.; Liu, C.; Zhu, Y. J.; Nelson, D. R.; Zhou, S. G.; Li, C. F.; Wang, L. Z.; Guo, X.; Sun, Y. Z.; Luo, H. M.; Li, Y.; Song, J. Y.; Henrissat, B.; Levasseur, A.; Qian, J.; Li, J. Q.; Luo, X.; Shi, L. C.; He, L.; Xiang, L.; Xu, X. L.; Niu, Y. Y.; Li, Q. S.; Han, M. V.; Yan, H. X.; Zhang, J.; Chen, H. M.; Lv, A. P.; Wang, Z.; Liu, M. Z.; Schwartz, D. C.; Sun, C. Genome sequence of the model medicinal mushroom Ganoderma lucidum. Nat. Commun. 2012, 3, 913. (12) Zhou, J. S.; Ji, S. L.; Ren, M. F.; He, Y. L.; Jing, X. R.; Xu, J. W. Enhanced accumulation of individual ganoderic acids in a submerged culture of Ganoderma lucidum by the overexpression of squalene synthase gene. Biochem. Eng. J. 2014, 90, 178−183. (13) Zhang, D. H.; Li, N.; Yu, X.; Zhao, P.; Li, T.; Xu, J. W. Overexpression of the homologous lanosterol synthase gene in ganoderic acid biosynthesis in Ganoderma lingzhi. Phytochemistry 2017, 134, 46−53. (14) Wagner, R.; Mitchell, D. A.; Sassaki, G. L.; De, A. A.; Berovic, M. Current techniques for the cultivation of Ganoderma lucidum for the production of biomass, ganoderic acid and polysaccharides. Food Technol. Biotechnol. 2003, 41, 371−382. (15) Xu, J. W.; Xu, Y. N.; Zhong, J. J. Production of individual ganoderic acids and expression of biosynthetic genes in liquid static and shaking cultures of Ganoderma lucidum. Appl. Microbiol. Biotechnol. 2010, 85, 941−948. (16) You, B. J.; Lee, H. Z.; Chung, K. R.; Lee, M. H.; Huang, M. J.; Tien, N.; Chan, C. W.; Kuo, Y. H. Enhanced production of ganoderic acids and cytotoxicity of Ganoderma lucidum using solid-medium culture. Biosci., Biotechnol., Biochem. 2012, 76, 1529−1534. 4689


Article

Journal of Agricultural and Food Chemistry and sterols in the basidiomycete H. sublateritium. Chem. Biol. 2007, 14, 1334−1346. (34) Cui, L. J.; Ni, X. L.; Ji, Q.; Teng, X. J.; Yang, Y. R.; Wu, C.; Zekria, D.; Zhang, D. S.; Kai, G. Y. Co-overexpression of geraniol-10hydroxylase and strictosidine synthase improves anti-cancer drug camptothecin accumulation in Ophiorrhiza pumila. Sci. Rep. 2015, 5, 8227. (35) Csernetics, A.; Nagy, G.; Iturriaga, E. A.; Szekeres, A.; Eslava, A. P.; Vágvölgyi, C.; Papp, T. Expression of three isoprenoid biosynthesis genes and their effects on the carotenoid production of the zygomycete Mucor circinelloides. Fungal Genet. Biol. 2011, 48, 696−703. (36) Zeng, J.; Wang, X. T.; Miao, Y. J.; Wang, C.; Zang, M. L.; Chen, X.; Li, M.; Li, X. Y.; Wang, Q.; Li, K. X. Metabolic engineering of wheat provitamin A by simultaneously overexpressing CrtB and silencing carotenoid hydroxylase (TaHYD). J. Agric. Food Chem. 2015, 63, 9083−9092. (37) Veen, M.; Stahl, U.; Lang, C. Combined overexpression of genes of the ergosterol biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae. FEMS Yeast Res. 2003, 4, 87−95. (38) Hampton, R.; Dimster-Denk, D.; Rine, J. The biology of HMGCoA reductase: the pros of contra-regulation. Trends Biochem. Sci. 1996, 21, 140−145. (39) Maury, J.; Asadollahi, M. A.; Møller, K.; Clark, A.; Nielsen, J. Microbial isoprenoid production: an example of green chemistry through metabolic engineering. Adv. Biochem. Eng. Biotechnol. 2005, 100, 19−51. (40) Kim, Y. K.; Kim, J. K.; Kim, Y. B.; Lee, S.; Kim, S. U.; Park, S. U. Enhanced accumulation of phytosterol and triterpene in hairy root cultures of platycodon grandiflorum by overexpression of Panax ginseng 3-hydroxy-3-methylglutaryl-coenzyme A reductase. J. Agric. Food Chem. 2013, 61, 1928−1934. (41) Cardoza, R. E.; Vizcaíno, J. A.; Hermosa, M. R.; Sousa, S.; González, F. J.; Llobell, A.; Monte, E.; Gutiérrez, S. Cloning and characterization of the erg1 gene of Trichoderma harzianum: Effect of the erg1 silencing on ergosterol biosynthesis and resistance to terbinafine. Fungal Genet. Biol. 2006, 43, 164−178. (42) Zhao, C. C.; Gao, X.; Liu, X. B.; Wang, Y.; Yang, S. L.; Wang, F. Q.; Ren, Y. H. Enhancing biosyntehsis of a ginsenoside precursor by self-assembly of two key enzymes in pichia pastoris. J. Agric. Food Chem. 2016, 64, 3380−3385. (43) Livak, K.; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001, 25, 402−408. (44) Gill, S.; Stevenson, J.; Kristiana, I.; Brown, A. J. Cholesteroldependent degradation of squalene monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase. Cell Metab. 2011, 13, 260−273. (45) Zhang, G. Refactoring β-Amyrin Synthesis in Saccharomyces cerevisiae. AIChE J. 2015, 61 (10), 3172−3179. (46) Li, H. J.; Zhang, D. H.; Yue, T. H.; Jiang, L. X.; Yu, X.; Zhao, P.; Li, T.; Xu, J. W. Improved polysaccharide production in a submerged culture of Ganoderma lucidum by the heterologous expression of Vitreoscilla hemoglobin gene. J. Biotechnol. 2016, 217, 132−137. (47) Zhang, W. X.; Tang, Y. J.; Zhong, J. J. Impact of oxygen level in gaseous phase ongene transcription and ganoderic acid biosynthesis in liquid static cultures of Ganoderma lucidum. Bioproc. Bioprocess Biosyst. Eng. 2010, 33, 683−690.

4690


Overexpression of the Squalene Epoxidase Gene Alone and in

Recommend Documents