Identi cation and Characterization of A Lanosterol synthase Gene from Sanghuangporus Baumii

Lanosterol synthase (LS) is a key enzyme involved in the mevalonate pathway (MVA pathway) to produce lanosterol, which is a precursor for synthesizing Sanghuangporus baumii triterpenoids. To research the characteristics and construction of LS, LS ORF and promoter were cloned from S. baumii. A 2,445 bp S. baumii LS sequence was obtained by rapid amplication of cDNA ends (RACE) technology and recombinant PCR. S. baumii LS sequence includes a 5’-untranslated region (129 bp), a 3’-untranslated region (87 bp), and an open reading frame (2,229 bp) encoding a 734 amino acids. The molecular weight of LS is 84.99 kDa, and transcription start site of S. baumii LS promoter sequence ranged from 1 740 bp to 1790 bp. LS promoter contained 12 CAAT-boxes, 5 ABREs, 6 G-Boxes, 6 CGTCA-motifs, and so on. The S. baumii LS protein was expressed in E. coli BL21 (DE3) (84.99 kDa + 21.15 kDa tag protein). The transcription level of S. baumii LS was the highest on day 11 in mycelia (1.6-fold).


Introduction
Sanghuangporus baumii, a traditional Chinese medicine, grows on the trunk of Syringa reticulat [1] . S. baumii used to belong to the genus of Inonotus or Phellinus and now belongs to the genus of Sanghuangporus [2] . Numerous studies have demonstrated that S. baumii possesses antitumor, antioxidant, and anti-in ammatory [3] . Besides, S. baumii contains many secondary metabolites, such as polysaccharides, avonoids, and terpenes [1] . Triterpenoids in S. baumii are important pharmacological active substances, which have the anti-tumor, anti-in ammatory, anti-bacterial and antiviral effect [4] .
Triterpenoids are complex mixtures, formed by six isoprene units and have a variety of structures.
Lanosterol synthase, a member of the OSC ((3S)-2, 3-oxidosqualene cyclase) family, is not only a key enzyme in cholesterol and steroid synthesis in animals but also in sterol and triterpenoids synthesis in plants and fungi. The enzyme activities are determined by a few key amino acids in the active site [5] .
Therefore, amino acids structure and activities are of great importance for the study of enzyme function and catalytic mechanism. In Sacchar cerevisiae, a variety of cyclization products are produced by mutating lanosterol synthase His234, which means the lanosterol synthase is related to the deprotonation of cations on the four-ring structure [12] . The swiss-models of OSC were studied in herbal plants, which shows the OSC structure in Panax ginseng, Panax notoginseng, Taraxacum mongolicum, Cimicifuga racemosa, and Lotus corniculatus are stable. There are some variations in the random curl, most of them are distributed on the surface of the protein [13] . In Siraitia grosvenorii, homology modeling has been used to predict the 3D structure of OSC (Cycloartenol Synthase, CAS). The interaction between CAS and substrates are analyzed by molecular docking, which shows that Asp491, Cys492, Cys570, Tyr540, and His265 are the key catalytic sites in CAS [14] .
At present, the LS gene has been cloned and examined in other organisms. For example, Ganoderma lucidum LS is cloned and transferred in an erg7 yeast strain lacking LS activity, which demonstrates that the cloned cDNA encodes a functional LS [15] . The ergosterol content in de cient mutant decreases to 42% than that of in wild strain after the Saccharomyces cerevisiae LS knockout cassette harboring the loxP-Marker-loxP element [16] . Poria cocos LS and promoter are cloned and then transformed into ERG7 hybrid diploid Saccharomyces cerevisiae strain YHR072W. The results show that the P. cocos LS gene mediates the formation of ergosterol in S. cerevisiae [17] . In S. baumii, acetyl-CoA acetyl transferase gene (AACT) [18] , 3-hydroxy-3-methylglutaryl-CoA synthase gene (HMGS) [19] , and squalene epoxidase gene (SE) [20] in MVA pathway have been cloned and expresses in E. coli, but S. baumi triterpenoids synthesis pathway is not fully understood. Therefore, it is highly important to analyze the characteristics of key genes at the beginning of the experiment.
S. baumii LS and promoter was analyzed for the rst time in this study. And we detected the transcription level of LS by real-time quantitative PCR further. Then, LS was constructed in pET-32a (+) and expressed in E. coli.

Materials And Methods
Strain and plasmid S. baumii was authenticated by visual observation and Internal Transcribed Spacer (ITS) identi cation. The pET-32a(+) vector was used as expression vectors. E. coli DH5α and BL21 (DE3) strains (Tiangen, Beijing, China) were purchased to expand reproduction and express recombinant vectors.
RNA, cDNA and DNA extraction S. baumii mycelia were collected and washed by distilled water. Then ground to powder by using liquid nitrogen. Next, S. baumii RNA, cDNA, and DNA were extracted according to Wang's description [18] .
Ampli cation of the full length of LS To obtain the full length of LS, the LS gene fragment should be cloned rstly. Primers (LS-S, LS-A; Table 1) were designed according to S. baumii transcription data [21] . Using cDNA as a template, LS 3 'and 5' gene fragments were ampli ed [18] . The 3' and 5' RACE PCR ampli cation products were veri ed by AGE (agarose gel electrophoresis) and sequencing (Boshi, Harbin, China). Subsequently, A 468 bp LS 5' fragment and a 2,011 bp 3' fragment were obtained, and the 5' and 3' cDNA fragments were spliced using software, a 2,445 bp S. baumii LS sequence was obtained.

Heterologous expression of LS in E. coli
Trelief™ SoSoo Cloning Kit was used to structure the expression vector to express in E. coli. Primers (LS-EcoRI, LS-HindIII; Table 1) (containing 20 bp homologous anks, which complementary with the ends of the pET-32a(+) linearized vector) were designed for PCR ampli cation [18] . The PCR product with homologous anks was puri ed using a MiniBEST DNA Fragment Puri cation Kit Ver.4.0 (TaKaRa). Then, the puri ed product and linearized vector were mixed according to the instructions. The recombinant vector was transferred to E. coli DH5α, and a single positive clone was inoculated in LB medium for extracting plasmid (pET-LS).
Bacterial liquid and gel were treated according to Wang's description [18] . At the end of running, the gel was stained with Coomassie Brilliant Blue Fast Staining solution (Solarbio).
Ampli cation of the LS promoter LS promoter primers (LS-P-f, LS-P-r; Table 1) were designed based on LS sequencing analysis and S. baumii genomic DNA. The PCR product was ampli ed and sequenced (Boshi, Harbin, China).

Sequence analysis
The LS ORF was obtained using ORF Finder (https://ncbiinsights.ncbi.nlm.nih.gov/tag/or nder/), and the LS sequence was compared using the NCBI database (https://www.ncbi.nlm.nih.gov/). Phylogenetic trees were constructed using MEGA 6.0 with the neighbor-joining method, and LS sequences from other species were downloaded from NCBI.

Transcription analysis of LS in the different development stage
Total RNA from mycelia, primordia, and young fruiting bodies were extracted, and cDNA was produced using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara). Quantitative real-time PCR (qRT-PCR) was performed on an Mx3000P Sequence Detection System (Agilent Technologies, California, USA). The reaction system was mixed as follow: 10 µL of SYBR GreenMaster Mix (Takara), 0.4 µL of LS-T-f (Table 1), 0.4 µL of LS-T-r (Table 1), 1 µL of cDNA from the different development stage and 8.2 µL of ddH 2 O. Each sample was analyzed in triplicate and repeated three times. Mycelia from day 9 served as a control sample, and β-tubulin was used as an internal reference for all qRT-PCR analyses. Relative transcription levels were calculated using the 2 − ΔΔCT method [23] . Variance (ANOVO) was used to analyze data, and P < 0.05 was considered statistically signi cant.
Data analysis LS transcription levels data was compared with total triterpenoids content [18] . Line chart was drawn using Excel software.

Sequence analysis of the LS sequence
The S. baumii LS sequence we obtained includes a 129 bp 5' UTR, an 87 bp 3' UTR, and a 2,229 bp ORF encoding a 734 amino acids. Next, it was submitted to NCBI GenBank to get accession number (Genbank accession number: MT108416). S. baumii LS sequence shared 89% identity and 99% query cover with Inonotus obliquus (Genbank accession number: QEP49720.1). The 20 homologous LS sequences from NCBI were used for phylogenetic tree construction, which showed that S. baumii LS was most similar to I. obliquus, but more distantly related to Amanita thiersii LS and Amanita muscaria LS (Fig. 2). The molecular weight was 84.99 kDa and the theoretical isoelectric point was 5.87. Alanine (Ala) was the most common (8.5%), and valine (Val) was the least (5.3%) in the amino acid sequence. Instability index of protein was 48.38, which means LS was unstable [24] . LS did not exist transmembrane helix and signal peptide through TMHMM Server (v.2.0) and SignalP 4.0 Server analysis. And LS was a hydrophilic protein by ProtScale software analysis. Prediction by solubility and subcellular localization showed LS was an insoluble protein in the cytoplasmic. When compared LS sequences with other fungi (Fig. 3) and predicted conserved domains, they were relatively conservative. The SQ Hop_cyclase_N was located in the 76-362 amino acid region of S.baumii LS, and the SQ Hop_cyclase_C was located in the 382-716 amino acid region of S.baumii LS.
The secondary structure of LS contained 46.36% α-helix, 2.56% β-sheet, and 51.08% random coil [25] . The three-dimensional structure of LS had 47.8% similarity with other LS by X-ray prediction [26−29] (Fig. 4). The three-dimensional structure model typically had 92% of residues in the allowed regions by PROCHECK soft, which suggested that the LS protein is theoretically reliable.
Analysis of the LS promoter A 1,854 bp sequence of LS upstream was ampli ed and submitted in NCBI (Genbank accession number: MT108416). Promoter sequence was analyzed by software, which found sequence transcription start site ranged from 1,740 bp to 1,790 bp. LS promoter contained many acting elements ( Table 2).

Prokaryotic expression
The prokaryotic expression of the pET-LS fusion protein was shown in a gel. Expected protein bands were consistent with software prediction (84.99 kDa + 21.15 kDa tag protein). The LS protease yield was correlated with induction time (Fig. 5). As shown in the gure, LS production was increasing with induction time.

Relationship between LS transcription level and triterpenoids content
The LS transcription level up-regulated 1.6-fold for the rst time on day 11 in mycelia ( Fig. 6), then decreased, and increased again to the primordial stage (1.5-fold). In the young fruit stage, the LS transcription level was reduced to a minimum (0.1-fold). The LS transcription level was highest on day 11 in mycelia and primordial stage, and it was signi cantly different from other stages.
The change trend of LS transcription level was opposite to that of triterpenoids content after 11 days. LS transcription level was lowest, but triterpenoids content was higher in young fruiting bodies phase.

Discussion
At present, the increased yield of Sanghuangporus triterpenoids is mainly by optimizing the extraction method and changing the inducer. Inonotus obliquus is extracted by 5% (v/w) Viscozyme L, and the total triterpenoids are the highest (24.3 mg/g) [30] . Methyl jasmonate (MeJA) (150 mmol/L) can induce Inonotus baumii to enhance triterpenoids yield, which is 4.05-fold higher than that in water [31] . Although these methods can increase triterpenoids yield, they are insu cient for the triterpenoids production in factories. Therefore, improving the triterpene yield by molecular biotechnology is a popular research method.
LS, a key enzyme in the MVA pathway, is a precursor of triterpenoid synthesis. A 2,229 bp S. baumii LS ORF sequence was obtained by PCR ampli cation and BLAST in NCBI. In Ganoderma lucidum, LS was found to contain a 2,181 bp ORF encoding a 726 amino acids [15] . In Saccharomyces cerevisiae, the LS gene coding region contains 2,901bp nucleotides [32] . In Poria cocos, a 2187 ORF was found out that it codes a 728 amino acid [17] . S. baumii LS ORF was longer than G. lucidum, S. cerevisiae, and P. cocos. In this study, we discovered S. baumii LS sequences and analyzed the molecular weight of S. baumii LS protein (84.99 kDa), and found the protein was unstable.
According to signal peptide analysis, subcellular localization, and prediction of the transmembrane domain, LS is an insoluble protein in the cytoplasmic, which is consistent with the site of S. baumii MVA pathway.
The site of transcription start in S. baumii LS promoter sequence ranged from 1 740 bp to 1790 bp. There are responsiveness acting elements contained by S. baumii LS promoter, which is similar to other species.
In the past, AACT promoters in the S. baumii MVA pathway were cloned [18] . LS and AACT promoter all contained ABRE (cis-acting element involved in the abscisic acid responsiveness), CGTCA-motif (cisacting regulatory element involved in the MeJA-responsiveness), LTR (cis-acting element involved in lowtemperature responsiveness), TGACG-motif (cis-acting regulatory element involved in the MeJAresponsiveness), which showed triterpenoids synthesis may relay to abscisic acid, low-temperature, and MeJA. LS promoter contained more ARE (essential cis-acting regulatory element for the anaerobic induction), ATC-motif (part of a conserved DNA module involved in light responsiveness), Box 4 (part of a conserved DNA module involved in light responsiveness), GT1-motif (light-responsive element), MRE (MYB binding site involved in light responsiveness) and TGA-element (auxin-responsive element) than AACT promoter. LS promoter elements are related to anaerobic induction and light responsiveness, so the reaction to catalyzed LS may require anaerobic and light stimuli.
In Poria cocos, the LS promoter region contains transcriptional sequesters associated with transcriptional regulation, including acid, light, methyl jasmine, etc [17] . In G. lucidum, the potential regulatory elements include the G-box/GT1-motif (light-responsive element), ABRE(cis-acting element involved in abscisic acid responsiveness), AuxRR-core (cis-acting regulatory element involved in auxin responsiveness), MBS (MYB binding site, involved in drought-inducibility), and Box-W1 (fungal elicitor responsive element). But no MeJA responsive element has been found in G. lucidum [15] . The S. baumii LS protein bands were consistent with software prediction (84.99 kDa + 21.15 kDa tag protein) and whose transcription level rst up-regulated 1.6-fold on day 11 in mycelia, then decreased, and increased again in the primordial stage (1.5-fold). The S. baumii LS transcription level was the highest on day 11 in mycelia. In G. lucidum, gene transcription level is relatively low in the mycelia and then increased to the primordial level, which is also the highest level (about 8.39 fold compared with 10 dold mycelia) [15] . This result is different from S. baumii LS expression, but they all have higher transcription level in the primordial stage.
The variation trend of LS transcriptional level was opposite to that of triterpenoids content. This may be because the LS transcriptional level was inhibited with the accumulation of triterpenoids in S. baumii intracellular. This result is similar to zhang 's [33] research, and the triterpenoids content is the highest in the S. baumii mycelia.
To sum up, S. baumii LS and promoter were cloned and analyzed for the rst time. Subsequently, LS was constructed into the vector and expressed in E. coli BL21. The transcriptional level of LS was explored at different development stages. These studies help us to understand the LS as a key enzyme gene in the triterpenoids synthesis pathway. However, to understand the mechanism of triterpenoids synthesis and gene function better, it is necessary to study the overexpression and suppression expression of LS in S. baumii. Moreover, the transcriptional regulatory factors of LS gene upstream may also be the key factors controlling triterpenoids synthesis.     Three-dimensional structure schematic representation of S. baumii LS.  LS transcriptional level and triterpenoids content in S. baumii in different development stages.