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 insufficient 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 amplification 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 (cis-acting regulatory element involved in the MeJA-responsiveness), LTR (cis-acting element involved in low-temperature responsiveness), TGACG-motif (cis-acting regulatory element involved in the MeJA-responsiveness), 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 first 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 d-old 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 first 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.