Research advances in methods of cyclezation mechanism of sesquiterpenes / 中国中药杂志

Terpenes are the largest group of natural products and contain the widest assortment of structural types. Terpene cyclization is also the most complex reaction found in nature. For a long time, terpenoids with diverse structures have attracted natural product chemists to explore their biosynthesis mechanism. Such a large number of terpene skeletons are catalyzed by enzymes called terpene synthase. Sesquiterpene synthase is a kind of terpene synthase, which can catalyze the cyclization of linear precursor farnesyl pyrophosphate(FPP) to sesquiterpene skeletons. Sesquiterpene synthase cyclize a single precursor FPP into many sesquiterpene skeletons. With the continuous discovery of sesquiterpene synthase, the cyclization mechanism of sesquiterpene synthase has been studied deeply. In recent years, with the development and improvement of isotope labeling of substrate FPP and structural analysis of sesquiterpene synthase, the structure and cyclization mechanism of sesquiterpene synthase have been studied more systematically and accurately. In this review, we reviewed the progress of the research methods on the mechanism of sesquiterpene cyclization by substrate isotope labeling and protein structure, as well as the summary and prospect of sesquiterpene synthase research.

Advances in biosynthesis of cadinane sesquiterpenes / 生物工程学报

Cadinanes are a class of bicyclic sesquiterpenes with complex stereochemistry and broad pharmacological activities, such as antibacterial, anti-inflammatory, and hypoglycemic activities. To date, structurally diverse and bioactive cadinane sesquiterpenes have been isolated and identified from a variety of plants and microorganisms. Moreover, deeper understandings on cadinane sesquiterpene synthases have been made. This article categorized the 124 new cadinanes which were published in the literatures in the past four years (2017-2020) into five structural types, and presented their pharmacological activities. We also illustrated the elucidation of the biosynthetic pathways for typical cadinanes, summarized the research progress on cadinane sesquiterpene synthases. Finally, current challenges and future prospects were proposed and discussed.

Functional Identification of a Multi-product Sesquiterpene Synthase from Ganoderma sinense / 中国实验方剂学杂志

Objective: Ganoderma sinense is one of the most famous medicinal fungi in China. Because it is a model medicinal fungus of basidiomycete,the functional identification of its sesquiterpene synthase is of great significance for the biosynthesis and regulation studies of fungal sesquiterpene. Method: A sesquiterpene synthase gene was discovered by mining the genome data of G. sinense. The sesquiterpene's conservative motifs was analyzed through multiple sequence alignment with two identified sesquiterpene synthases of G. sinense and three terpenoid synthases in the Nr database,which have the highest similarity to it. Subsequently,heterologous expression was observed in Escherichia coli,and protein expression was detected by SDS-PAGE. Volatile compounds were collected by solid phage microextraction (SPME) and detected by GC-MS. Result: The enzyme containing sesquiterpene conserved motifs DDXXDE and NSE/DTE were efficiently expressed in E. coli,and 11 sesquiterpenes were synthesized with endogenous FPP as the substrate. The product α-cadinol at 18.6 min was considered to be the main product,and previous studies showed a significant anticancer activity. According to the comparison with chemical standards,three products were identified as γ-muurlene,α-muurolene and δ-cadinene,respectively. Conclusion: The functional identification of multi-product sesquiterpene synthase from G. sinense can promote the study on the mechanism underlying its product diversity,and lay a foundation for the production of rare or novel sesquiterpenes by improving the catalytic activity of enzymes with enzyme engineering technology.

Cloning and expression analysis of sesquiterpene synthase gene in Dendrobium officinale / 中草药

Objective To clone the full-length cDNA encoding sesquiterpene synthase (SES) gene from Dendrobium officinale (DoSES), then to analyze the expression difference in different tissues and expression patterns of DoSES induced by methyl jasmonate (MeJA). Methods RACE was used to clone the full length cDNA of DoSES. Bioinformatics analysis was carried out by using related software and online website. Then, the expression patterns of DoSES were studied by quantitative real-time PCR (qRT-PCR). Results The DoSES gene was obtained (GenBank accession number KX278311), and the full-length cDNA was 1 890 bp, encoding the protein of 520 amino acids. DoSES had highest homology ( ≈ 60%) with SES proteins from other plants. Tissue expression analysis showed that DoSES had the highest expression in the stems, followed by leaves, flowers, protocorm and roots. The results of qRT-PCR analysis suggested that DoSES could be induced by MeJA. Conclusion The cDNA encoding DoSES was cloned. The research results provide basic materials for further function alanalysis of this gene in the future.

Cloning, bioinformatics, and expression analysis of sesquiterpene synthase gene As-SesTPS1 from Aquilaria sinensis / 中草药

To clone a sesquiterpene synthase gene denoted As-SesTPS1 from total RNA of Aquilaria sinensis based on previous transcriptome sequencing data and analyze the bioinformatics and expression of the gene. The full length cDNA of As-SesTPS1 was obtained by reverse transcription-PCR (RT-PCR). The sequence similarity and homology were analyzed, and the structure and function of the coding protein were predicted by bioinformatics method. The gene expression levels in different samples of A. sinensis were quantified using qRT-PCR technique. The sesquiterpene synthase gene As-SesTPS1 was cloned, which contained an ORF of 1 671 bp, encoding 556 amino acids, and shared high similarity to multiple sesquiterpene synthase genes. The protein encoded by this gene had conserved RRx8W and DDxxD motifs. It was confirmed the gene was highly expressed in agarwood sample through qRT-PCR technique. The sesquiterpene synthase gene As-SesTPS1 is cloned, which will lay a foundation for further study of sesquiterpene biosynthase pathway in A. sinensis.

Subcellular localization of a sesquiterpene synthase from Aquilaria sinensis utilizing three transient expression systems / 中草药

Objective: To investigate the subcellular localization of a sesquiterpene synthase from Aquilaria sinensis (ASS) and compare three transient expression systems in the subcellular localization study. Methods: An ASS gene was amplified by RT-PCR using specific primers and cloned into the pEZS-NL and pFGC5941GFP to generate two plant expression vectors: pEZS-NL-ASS- GFP and p5941-GFP-ASS. Three transient expression systems, agroinfiltration of tobacco leaves, particle bombardment of onion epidermal cells, and PEG transformation of protoplasts isolated from ASS calli were adopted and compared. The expression of the GFP fusion proteins were observed by a confocal laser scanning microscopy. Results: The green fluorescence could be observed in all the three systems. However, the results were comparatively different. The cytoplasm and plastid localization of the GFP fusion protein observed in protoplasts was comparatively clear and was consistent with the studies in other plant species. Conclusion: The results demonstrate that the ASS is located in the cytoplasm and plastid in the ASS protoplasts and the different locations in three systems might be caused by the distinct characters of the heterologous or homologous cells.