Chromosome-level genome assembly of Prunella vulgaris L. provides insights into pentacyclic triterpenoid biosynthesis.

Prunella vulgaris is one of the bestselling and widely used medicinal herbs. It is recorded as an ace medicine for cleansing and protecting the liver in Chinese Pharmacopoeia and has been used as the main constitutions of many herbal tea formulas in China for centuries. It is also a traditional folk medicine in Europe and other countries of Asia. Pentacyclic triterpenoids are a major class of bioactive compounds produced in P. vulgaris. However, their biosynthetic mechanism remains to be elucidated. Here, we report a chromosome-level reference genome of P. vulgaris using an approach combining Illumina, ONT, and Hi-C technologies. It is 671.95 Mb in size with a scaffold N50 of 49.10 Mb and a complete BUSCO of 98.45%. About 98.31% of the sequence was anchored into 14 pseudochromosomes. Comparative genome analysis revealed a recent WGD in P. vulgaris. Genome-wide analysis identified 35 932 protein-coding genes (PCGs), of which 59 encode enzymes involved in 2,3-oxidosqualene biosynthesis. In addition, 10 PvOSC, 358 PvCYP, and 177 PvUGT genes were identified, of which five PvOSCs, 25 PvCYPs, and 9 PvUGTs were predicted to be involved in the biosynthesis of pentacyclic triterpenoids. Biochemical activity assay of PvOSC2, PvOSC4, and PvOSC6 recombinant proteins showed that they were mixed amyrin synthase (MAS), lupeol synthase (LUS), and ß-amyrin synthase (BAS), respectively. The results provide a solid foundation for further elucidating the biosynthetic mechanism of pentacyclic triterpenoids in P. vulgaris.

De Novo Transcriptome Sequencing of Codonopsis lanceolata for Identification of Triterpene Synthase and Triterpene Acetyltransferase.

Codonopsis lanceolata (Campanulaceae) is a perennial plant commonly known as the bonnet bellflower. This species is widely used in traditional medicine and is considered to have multiple medicinal properties. In this study, we found that shoots and roots of C. lanceolata contained various types of free triterpenes (taraxerol, ß-amyrin, α-amyrin, and friedelin) and triterpene acetates (taraxerol acetate, ß-amyrin acetate, and α-amyrin acetate). The content of triterpenes and triterpene acetates by GC analysis was higher in the shoot than in the roots. To investigate the transcriptional activity of genes involved in triterpenes and triterpene acetate biosynthesis, we performed de novo transcriptome analysis of shoots and roots of C. lanceolata by sequencing using the Illumina platform. A total of 39,523 representative transcripts were obtained. After functional annotation of the transcripts, the differential expression of genes involved in triterpene biosynthetic pathways was investigated. Generally, the transcriptional activity of unigenes in the upstream region (MVA and MEP pathway) of triterpene biosynthetic pathways was higher in shoots than in roots. Various triterpene synthases (2,3-oxidosqualene cyclase, OSC) participate to produce triterpene skeletons by the cyclization of 2,3-oxidosqualene. A total of fifteen contigs were obtained in annotated OSCs in the representative transcripts. Functional characterization of four OSC sequences by heterologous expression in yeast revealed that ClOSC1 was determined as taraxerol synthase, and ClOSC2 was a mixed-amyrin synthase producing α-amyrin and ß-amyrin. Five putative contigs of triterpene acetyltransferases showed high homology to the lettuce triterpene acetyltransferases. Conclusively, this study provides the basis of molecular information, particularly for the biosynthesis of triterpenes and triterpene acetates in C. lanceolata.

[Cloning and bioinformatics analysis of ß-amyrin synthase in Dipsacus asper].

Dipsaci Radix is one of the commonly used Chinese medicinal materials in China, with a long history. It has the medicinal activities of nourishing liver and kidney, recovering from broken sinews, and treating bone fracture. Triterpenoid saponins are the main functional ingredients of Dipsacus asper. ß-Amyrin synthases(ß-AS) as a superfamily of oxidosqualene cyclases(OSCs) can catalyze the construction of the skeleton structure of oleanane-type triterpenoid saponins. There are only a few studies about the ß-AS in D. asper, and the catalytic mechanism of this enzyme remains to be explored. To enrich the information of ß-AS, according to the transcriptome sequencing results, we cloned DaWß-AS gene from D. asper into a specific vector for heterologous expression in Escherichia coli. In the meantime, real-time PCR was performed to analyze the relative expression of DaWß-AS in four different tissues of D. asper. The results of RT-qPCR showed DaWß-AS had the highest expression level in leaves. Bioinformatics results indicated that DaWß-AS had a conserved domain of PLN03012 superfamily, belonging to the cl31551 superfamily. There was no transmembrane domain or signal peptide in DaWß-AS. This study provides a scientific basis for revealing the biological pathways of triterpenoid saponins in D. asper, which will facilitate the biosynthesis of the associated saponins and afford reference for the cultivation and development of high-quality resources of D. asper.

Biosynthesis of TiO2 nanoparticles by Caricaceae (Papaya) shell extracts for antifungal application.

Titanium dioxide nanoparticles (TiO2 NPs) were prepared by Caricaceae (Papaya) Shell extracts. The Nanoparticles were analyzed by UV-Vis spectrums, X-ray diffractions, and energy-dispersive X-rays spectroscopy analyses with a scanning electron microscope. An antifungal study was carried out for TiO2 NP in contradiction of S. sclerotiorums, R. necatrixs and Fusarium classes that verified a sophisticated inhibitions ratio for S. sclerotiorums (60.5%). Germs of pea were individually preserved with numerous concentrations of TiO2 NPs. An experience of TiO2 NPs (20%, 40%, 80% and 100%), as well as mechanisms that instigated momentous alterations in seed germinations, roots interval, shoot lengths, and antioxidant enzymes, were investigated. Associated with controls, the supreme seeds germinations, roots and plant growth were perceived with the treatments of TiO2 NPs. Super-oxide dis-mutase and catalase activities increased because of TiO2 NPs treatments. This advocates that TiO2 Nanoparticles may considerably change antioxidant metabolisms in seed germinations.

Metabolic stimulation-elicited transcriptional responses and biosynthesis of acylated triterpenoids precursors in the medicinal plant Helicteres angustifolia.

BACKGROUND: Helicteres angustifolia has long been used in Chinese traditional medicine. It has multiple pharmacological benefits, including anti-inflammatory, anti-viral and anti-tumor effects. Its main active chemicals include betulinic acid, oleanolic acid, helicteric acid, helicterilic acid, and other triterpenoid saponins. It is worth noting that some acylated triterpenoids, such as helicteric acid and helicterilic acid, are characteristic components of Helicteres and are relatively rare among other plants. However, reliance on natural plants as the only sources of these is not enough to meet the market requirement. Therefore, the engineering of its metabolic pathway is of high research value for enhancing the production of secondary metabolites. Unfortunately, there are few studies on the biosynthetic pathways of triterpenoids in H. angustifolia, hindering its further investigation. RESULTS: Here, the RNAs of different groups treated by metabolic stimulation were sequenced with an Illumina high-throughput sequencing platform, resulting in 121 gigabases of data. A total of 424,824 unigenes were obtained after the trimming and assembly of the raw data, and 22,430 unigenes were determined to be differentially expressed. In addition, three oxidosqualene cyclases (OSCs) and four Cytochrome P450 (CYP450s) were screened, of which one OSC (HaOSC1) and one CYP450 (HaCYPi3) achieved functional verification, suggesting that they could catalyze the production of lupeol and oleanolic acid, respectively. CONCLUSION: In general, the transcriptomic data of H. angustifolia was first reported and analyzed to study functional genes. Three OSCs, four CYP450s and three acyltransferases were screened out as candidate genes to perform further functional verification, which demonstrated that HaOSC1 and HaCYPi3 encode for lupeol synthase and ß-amyrin oxidase, which produce corresponding products of lupeol and oleanolic acid, respectively. Their successful identification revealed pivotal steps in the biosynthesis of acylated triterpenoids precursors, which laid a foundation for further study on acylated triterpenoids. Overall, these results shed light on the regulation of acylated triterpenoids biosynthesis.

Reciprocal mutations of two multifunctional ß-amyrin synthases from Barbarea vulgaris shift α/ß-amyrin ratios.

In the wild cruciferous wintercress (Barbarea vulgaris), ß-amyrin-derived saponins are involved in resistance against insect herbivores like the major agricultural pest diamondback moth (Plutella xylostella). Enzymes belonging to the 2,3-oxidosqualene cyclase family have been identified and characterized in B. vulgaris G-type and P-type plants that differ in their natural habitat, insect resistance and saponin content. Both G-type and P-type plants possess highly similar 2,3-oxidosqualene cyclase enzymes that mainly produce ß-amyrin (Barbarea vulgaris Lupeol synthase 5 G-Type; BvLUP5-G) or α-amyrin (Barbarea vulgaris Lupeol synthase 5 P-Type; BvLUP5-P), respectively. Despite the difference in product formation, the two BvLUP5 enzymes are 98% identical at the amino acid level. This provides a unique opportunity to investigate determinants of product formation, using the B. vulgaris 2,3-oxidosqualene cyclase enzymes as a model for studying amino acid residues that determine differences in product formation. In this study, we identified two amino acid residues at position 121 and 735 that are responsible for the dominant changes in generated product ratios of ß-amyrin and α-amyrin in both BvLUP5 enzymes. These amino acid residues have not previously been highlighted as directly involved in 2,3-oxidosqualene cyclase product specificity. Our results highlight the functional diversity and promiscuity of 2,3-oxidosqualene cyclase enzymes. These enzymes serve as important mediators of metabolic plasticity throughout plant evolution.

Probing the functions of friedelane-type triterpene cyclases from four celastrol-producing plants.

Triterpenes are among the most diverse plant natural products, and their diversity is closely related to various triterpene skeletons catalyzed by different 2,3-oxidosqualene cyclases (OSCs). Celastrol, a friedelane-type triterpene with significant bioactivities, is specifically distributed in higher plants, such as Celastraceae species. Friedelin is an important precursor for the biosynthesis of celastrol, and it is synthesized through the cyclization of 2,3-oxidosqualene, with the highest number of rearrangements being catalyzed by friedelane-type triterpene cyclases. However, the molecular mechanisms underlying the catalysis of friedelin production by friedelane-type triterpene cyclases have not yet been fully elucidated. In this study, transcriptome data of four celastrol-producing plants from Celastraceae were used to identify a total of 21 putative OSCs. Through functional characterization, the friedelane-type triterpene cyclases were separately verified in the four plants. Analysis of the selection pressure showed that purifying selection acted on these OSCs, and the friedelane-type triterpene cyclases may undergo weaker selective restriction during evolution. Molecular docking and site-directed mutagenesis revealed that changes in some amino acids that are unique to friedelane-type triterpene cyclases may lead to variations in catalytic specificity or efficiency, thereby affecting the synthesis of friedelin. Our research explored the functional diversity of triterpene synthases from a multispecies perspective. It also provides some references for further research on the relative mechanisms of friedelin biosynthesis.

Expression Analysis of Lanosterol Synthase Gene in Dynamic Accumulation of Triterpenoids in Sanghuangporus baumii.

BACKGROUND: Sanghuangporus baumii is a traditional Chinese medicine with anti- cancer, anti-tumor, and anti-inflammatory effects. Triterpenoids are one of the main medicinal ingredients found in S. baumii. However, the dynamic changes of triterpenoids content and its molecular regulation mechanism are still unclear. OBJECTIVE: Some studies have shown that Lanosterol synthase ( LS) is a key enzyme involved in the mevalonate pathway (MVA pathway) to produce lanosterol, which is a precursor for synthesizing S. baumii triterpenoids. Therefore, the study of LS gene and expression characteristics can provide clues for the further study of triterpenoids synthesis. METHODS: The PCR, RACE PCR, RT-PCR, homologous recombination and prokaryotic expression technology were used to research the gene characteristic and dynamic changes of LS transcription level. RESULTS: The S. baumii LS sequence included a 5'-untranslated region (129 bp), a 3'-untranslated region (87 bp), and an open reading frame (2,229 bp) encoding 734 amino acids. The S. baumii LS protein was expressed in E. coli BL21 (DE3). The transcription start site of the S. baumii LS promoter sequence ranged from 1 740 bp to 1790 bp. The LS promoter contained 12 CAAT-boxes, 5 ABREs, 6 G-Boxes, 6 CGTCA-motifs, and so on. The LS transcription levels were the highest on day 11 in mycelia (1.6-fold), and the triterpenoids content also gradually increased. The transcription levels began to decrease on day 13, but the triterpenoids content still increased. CONCLUSION: The S. baumii LS was cloned and characterized to help to understand the mechanism of triterpenoids synthesis. In addition, we studied the relationship between LS transcription level and triterpenoid dynamic accumulation, and we found that they had a certain correlation.

Comparative transcriptome analysis reveals sesquiterpenoid biosynthesis among 1-, 2- and 3-year old Atractylodes chinensis.

BACKGROUND: Atractylodes chinensis (DC.) Koidz is a well-known medicinal plant containing the major bioactive compound, atractylodin, a sesquiterpenoid. High-performance liquid chromatography (HPLC) analysis demonstrated that atractylodin was most abundant in 3-year old A. chinensis rhizome, compared with those from 1- and 2-year old rhizomes, however, the molecular mechanisms underlying accumulation of atractylodin in rhizomes are poorly understood. RESULTS: In this study, we characterized the transcriptomes from rhizomes of 1-, 2- and 3-year old (Y1, Y2 and Y3, respectively) A. chinensis, to identify differentially expressed genes (DEGs). We identified 240, 169 and 131 unigenes encoding the enzyme genes in the mevalonate (MVA), methylerythritol phosphate (MEP), sesquiterpenoid and triterpenoid biosynthetic pathways, respectively. To confirm the reliability of the RNA sequencing analysis, eleven key gene encoding factors involved in the sesquiterpenoid and triterpenoid biosynthetic pathway, as well as in pigment, amino acid, hormone and transcription factor functions, were selected for quantitative real time PCR (qRT-PCR) analysis. The results demonstrated similar expression patterns to those determined by RNA sequencing, with a Pearson's correlation coefficient of 0.9 between qRT-PCR and RNA-seq data. Differential gene expression analysis of rhizomes from different ages revealed 52 genes related to sesquiterpenoid and triterpenoid biosynthesis. Among these, seven DEGs were identified in Y1 vs Y2, Y1 vs Y3 and Y2 vs Y3, of which five encoded four key enzymes, squalene/phytoene synthase (SS), squalene-hopene cyclase (SHC), squalene epoxidase (SE) and dammarenediol II synthase (DS). These four enzymes directly related to squalene biosynthesis and subsequent catalytic action. To validate the result of these seven DEGs, qRT-PCR was performed and indicated most of them displayed lower relative expression in 3-year old rhizome, similar to transcriptomic analysis. CONCLUSION: The enzymes SS, SHC, SE and DS down-regulated expression in 3-year old rhizome. This data corresponded to the higher content of sesquiterpenoid in 3-year old rhizome, and confirmed by qRT-PCR. The results of comparative transcriptome analysis and identified key enzyme genes laid a solid foundation for investigation of production sesquiterpenoid in A. chinensis.

De novo transcriptome analysis and identification of candidate genes associated with triterpenoid biosynthesis in Trichosanthes cucumerina L.

KEY MESSAGE: De novo transcriptome analysis from callus, leaf, and fruit of Trichosanthes cucumerina L. for the identification of genes associated with triterpenoid biosynthesis, especially bryonolic acid and cucurbitacin B. Trichosanthes cucumerina L. (TC) has been used as a medicinal plant in Thailand with various potential functions. Two major triterpenoids found in this plant, bryonolic acid and cucurbitacin B, are receiving increased attention for their activities. Here, we provide TC transcriptome data to identify genes involved in the triterpenoid biosynthetic pathway through callus, where was previously suggested as a novel source for bryonolic acid production as opposed to leaf and fruit. A de novo assembly of approximately 290-thousand transcripts generated from these tissues led to two putative oxidosqualene cyclases: isomultiflorenol synthase (IMS) and cucurbitadienol synthase (CBS). TcIMS and TcCBS, genes that encode substrates for two characteristic triterpenoids in cucurbitaceous plants, were identified as isomultiflorenol synthase and cucurbitadienol synthase, respectively. These two genes were functionally characterised in mutant yeast Gil77 systems, which led to the productions of isomultiflorenol and cucurbitadienol. Moreover, the callus-specific gene expression profiles were also presented. These obtained information showed candidate cytochrome P450s with predicted full-length sequences, which were most likely associated with triterpenoid biosynthesis, especially bryonolic acid. Our study provides useful information and a valuable reference for the further studies on cucurbitaceous triterpenoids.

ARPI, ß-AS, and UGE regulate glycyrrhizin biosynthesis in Glycyrrhiza uralensis hairy roots.

KEY MESSAGE: ARPI, ß-AS, and UGE were cloned from G. uralensis and their regulatory effects on glycyrrhizin biosynthesis were investigated. ß-AS and UGE but not ARPI positively regulate the biosynthesis of glycyrrhizin. Glycyrrhiza uralensis Fisch. has been used to treat respiratory, gastric, and liver diseases since ancient China. The most important and widely studied active component in G. uralensis is glycyrrhizin (GC). Our pervious RNA-Seq study shows that GC biosynthesis is regulated by multiple biosynthetic pathways. In this study, three target genes, ARPI, ß-AS, and UGE from different pathways were selected and their regulatory effects on GC biosynthesis were investigated using G. uralensis hairy roots. Our data show that hairy roots knocking out ARPI or UGE died soon after induction, indicating that the genes are essential for the growth of G. uralensis hairy roots. Hairy roots with ß-AS knocked out grew healthily. However, they failed to produce GC, suggesting that ß-AS is required for triterpenoid skeleton formation. Conversely, overexpression of UGE or ß-AS significantly increased the GC content, whereas overexpression of ARPI had no obvious effects on GC accumulation in G. uralensis hairy roots. Our findings demonstrate that ß-AS and UGE positively regulate the biosynthesis of GC.

Integrative transcriptome analysis identifies new oxidosqualene cyclase genes involved in ginsenoside biosynthesis in Jilin ginseng.

BACKGROUND: Jilin ginseng, Panax ginseng, is a valuable medicinal herb whose ginsenosides are its major bioactive components. The ginseng oxidosqualene cyclase (PgOSC) gene family is known to play important roles in ginsenoside biosynthesis, but few members of the gene family have been functionally studied. METHODS: The PgOSC gene family has been studied by an integrated analysis of gene expression-ginsenoside content correlation, gene mutation-ginsenoside content association and gene co-expression network, followed by functional analysis through gene regulation. RESULTS: We found that five of the genes in the PgOSC gene family, including two published ginsenoside biosynthesis genes and three new genes, were involved in ginsenoside biosynthesis. Not only were the expressions of these genes significantly correlated with ginsenoside contents, but also their nucleotide mutations significantly influenced ginsenoside contents. These results were further verified by regulation analysis of the genes by methyl jasmonate (MeJA) in ginseng hairy roots. Four of these five PgOSC genes were mapped to the ginsenoside biosynthesis pathway. These PgOSC genes expressed differently across tissues, but relatively consistent across developmental stages. These PgOSC genes formed a single co-expression network with those published ginsenoside biosynthesis genes, further confirming their roles in ginsenoside biosynthesis. When the network varied, ginsenoside biosynthesis was significantly influenced, thus revealing the molecular mechanism of ginsenoside biosynthesis. CONCLUSION: At least five of the PgOSC genes, including the three newly identified and two published PgOSC genes, are involved in ginsenoside biosynthesis. These results provide gene resources and knowledge essential for enhanced research and applications of ginsenoside biosynthesis in ginseng.

[Synergistic effect on biosynthesis of Panax notoginseng saponins by overexpressing a transcription factor PnbHLH and RNA interference of cycloartenol synthase gene].

This study cloned the transcription factor gene PnbHLH which held an open reading frame of 966 bp encoding 321 amino acids. This study constructed the overexpression vector of transcription factor PnbHLH of Panax notoginseng. The combination of PnbHLH overexpression and RNAi of the key enzyme gene PnCAS involved in the phytosterol biosynthesis was achieved in P. notoginseng cells, thus exploring the biosynthetic regulation of P. notoginseng saponins(PNS) by the synergistic effect of PnbHLH overexpression and PnCAS RNAi. The results showed that the PnbHLH transcription factor interacted with the promoters of key enzyme genes PnDS, PnSS and PnSE in the biosynthetic pathway of PNS, and then regulated the expression levels of key enzyme genes and affected the biosynthesis of saponins indirectly. Further study indicated that the synergistic effect of PnbHLH overexpression and PnCAS RNAi was a more effective approach to regulate the biosynthesis of saponins. Compared with the wild type and PnCAS RNAi cells of P. notoginseng, the contents of total saponins and monomeric saponins(Rd, Rb_1, Re, Rg_1 and R_1) were increased to some extent in the cell lines of PnbHLH overexpression and PnCAS RNAi. This indicated that the two ways of forward regulation and reverse regulation of saponin biosynthesis showed superposition effect. This study explored a more rational and efficient regulation strategy of PNS biosynthesis based on the advantages of multi-point regulation of transcription factors as well as the down-regulation of by-product synthesis of saponins.

Characterization of a 2,3-oxidosqualene cyclase in the toosendanin biosynthetic pathway of Melia toosendan.

Toosendanin, bearing a furan ring, is a limonoid belonging to the group of tetranortriterpenoids. Toosendanin is a phytochemical found in the medicinal plant Melia toosendan Sieb. et Zucc. of the Meliaceae family. Toosendanin and its derivatives demonstrate high insecticidal activity and are important pesticides derived from plants. Despite intensive investigation of limonoids over several decades, the biosynthetic pathway of these triterpenoids is less understood. To identify the key enzymes involved in the toosendanin biosynthetic pathway, we analyzed the contents of toosendanin in various plant tissues and parts and found that the highest level of toosendanin was found in the developing fruit and gradually decreased as the fruit matured. More than 346 116 transcripts were assembled based on 394 million paired-end Illumina reads and 6 million PacBio reads from the pooled RNA samples of fruits, leaves and young barks. A total of 186 263 genes were predicted. Six 2,3-oxidosqualene cyclase (OSC) genes were identified by analyzing the association between gene expression and metabolite profiles. Functional analyses using the Nicotiana benthamiana transient expression assay showed that MtOSC1 catalyzed 2,3-oxidosqualene to produce a tetracyclic triterpene skeleton, tirucalla-7,24-dien-3ß-ol, which is predicted as the precursor for toosendanin biosynthesis. We identified another OSC, MtOSC6, which is a lupeol synthase. Using synthetic biology methods, these identified enzymes could be used to model a biosynthetic pathway to produce large quantities of toosendanin.

Citrus reticulata peel oil as an antiatherogenic agent: Hypolipogenic effect in hepatic cells, lipid storage decrease in foam cells, and prevention of LDL oxidation.

BACKGROUND AND AIMS: Hypercholesterolemia and oxidative stress are two of the most important risk factors for atherosclerosis. The aim of the present work was to evaluate mandarin (Citrus reticulata) peel oil (MPO) in cholesterol metabolism and lipid synthesis, and its antioxidant capacity. METHODS AND RESULTS: Incubation of hepatic HepG2 cells with MPO (15-60 µL/L) reduced cholesterogenesis and saponifiable lipid synthesis, demonstrated by [14C]acetate radioactivity assays. These effects were associated with a decrease in a post-squalene reaction of the mevalonate pathway. Molecular docking analyses were carried out using three different scoring functions to examine the cholesterol-lowering property of all the components of MPO against lanosterol synthase. Docking simulations proposed that minor components of MPO monoterpenes, like alpha-farnesene and neryl acetate, as well the major component, limonene and its metabolites, could be partly responsible for the inhibitory effects observed in culture assays. MPO also decreased RAW 264.7 foam cell lipid storage and its CD36 expression, and prevented low-density lipoprotein (LDL) lipid peroxidation. CONCLUSION: These results may imply a potential role of MPO in preventing atherosclerosis by a mechanism involving inhibition of lipid synthesis and storage and the decrease of LDL lipid peroxidation.

Synthesis and evaluation of heterocycle structures as potential inhibitors of Mycobacterium tuberculosis UGM.

In this study, we screen three heterocyclic structures as potential inhibitors of UDP-galactopyranose mutase (UGM), an enzyme involved in the biosynthesis of the cell wall of Mycobacterium tuberculosis. In order to understand the binding mode, docking simulations are performed on the best inhibitors. Their activity on Mycobacterium tuberculosis is also evaluated. This study made it possible to highlight an "oxazepino-indole" structure as a new inhibitor of UGM and of M. tuberculosis growth in vitro.

Comparative Studies on Bioactive Compounds, Ganoderic Acid Biosynthesis, and Antioxidant Activity of Pileus and Stipes of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes) Fruiting Body at Different Growth Stages.

Total phenolics, flavonoids, and polysaccharides, and individual ganoderic acid (GA) contents, antioxidant capacity, and transcription levels of key enzyme genes involved in GA biosynthesis in pileus and stipes of Ganoderma lucidum fruiting body at different growth stages were investigated in this study. Results showed that the highest total phenolics and total flavonoids contents were determined in stipes at spore maturity stage, resulting in high antioxidant activity, while the highest total polysaccharide content was found in pileus at the same stage. The pileus contained more GA than the stipes, and higher contents of ganoderic acid A and D were found at fruiting body mature stage while that of ganoderic acid B, C2, and G were found at bud elongation stage. Results from quantitative real-time PCR indicated that higher gene transcription levels of hydroxyl methylglutaryl-CoA reductase (hmgr), farnesyl pyrophosphate synthase (fps), squalene synthase (sqs), and oxidosqualene cyclase (osc) were found in pileus at bud elongation stage. Our findings will be helpful for understanding the biosynthesis of bioactive components and determining the harvest time for the desired G. lucidum fruiting bodies.

Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice.

Skin lesions, cataracts, and congenital anomalies have been frequently associated with inherited deficiencies in enzymes that synthesize cholesterol. Lanosterol synthase (LSS) converts (S)-2,3-epoxysqualene to lanosterol in the cholesterol biosynthesis pathway. Biallelic mutations in LSS have been reported in families with congenital cataracts and, very recently, have been reported in cases of hypotrichosis. However, it remains to be clarified whether these phenotypes are caused by LSS enzymatic deficiencies in each tissue, and disruption of LSS enzymatic activity in vivo has not yet been validated. We identified two patients with novel biallelic LSS mutations who exhibited congenital hypotrichosis and midline anomalies but did not have cataracts. We showed that the blockade of the LSS enzyme reaction occurred in the patients by measuring the (S)-2,3-epoxysqualene/lanosterol ratio in the forehead sebum, which would be a good biomarker for the diagnosis of LSS deficiency. Epidermis-specific Lss knockout mice showed neonatal lethality due to dehydration, indicating that LSS could be involved in skin barrier integrity. Tamoxifen-induced knockout of Lss in the epidermis caused hypotrichosis in adult mice. Lens-specific Lss knockout mice had cataracts. These results confirmed that LSS deficiency causes hypotrichosis and cataracts due to loss-of-function mutations in LSS in each tissue. These mouse models will lead to the elucidation of the pathophysiological mechanisms associated with disrupted LSS and to the development of therapeutic treatments for LSS deficiency.

Transcriptome analysis and functional characterization of oxidosqualene cyclases of the arjuna triterpene saponin pathway.

Arjuna (Terminalia arjuna) tree has been popular in Indian traditional medicine to treat cardiovascular ailments. The tree accumulates bioactive triterpene glycosides (saponins) and aglycones (sapogenins), in a tissue-preferential manner. Oleanane triterpenes/saponins (derived from ß-amyrin) with potential cardioprotective function predominantly accumulate in the bark. However, arjuna triterpene saponin pathway enzymes remain to be identified and biochemically characterized. Here, we employed a combined transcriptomics, metabolomics and biochemical approach to functionally define a suite of oxidosqualene cyclases (OSCs) that catalyzed key reactions towards triterpene scaffold diversification. De novo assembly of 131 millions Illumina NextSeq500 sequencing reads obtained from leaf and stem bark samples led to a total of 156,650 reference transcripts. Four distinct OSCs (TaOSC1-4) with 54-71 % sequence identities were identified and functionally characterized. TaOSC1, TaOSC3 and TaOSC4 were biochemically characterized as ß-amyrin synthase, cycloartenol synthase and lupeol synthase, respectively. However, TaOSC2 was found to be a multifunctional OSC producing both α-amyrin and ß-amyrin, but showed a preference for α-amyrin product. Both TaOSC1 and TaOSC2 produced ß-amyrin, the direct precursor for oleanane triterpene/saponin biosynthesis; but, TaOSC1 transcript expressed preferentially in bark, suggesting a major role of TaOSC1 in the biosynthesis of oleanane triterpenes/saponins in bark.

Identification and functional characterization of squalene epoxidases and oxidosqualene cyclases from Tripterygium wilfordii.

KEY MESSAGE: We cloned two squalene epoxidases and five oxidosqualene cyclases, and identified their function using CRISPR/Cas9 tool and yeast heterologous expression. Triterpenes are the main active ingredients of Tripterygium wilfordii Hook.f., a traditional Chinese medicinal plant with many encouraging preclinical applications. However, the biosynthetic pathways of triterpenes in this plant are poorly understood. Here, we report on the isolation and identification of two squalene epoxidases (SQE6 and SQE7) and five oxidosqualene cyclases (OSC4-8) from T. wilfordii. Yeast complementation assays showed that TwSQE6 and TwSQE7 can functionally complement an erg1 yeast mutant that was constructed using the CRISPR/Cas9 system. The putative OSC genes were functionally characterized by heterologous expression in yeast. GC/MS analysis of the fermentation products of the transgenic yeast showed that both TwOSC4 and TwOSC6 are cycloartenol synthases, while TwOSC8 is a ß-amyrin synthase. The discovery of these genes expands our knowledge of key enzymes in triterpenoid biosynthesis, and provides additional target genes for increasing the production of triterpenes in T. wilfordii tissue cultures by disrupting competing pathways, or in chassis cells by reconstituting the triterpenoid biosynthetic pathway.