Transcriptomic Analysis of Kalopanax septemlobus and Characterization of KsBAS, CYP716A94 and CYP72A397 Genes Involved in Hederagenin Saponin Biosynthesis.

Kalopanax septemlobus, commonly named the castor aralia tree, is a highly valued woody medicinal tree belonging to the family Araliaceae. Kalopanax septemlobus contains approximately 15 triterpenoid saponins primarily constituted of hederagenin aglycones. Hederagenin is a representative precursor for hemolytic saponin in plants. In the present study, transcriptome analysis was performed to discover genes involved in hederagenin saponin biosynthesis in K. septemlobus. De novo assembly generated 82,698 unique sequences, including 17,747 contigs and 64,951 singletons, following 454 pyrosequencing. Oxidosqualene cyclases (OSCs) are enzymes that catalyze the formation of diverse triterpene skeletons from 2,3-oxidosqualene. Heterologous expression of an OSC sequence in yeast revealed that KsBAS is a ß-amyrin synthase gene. Cytochrome P450 genes (CYPs) make up a supergene family in the plant genome and play a key role in the biosynthesis of sapogenin aglycones. In total, 95 contigs and 110 singletons annotated as CYPs were obtained by sequencing the K. septemlobus transcriptome. By heterologous expression in yeast, we found that CYP716A94 was ß-amyrin 28-oxidase involved in oleanolic acid production from ß-amyrin, and CYP72A397 was oleanolic acid 23-hydroxylase involved in hederagenin production from oleanolic acid. Engineered yeast co-expressing KsBAS, CYP716A94 and CYP72A397 produced hederagenin. Kalopanax septemlobus CYP72A397 is a novel CYP enzyme that synthesizes hederagenin aglycone from oleanolic acid as a single product. In conclusion, we characterized three genes participating in sequential steps for hederagenin biosynthesis from ß-amyrin, which are likely to play a major role in hederagenin saponin biosynthesis in K. septemlobus.

Exploring genes involved in benzoic acid biosynthesis in the Populus davidiana transcriptome and their transcriptional activity upon methyl jasmonate treatment.

Benzoic acids (BAs) are important structural elements in a wide variety of essential compounds and natural products, and play crucial roles in plant fitness. BA is a precursor of diverse benzenoid compounds, including the hormone salicylic acid (SA) and the aglycone moiety of salicin, which is particularly important in the Salicaceae family. The biosynthetic pathways leading to BA formation in plants are largely unknown. Recently, the CoA-dependent ß-oxidative BA biosynthesis pathway, which occurs in peroxisomes, has been characterized in petunia. The core of this pathway is cinnamic acid → cinnamoyl-CoA â†’ 3-hydroxy-3-phenylpropanoyl-CoA â†’ 3-oxo-3-phenylpropanoyl-CoA â†’ benzoyl-CoA. Here, we used 454 pyrosequencing to analyze the transcriptome of Populus davidiana and isolate putative genes involved in BA biosynthesis. De novo assembly generated 57,322 unique sequences, including 15,217 contigs and 42,105 singletons. From the unique sequences, we selected six genes exhibiting high similarity to genes encoding L-phenylalanine ammonia lyase, cinnamate:CoA ligase, cinnamoyl-CoA hydratase-dehydrogenase, 3-ketoacyl-CoA thiolase, benzoyl-CoA:benzyl alcohol O-benzoyltransferase, and benzaldehyde dehydrogenase. Each of these enzymes might be involved in BA biosynthesis. Real-time PCR (qPCR) analysis revealed that these six genes were highly transcribed in the aerial organs of P. davidiana, particularly in leaves. Treating the leaves of in vitro cultured plants with methyl jasmonate (MeJA) strongly enhanced the mRNA accumulation of all 6 genes, and this treatment also clearly enhanced the accumulation of BA, SA, salicyl alcohol, benzyl alcohol, benzyl benzoate, and benzaldehyde but not salicin. Our study shows that P. davidiana may possess a CoA-dependent ß-oxidative BA synthesis pathway. We also identified a relationship between the transcription of these genes and the accumulation of benzenoids, including BA and SA, which are highly responsive to the defense signaling molecule (MeJA).

Alteration of Panax ginseng saponin composition by overexpression and RNA interference of the protopanaxadiol 6-hydroxylase gene (CYP716A53v2).

BACKGROUND: The roots of Panax ginseng contain noble tetracyclic triterpenoid saponins derived from dammarenediol-II. Dammarene-type ginsenosides are classified into the protopanaxadiol (PPD) and protopanaxatriol (PPT) groups based on their triterpene aglycone structures. Two cytochrome P450 (CYP) genes (CYP716A47 and CYP716A53v2) are critical for the production of PPD and PPT aglycones, respectively. CYP716A53v2 is a protopanaxadiol 6-hydroxylase that catalyzes PPT production from PPD in P. ginseng. METHODS: We constructed transgenic P. ginseng lines overexpressing or silencing (via RNA interference) the CYP716A53v2 gene and analyzed changes in their ginsenoside profiles. RESULT: Overexpression of CYP716A53v2 led to increased accumulation of CYP716A53v2 mRNA in all transgenic roots compared to nontransgenic roots. Conversely, silencing of CYP716A53v2 mRNA in RNAi transgenic roots resulted in reduced CYP716A53v2 transcription. HPLC analysis revealed that transgenic roots overexpressing CYP716A53v2 contained higher levels of PPT-group ginsenosides (Rg1, Re, and Rf) but lower levels of PPD-group ginsenosides (Rb1, Rc, Rb2, and Rd). By contrast, RNAi transgenic roots contained lower levels of PPT-group compounds and higher levels of PPD-group compounds. CONCLUSION: The production of PPD- and PPT-group ginsenosides can be altered by changing the expression of CYP716A53v2 in transgenic P. ginseng. The biological activities of PPD-group ginsenosides are known to differ from those of the PPT group. Thus, increasing or decreasing the levels of PPT-group ginsenosides in transgenic P. ginseng may yield new medicinal uses for transgenic P. ginseng.

Production of the dammarene sapogenin (protopanaxadiol) in transgenic tobacco plants and cultured cells by heterologous expression of PgDDS and CYP716A47.

KEY MESSAGE: Protopanaxadiol (PPD) is an aglycone of dammarene-type ginsenoside and has high medicinal values. In this work, we reported the PPD production in transgenic tobacco co-overexpressing PgDDS and CYP716A47. PPD is an aglycone of ginsenosides produced by Panax species and has a wide range of pharmacological activities. PPD is synthesized via the hydroxylation of dammarenediol-II (DD) by CYP716A47 enzyme. Here, we established a PPD production system via cell suspension culture of transgenic tobacco co-overexpressing the genes for PgDDS and CYP716A47. The concentration of PPD in transgenic tobacco leaves was 2.3-5.7 µg/g dry weight (DW), depending on the transgenic line. Leaf segments were cultured on medium with various types of hormones to induce callus. Auxin treatment, particularly 2,4-D, strongly enhanced the production of DD (783.8 µg g(-1) DW) and PPD (125.9 µg g(-1) DW). Treatment with 2,4-D enhanced the transcription of the HMG-Co reductase (HMGR) and squalene epoxidase genes. PPD production reached 166.9 and 980.9 µg g(-1) DW in a 250-ml shake flask culture and in 5-l airlift bioreactor culture, respectively.