Ginseng Powerpoint Template Free Download
Pinkie Mclucas
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Country of Origin Labeling (COOL) is a labeling law that requires retailers, such as full-line grocery stores, supermarkets and club warehouse stores, to notify their customers with information regarding the source of certain foods. Food products covered by the law include muscle cut and ground meats: lamb, goat, and chicken; wild and farm-raised fish and shellfish; fresh and frozen fruits and vegetables; peanuts, pecans, and macadamia nuts; and ginseng.
In the present study, we sought to develop a high-titer microbial fermentation-based platform for the production of Rg2 and Re. We combined gene discovery and functional assays to characterize a UGT94-familiy gene (PgURT94) from P. ginseng and completely resolve the biosynthetic pathway of Rg2 and Re. Based on a previously constructed PPT- and Rg1-producing yeast chassis23, the complete biosynthetic pathway of Rg2 and Re was reconstructed in yeast. De novo high-level production of Rg2 and Re was achieved through strain engineering and fed-batch fermentation. This work also establishes a successful example of the high-level production of rare natural plant products, especially those with rhamnosylation modifications.
a Heat-map analysis of the relative abundance of PgURT94 expression, along with PgDDS, CYP716A47, CYP716A53v2, and PgUGT71A53 in different parts of P. ginseng. b HPLC analysis of the in vitro reaction products catalyzed by PgURT94 crude enzyme using Rh1 as sugar acceptor and UDP-rhamnose as sugar donor. c HPLC analysis of the in vitro reaction products catalyzed by PgURT94 crude enzyme using Rg1 as sugar acceptor and UDP-rhamnose as sugar donor. Crude enzymes of E. coli strain harboring pET28a empty vector were used as a negative control for above assays and authentic ginsenoside samples Rh1, Rg1, Rg1, and Re were monitored as standards.
Re production in strain Re-01 was much higher than Rg2 production in strain Rg2-04, despite the fact that Re has a more complicated biosynthetic pathway. The Re content is also much higher than that of Rg2 in Panax plants, including P. ginseng and P. notoginseng1,2. To test whether this phenomenon is determined by some intrinsic factors or just a coincidence, we then examined the difference between the two yeast strains. Re-01 and Rg2-04 share the same PPT-producing background strain and downstream rhamnosylation pathway (Fig. 1), thus PgUGT71A53 and PgUGT71A54 may contribute to different production between the two strains.
Authentic ginsenoside standards protopanaxadiol (PPD), protopanaxatriol (PPT), compound K, ginsenoside F1, Rh1, Rg2, Rf, Rg1, and Re were purchased from Nantong Feiyu Biological Technology (Jiangsu, China). Plasmids pMD18-T (TaKaRa, Dalian, China) and pET28a (Merck, Germany) were used for UGTs cloning and expressing, respectively. E. coli strain TOP10 was used for gene cloning, and BL21 (DE3) was used for UGTs heterologous expression. Plasmids pUG66, pAG25 and pSH69 obtained from EUROSCARF were used as the template for the amplification of ble, NatMX and hygroB selection marker, respectively. S. cerevisiae strain PPT-10 and Rg1-0223 constructed in previous study were used as the parent strain for all engineering. The genes PgURT94, PgUGT71A54 (Genbank accession No. KP795113.1), PgUGT71A53 (KF377585.1), PgUGT94Q330, AtRHM2 (Q9LPG6.1), and VvRHM-NRS36 were codon-optimized and synthesized by Genscript Corporation (Nanjing, China).
Panax ginseng C. A. Mey is one of famous medicinal herb plant species. Its major bioactive compounds are various ginsenosides in roots and rhizomes. It is commonly accepted that ginsenosides are synthesized from terpene precursors, IPP and DMAPP, through the cytoplasmic mevalonate (MVA) pathway. Another plastic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway was proved also contributing to ginsenoside generation in the roots of P. ginseng by using specific chemical inhibitors recently. But their gene expression characteristics are still under reveal in P. ginseng. With the development of the high-throughput next generation sequencing (NGS) technologies, we have opportunities to discover more about the complex ginsenoside biosynthesis pathways in P. ginseng.
At the transcriptional level, the MEP pathway has similar contribution to ginsenoside biosynthesis in ginseng roots, but much higher in ginseng leaves, compared with the MVA pathway. The IspD might be the key enzyme for ginsenoside generation through the MEP pathway. These results provide new information for further synthetic biology study on ginsenoside metabolic regulation.
In this work, the sequencing on each ginseng mRNA sample generated more than 20 million reads (Additional file 1: Table S1). After filtering adaptors, low quality reads, rRNA reads, mitochondrial and plastic RNA reads and bacterial reads, we got high-quality clean reads, accounting for more than 85% of the raw reads.
According to previous studies, the ginsenoside biosynthesis process includes three metabolic modules: terpene precursor synthesis, triterpene skeleton synthesis and various-type ginsenoside synthesis [13, 24, 25]. The terpene precursor biosynthesis module consists the MVA pathway and the MEP pathway to generate terpene precursor, IPP or DAMPP [8, 9]. Based on our annotation results, the genes encoding the enzymes involved in the three metabolic modules were all retrieved from our assembled ginseng RNA-seq datasets (Table 2). Particularly for the MEP pathway, it was for the first time to identify all the genes along the whole pathway based on transcriptomic data of ginseng samples. Its largest enzyme family was 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and consisted of about 13 sub-families, 20 genes and 56 isoforms, while its smallest enzyme family was 2-C-methyl-D-erythritol 4-phosphate cytidylyl transferase (IspD), only 2 sub-families, 2 genes and 10 isoforms identified. In addition, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) in the MVA pathway, geranylgeranyl diphosphate synthase (GGR) in the triterpene skeleton biosynthesis module, as well as glycosyl transferase (GT) and cytochrome P450 (CYP450) in the ginsenoside biosynthesis module were also found being the largest protein family in their corresponding metabolic modules, respectively. Furthermore, the gene diversity happened not only in root tissues but also in other tissues of ginseng plants (Table 2).
Therefore, the gene transcription levels related to the MVA pathway, triterpene skeleton biosynthesis and the final steps of ginsenoside biosynthesis were generally higher in the underground tissues, especially in the roots and rhizomes of P. ginseng. But the genes involved in the MEP pathway transcribed at high levels predominantly in the leaves, followed by in the lateral roots, but at low levels in the vascular and xylem tissues (root cores and stems), whose phenomenon consisted with its plastic origin [9]. The gene expression levels in the final steps of ginsenoside biosynthesis, mostly consisting of GT and CYP450 super gene families, were also higher in the living cells (such as in the lateral roots, rhizomes and leaves), but much lower in the vascular and xylem cells (such as in the root cores and stems) of ginseng plants (Fig. 1b).
Further detection and comparison on the gene transcript abundance between the MEP pathway and the MVA pathway (Fig. 3a, b) showed that the gene transcription levels of the two pathways in general were not significantly different in ginseng root samples, indicating that the MEP pathway also contributed to ginsenoside synthesis in the roots of P. ginseng. However, the IspD gene of the MEP pathway transcribed at very low abundance no matter in ginseng roots or in other ginseng tissues (Fig. 3a, c), while the MVK, PMK and MVD genes of the MVA pathway also transcribed at low levels in all the ginseng tissue samples (Fig. 3b, d). These four low-transcribed genes might have essential impact on IPP or IPP-derived metabolite generation, speculating that they might be the potential targets for metabolic manipulation of ginsenoside production using synthetic biology techniques in the future.
The terpene precursors, IPP and DMAPP, either generated by the MVA pathway or the MEP pathway, can be transformed each other by IDI then go into the triterpene skeleton biosynthesis pathway. Based on the gene expression profiling from the whole point of view, the genes of the triterpene skeleton synthesis pathway transcribed constantly among all the tissues and all the ages in our ginseng samples (Fig. 4), showing that they might have little suppression effect on ginsenoside biosynthesis.
Due to the high sensitivity of RNA sequencing technology, we finally examined the gene expression levels in different ginseng tissue samples by random selection of several genes of the MEP pathway, including IspD gene, to carry out quantitative RT-PCR (qPCR) validation experiments. The gene names and the corresponding primers were listed in Additional file 2: Table S2. The gene expression correlation between RNA-seq results and qPCR verification was shown in Fig. 6. The correlation coefficient (R) was as high as above 0.825, explaining the reliability of gene expression results in the RNA-seq data of ginseng samples in this study.
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