Deok-Chun Yang

Biological Synthesis of Nanoparticles from Plants and Microorganisms

Nanotechnology has become one of the most promising technologies applied in all areas of science. Metal nanoparticles produced by nanotechnology have received global attention due to their extensive applications in the biomedical and physiochemical fields. Recently, synthesizing metal nanoparticles using microorganisms and plants has been extensively studied and has been recognized as a green and efficient way for further exploiting microorganisms as convenient nanofactories. Here, we explore and detail the potential uses of various biological sources for nanoparticle synthesis and the application of those nanoparticles. Furthermore, we highlight recent milestones achieved for the biogenic synthesis of nanoparticles by controlling critical parameters, including the choice of biological source, incubation period, pH, and temperature.

Conversion of Major Ginsenoside Rb1 to Ginsenoside F2 by Caulobacter leidyia

Ginsenoside Rb1 is the most predominant ginsenoside in Panax species (ginseng) and the hydrolysis of this ginsenoside produces pharmaceutically active compounds. Caulobacter leidyia GP45, one of the isolates having strong β-glucosidase-producing activity, converted ginsenoside Rb1 to the active metabolites by 91%. The structures of the resultant metabolites were identified by NMR. Ginsenoside Rb1 had been consecutively converted to ginsenoside Rd (1), F2 (2) and compound K (3) via the hydrolyses of 20-C β-(1→6)-glucoside, 3-C β-(1→2)-glucoside, and 3-C β-glucose of ginsenoside Rb1.

Biosynthesis and biotechnological production of ginsenosides

Medicinal plants are essential for improving human health, and around 75% of the population in developing countries relies mainly on herb-based medicines for health care. As the king of herb plants, ginseng has been used for nearly 5,000 years in the oriental and recently in western medicines. Among the compounds studied in ginseng plants, ginsenosides have been shown to have multiple medical effects such as anti-oxidative, anti-aging, anti-cancer, adaptogenic and other health-improving activities. Ginsenosides belong to a group of triterpene saponins (also called ginseng saponins) that are found almost exclusively in Panax species and accumulated especially in the plant roots. In this review, we update the conserved and diversified pathway/enzyme biosynthesizing ginsenosides which have been presented. Particularly, we highlight recent milestone works on functional characterization of key genes dedicated to the production of ginsenosides, and their application in engineering plants and yeast cells for large-scale production of ginsenosides.

Ginsenoside profiles and related gene expression during foliation in Panax ginseng Meyer

Panax ginseng is one of the most important medicinal plants in Asia. Triterpene saponins, known as ginsenosides, are the major pharmacological compounds in P. ginseng. The present study was conducted to evaluate the changes in ginsenoside composition according to the foliation stage of P. ginseng cultured in a hydroponic system. Among the three tested growth stages (closed, intermediate, and opened), the highest amount of total ginsenoside in the main and fine roots was in the intermediate stage. In the leaves, the highest amount of total ginsenoside was in the opened stage. The total ginsenoside content of the ginseng leaf was markedly increased in the transition from the closed to intermediate stage, and increased more slowly from the intermediate to opened leaf stage, suggesting active biosynthesis of ginsenosides in the leaf. Conversely, the total ginsenoside content of the main and fine roots decreased from the intermediate to opened leaf stage. This suggests movement of ginsenosides during foliation from the root to the leaf, or vice versa. The difference in the composition of ginsenosides between the leaf and root in each stage of foliation suggests that the ginsenoside profile is affected by foliation stage, and this profile differs in each organ of the plant. These results suggest that protopanaxadiol- and protopanaxatriol (PPT)-type ginsenosides are produced according to growth stage to meet different needs in the growth and defense of ginseng. The higher content of PPT-type ginsenosides in leaves could be related to the positive correlation between light and PPT-type ginsenosides.

Comparative analysis of expressed sequence tags (ESTs) of ginseng leaf.

The expressed sequence tags (ESTs) referenced in this report are the first transcriptomes in a leaf from a half-shade ginseng plant. A cDNA library was constructed from samples of the leaves of 4-year-old Panax ginseng plants, which were cultured in a field. The 2,896 P. ginseng cDNA clones represent 1,576 unique sequences, consisting of 1,167 singletons and 409 contig sequences. BLAST comparisons of the cDNAs in GenBanks non-redundant databases revealed that 2,579 of the 2,896 cDNAs (89.1%) exhibited a high degree of sequence homology to genes from other organisms. The majority of the identified transcripts were found to be genes related with energy, metabolism, subcellular localization, and protein synthesis and transport. The chlorophyll a/b-binding protein ESTs in the ginseng leaf samples manifested a substantially higher level of expression than was observed in other plant leaves. The ESTs involved in ginsenoside biosynthesis were also identified and discussed.

Functional Analysis of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Encoding Genes in Triterpene Saponin-Producing Ginseng

Coenzyme A reductases contribute to the production of triterpene saponin in ginseng. Ginsenosides are glycosylated triterpenes that are considered to be important pharmaceutically active components of the ginseng (Panax ginseng ‘Meyer’) plant, which is known as an adaptogenic herb. However, the regulatory mechanism underlying the biosynthesis of triterpene saponin through the mevalonate pathway in ginseng remains unclear. In this study, we characterized the role of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) concerning ginsenoside biosynthesis. Through analysis of full-length complementary DNA, two forms of ginseng HMGR (PgHMGR1 and PgHMGR2) were identified as showing high sequence identity. The steady-state mRNA expression patterns of PgHMGR1 and PgHMGR2 are relatively low in seed, leaf, stem, and flower, but stronger in the petiole of seedling and root. The transcripts of PgHMGR1 were relatively constant in 3- and 6-year-old ginseng roots. However, PgHMGR2 was increased five times in the 6-year-old ginseng roots compared with the 3-year-old ginseng roots, which indicates that HMGRs have constant and specific roles in the accumulation of ginsenosides in roots. Competitive inhibition of HMGR by mevinolin caused a significant reduction of total ginsenoside in ginseng adventitious roots. Moreover, continuous dark exposure for 2 to 3 d increased the total ginsenosides content in 3-year-old ginseng after the dark-induced activity of PgHMGR1. These results suggest that PgHMGR1 is associated with the dark-dependent promotion of ginsenoside biosynthesis. We also observed that the PgHMGR1 can complement Arabidopsis (Arabidopsis thaliana) hmgr1-1 and that the overexpression of PgHMGR1 enhanced the production of sterols and triterpenes in Arabidopsis and ginseng. Overall, this finding suggests that ginseng HMGRs play a regulatory role in triterpene ginsenoside biosynthesis.

Rapid propagation of Eleutherococcus senticosus via direct somatic embryogenesis from explants of seedlings

Explants from three different parts (cotyledon, hypocotyl or root) of one week-old seedlings of Eleutherococcus senticosus were cultured on Murashige and Skoog (MS) medium with 1.0 mg l-1 2,4-D. Somatic embryos were formed directly from the surfaces of explants. The frequency of direct somatic embryo formation was the highest in the hypocotyl segments (75%) as compared to cotyledon (56%) or root segments (12%). When hypocotyl explants from 3 different stages of seedlings (zero, one or three week-old) were cultured on MS medium with 1.0 mg l-1 2,4-D, the frequency of somatic embryo formation rapidly declined as the zygotic embryos germinated. However most somatic embryos (93%) from explants of zygotic embryos developed as fused state (multiple embryo), whereas somatic embryos (over 89%) from more developed seedlings developed into single state (single embryo). Single embryos germinated and regenerated into plantlets with both shoots and roots, while multiple embryos only regenerated into only multiple shoots. Plantlets that regenerated from single embryos of E. senticosus were acclimatized in a greenhouse.

Spermidine alleviates the growth of saline-stressed ginseng seedlings through antioxidative defense system

Protective effects of exogenous spermidine (Spd), activity of antioxygenic enzymes, and levels of free radicals in a well-known medicinal plant, Panax ginseng was examined. Seedlings grown in salinized nutrient solution (150 mM NaCl) for 7d exhibited reduced relative water content, plant growth, increased free radicals, and showing elevated lipid peroxidation. Application of Spd (0.01, 0.1, and 1mM) to the salinized nutrient solution showed increased plant growth by preventing chlorophyll degradation and increasing PA levels, as well as antioxidant enzymes such as CAT, APX, and GPX activity in the seedlings of ginseng. During salinity stress, Spd was effective for lowering the accumulation of putrescine (Put), with a significant increase in the spermidine (Spd) and spermine (Spm) levels in the ginseng seedlings. A decline in the Put level ran parallel to the higher accumulation of proline (Pro), and exogenous Spd also resulted in the alleviation of Pro content under salinity. Hydrogen peroxide (H2O2) and superoxide (O2(-)) production rates were also reduced in stressed plants after Spd treatment. Furthermore, the combined effect of Spd and salt led to a significant increase in diamine oxidase (DAO), and subsequent decline in polyamine oxidase (PAO). These positive effects were observed in 0.1 and 1mM Spd concentrations, but a lower concentration (0.01 mM) had a very limited effect. In summary, application of exogenous Spd could enhance salt tolerance of P. ginseng by enhancing the activities of enzyme scavenging system, which influence the intensity of oxidative stress.

Expression and stress tolerance of PR10 genes from Panax ginseng C. A. Meyer

Pathogenesis-related 10 protein families (PgPR10 proteins) from ginseng are reported to have ribonuclease activity, conferring defense-related resistance against various stresses. Homology-based PCR using PgPR10-2 specific primers allowed for the isolation of two additional PgPR10 genes. PgPR10-1 is identical to the previously reported ribonuclease 1, while PgPR10-3 is a newly-discovered protein, suggesting that the PgPR10s are a multi-gene family. Differential organ-specific transcripts of PgPR10-1 and PgPR10-2 in the flower bud and root, respectively, indicate that there are tissue-specific functional roles for this gene family. Overexpression of PgPR10-2 in Arabidopsis conferred longer root length and a tolerant growth phenotype on NaCl-supplemented media. Further changes in transcriptional levels against sets of abiotic stressors suggest similar functional roles of PgPR10-1 in the root and predominantly in the flower organ based on its higher expression levels. Overall, this suggests that the manipulation of PgPR10 genes in plants can be used as valuable tool to enhance its physiological status.

Enzymatic biotransformation of ginsenoside Rb1 to compound K by recombinant β-glucosidase from Microbacterium esteraromaticum.

We cloned and characterized a β-glucosidase (bgp3) gene from Microbacterium esteraromaticum isolated from ginseng field. The bgp3 gene consists of 2,271 bp encoding 756 amino acids which have homology to the glycosyl hydrolase family 3 protein domain. The molecular mass of purified Bgp3 was 80 kDa, as determined by SDS-PAGE. The enzyme (Bgp3) catalyzed the conversion of ginsenoside Rb1 to the more pharmacologically active minor ginsenoside Rd and compound K. The Bgp3 hydrolyzed the outer glucose moiety attached to the C-20 position of ginsenoside Rb1, followed by hydrolysis of the inner glucose moiety attached to the C-3 position. Using 0.1 mg mL(-1) enzyme in 20 mM sodium phosphate buffer at 40 °C and pH 7.0, 1.0 mg mL(-1) ginsenoside Rb1 was transformed into 0.46 mg mL(-1) compound K within 60 min with a corresponding molar conversion yield of 77%. Bgp3 hydrolyzed the ginsenoside Rb1 along the following pathway: Rb1 → Rd → compound K.