Production of salidroside and tyrosol in cell suspension cultures of Rhodiola crenulata

Salidroside and its aglycone tyrosol are important compounds found in Rhodiola plants. In this study, callus derived from Rhodiola crenulata was induced and grown when explants were incubated on a Murashige and Skoog (MS) medium containing various concentrations of 6-benzyaldenine (BA), naphthalene acetic acid (NAA) and thidiazuron (TDZ). Callus was easily initiated from juvenile leaves in half strength MS medium supplemented with 0.5 mg/L BA and 3.0 mg/L NAA, while full strength MS containing 0.5 mg/L TDZ and 0.5 mg/L NAA was the best for callus subculture and subsequent cell suspension culture. The activities of l-phenylalanine ammonia lyase (PAL) and β-d-glucosidase, two key enzymes in salidroside synthesis, increased at first and subsequently decreased in cell suspension cultures. The salidroside and tyrosol levels in the cell suspension cultures were determined using high-performance liquid chromatography. High levels of salidroside and tyrosol were detected in cell suspension cultures of R. crenulata extracted with 75 % methanol, demonstrating that the biotechnological production of these compounds using plant cell suspension cultures derived from R. crenulata may be an attractive alternative to harvest-based production.     

红景天甙生物合成途径：酪醇合成的起始反应及其糖基化

红景天甙(Salidroside)生源途径分子机制的解析是利用基因工程、代谢工程技术合成目标化合物的基础。糖基化是红景天甙生物合成的最后一步反应。在前期工作中,本课题组率先报道了与红景天甙生物合成相关的3个尿苷二磷酸葡萄糖基转移酶(UGTs)基因,在体外酶学性质研究的基础上,利用根癌农杆菌和发根农杆菌介导分别建立了相关转基因体系,鉴别了红景天甙生物合成最适UGT及植物和毛状根生物反应器系统合成红景天甙的效率差异;酪醇(Tyrosol)是红景天甙糖基化反应的甙元底物分子,其具体的代谢通路及其调控机制仍不明确。针对酪醇生物合成来源主要存在两种观点:一是酪醇可能来自于苯丙烷代谢途径产生的4-香豆酸,该途径起源于苯丙氨酸;二是生物碱代谢途径的中间产物酪胺可能是酪醇生物合成的前体,该途径则起源于酪氨酸。在后续工作中,否定了酪醇来源于苯丙烷代谢途径的可能性,进一步的工作证实酪氨酸脱羧酶(TyrDC)在酪醇生物合成的起始反应中担负着重要功能,酪醇作为一种苯乙烷类化合物衍生物,其生物合成来源于生物碱代谢途径。     

Enhanced production of flavonolignans in hairy root cultures of Silybum marianum by over-expression of chalcone synthase gene

In the present study, metabolic engineering approach was used through over-expressing the Petunia chalcone synthase (chsA) gene in order to enhance the silymarin production level in the hairy root cultures of Silybum marianum. Molecular analysis confirmed the presence and integration of chsA transgene in transgenic hairy roots. Chemical analysis indicated that the over-expression of chsA gene enhanced the silymarin production level in the transgenic line as much as 7-folds than the non-transgenic hairy roots. Moreover, the silybin content, the main active component of silymarin, was proved to be 10 times higher in transgenic hairy roots than those of the non-transgenic ones. Therefore, the over-expression of petunia chsA gene in S. marianum hairy roots did not result in gene silencing, but led to an enhanced biosynthesis of the flavonolignans.     

Rewiring central carbon metabolism for tyrosol and salidroside production in Saccharomyces cerevisiae

Metabolic engineering of Saccharomyces cerevisiae for high-level production of aromatic chemicals has received increasing attention in recent years. Tyrosol production from glucose by S. cerevisiae is considered an environmentally sustainable and safe approach. However, the production of tyrosol and salidroside by engineered S. cerevisiae has been reported to be lower than 2 g/L to date. In this study, S. cerevisiae was engineered with a push-pull-restrain strategy to efficiently produce tyrosol and salidroside from glucose. The biosynthetic pathways of ethanol, phenylalanine, and tryptophan were restrained by disrupting PDC1, PHA2, and TRP3. Subsequently, tyrosol biosynthesis was enhanced with a metabolic pull strategy of introducing PcAAS and EcTyrA^M53I/A354V. Moreover, a metabolic push strategy was implemented with the heterologous expression of phosphoketolase (Xfpk), and then erythrose 4-phosphate was synthesized simultaneously by two pathways, the Xfpk-based pathway and the pentose phosphate pathway, in S. cerevisiae. Furthermore, the heterologous expression of Xfpk alone in S. cerevisiae efficiently improved tyrosol production compared with the coexpression of Xfpk and phosphotransacetylase. Finally, the tyrosol yield increased by approximately 135-folds, compared with that of parent strain. The total amount of tyrosol and salidroside with glucose fed-batch fermentation was over 10 g/L and reached levels suitable for large-scale production.     

Molecular characterization of flavonoid biosynthetic genes and accumulation of baicalin,baicalein, and wogonin in plant and hairy root of Scutellaria lateriflora

Scutellaria lateriflora is well known for its medical applications because of the presence of flavanoids and alkaloids. The present study aimed to explore the molecular aspects and regulations of flavanoids. Five partial cDNAs encoding genes that are involved in the flavonoid biosynthetic pathway: phenylalanine ammonia lyase (SlPAL), cinnamate 4-hydroxylase (SlC4H), 4-coumaroyl CoA ligase (Sl4CL), chalcone synthase (SlCHS), and chalcone isomerase (SlCHI) were isolated from S. lateriflora. Organ expression analysis showed that these genes were expressed in all organs analyzed with the highest levels correlating with the richest accumulation of wogonin in the roots. Baicalin and baicalein differentially accumulated in S. lateriflora plants, with the highest concentration of baicalin and baicalein detected in the leaves and stems, respectively. Exogenous methyl jasmonate (MeJA) significantly enhanced the expression of SlCHS and SlCHI, and accumulation of baicalin (22.54 mg/g), baicalein (1.24 mg/g), and wogonin (5.39 mg/g) in S. lateriflora hairy roots. In addition, maximum production of baicalin, baicalein, and wogonin in hairy roots treated with MeJA was approximately 7.44-, 2.38-, and 2.12-fold, respectively. Light condition increased the expression level of SlCHS, the first committed step in flavonoid biosynthesis in hairy roots of S. lateriflora after 3 and 4 weeks of development compared to the dark condition. Dark-grown hairy roots contained a higher content of baicalin and baicalein than light-grown hairy roots, while light-grown hairy roots accumulated more wogonin than dark-grown hairy roots. These results may helpful for the metabolic engineering of flavonoids biosynthesis in S. lateriflora.     

Tryptophan decarboxylase plays an important role in ajmalicine biosynthesis in Rauvolfia verticillata

Tryptophan decarboxylase (TDC) converts tryptophan into tryptamine that is the indole moiety of ajmalicine. The full-length cDNA of Rauvolfia verticillata (RvTDC) was 1,772 bps that contained a 1,500-bp ORF encoding a 499-amino-acid polypeptide. Recombinant 55.5 kDa RvTDC converted tryptophan into tryptamine. The K _m of RvTDC for tryptophan was 2.89 mM, higher than those reported in other TIAs-producing plants. It demonstrated that RvTDC had lower affinity to tryptophan than other plant TDCs. The K _m of RvTDC was also much higher than that of strictosidine synthase and strictosidine glucosidase in Rauvolfia. This suggested that TDC might be the committed-step enzyme involved in ajmalicine biosynthesis in R. verticillata. The expression of RvTDC was slightly upregulated by MeJA; the five MEP pathway genes and SGD showed no positive response to MeJA; and STR was sharply downregulated by MeJA. MeJA-treated hairy roots produced higher level of ajmalicine (0.270 mg g^?1 DW) than the EtOH control (0.183 mg g^?1 DW). Highest RvTDC expression level was detected in hairy root, about respectively 11, 19, 65, and 109-fold higher than in bark, young leaf, old leaf, and root. Highest ajmalicine content was also found in hairy root (0.249 mg g^?1 DW) followed by in bark (0.161 mg g^?1 DW) and young leaf (0.130 mg g^?1 DW), and least in root (0.014 mg g^?1 DW). Generally, the expression level of RvTDC was positively consistent with the accumulation of ajmalicine. Therefore, it could be deduced that TDC might be the key enzyme involved in ajmalicine biosynthesis in Rauvolfia.     

Characterization of UDP-glucose dehydrogenase isoforms in the medicinal legume Glycyrrhiza uralensis

Uridine 5′-diphosphate (UDP)-glucose dehydrogenase (UGD) produces UDP-glucuronic acid from UDP-glucose as a precursor of plant cell wall polysaccharides. UDP-glucuronic acid is also a sugar donor for the glycosylation of various plant specialized metabolites. Nevertheless, the roles of UGDs in plant specialized metabolism remain poorly understood. Glycyrrhiza species (licorice), which are medicinal legumes, biosynthesize triterpenoid saponins, soyasaponins and glycyrrhizin, commonly glucuronosylated at the C-3 position of the triterpenoid scaffold. Often, several different UGD isoforms are present in plants. To gain insight into potential functional differences among UGD isoforms in triterpenoid saponin biosynthesis in relation to cell wall component biosynthesis, we identified and characterized Glycyrrhiza uralensis UGDs (GuUGDs), which were discovered to comprise five isoforms, four of which (GuUGD1–4) showed UGD activity in vitro. GuUGD1–4 had different biochemical properties, including their affinity for UDP-glucose, catalytic constant, and sensitivity to feedback inhibitors. GuUGD2 had the highest catalytic constant and highest gene expression level among the GuUGDs, suggesting that it is the major isoform contributing to the transition from UDP-glucose to UDP-glucuronic acid in planta. To evaluate the contribution of GuUGD isoforms to saponin biosynthesis, we compared the expression patterns of GuUGDs with those of saponin biosynthetic genes in methyl jasmonate (MeJA)-treated cultured stolons. GuUGD1–4 showed delayed responses to MeJA compared to those of saponin biosynthetic genes, suggesting that MeJA-responsive expression of GuUGDs compensates for the decreased UDP-glucuronic acid pool due to consumption during saponin biosynthesis.     

Arbuscular mycorrhizal symbiosis and methyl jasmonate avoid the inhibition of root hydraulic conductivity caused by drought

Hormonal regulation and symbiotic relationships provide benefits for plants to overcome stress conditions. The aim of this study was to elucidate the effects of exogenous methyl jasmonate (MeJA) application on root hydraulic conductivity (L) of Phaseolus vulgaris plants which established arbuscular mycorrhizal (AM) symbiosis under two water regimes (well-watered and drought conditions). The variation in endogenous contents of several hormones (MeJA, JA, abscisic acid (ABA), indol-3-acetic acid (IAA), salicylic acid (SA)) and the changes in aquaporin gene expression, protein abundance and phosphorylation state were analyzed. AM symbiosis decreased L under well-watered conditions, which was partially reverted by the MeJA treatment, apparently by a drop in root IAA contents. Also, AM symbiosis and MeJA prevented inhibition of L under drought conditions, most probably by a reduction in root SA contents. Additionally, the gene expression of two fungal aquaporins was upregulated under drought conditions, independently of the MeJA treatment. Plant aquaporin gene expression could not explain the behaviour of L. Conversely, evidence was found for the control of L by phosphorylation of aquaporins. Hence, MeJA addition modified the response of L to both AM symbiosis and drought, presumably by regulating the root contents of IAA and SA and the phosphorylation state of aquaporins.     

地黄RgMYB10基因的克隆与表达分析

MYB转录因子是植物最大的转录因子家族之一,广泛参与植物的生长发育、逆境胁迫和次生代谢产物积累。该研究通过同源比对和功能注释,在地黄(Rehmannia glutinosa)转录组中筛选出MYB的转录本,设计特异性引物对MYB基因的cDNA序列进行PCR扩增,用水杨酸(SA)、Ag⁺、茉莉酸甲酯(MeJA)和腐胺(Put)这4种诱导子处理地黄毛状根,并通过实时荧光定量PCR(qRT-PCR)检测候选MYB基因的表达。结果显示:(1)成功克隆到1个地黄MYB基因,命名为RgMYB10;该基因编码247个氨基酸残基,蛋白质相对分子质量28.48 kD,等电点为5.14,属于R2R3-MYB转录因子。(2)qRT-PCR结果显示,RgMYB10在须根中表达量最高,其次为茎,块根中的表达量最低。(3)RgMYB10在MeJA处理后的毛状根中显著上调表达,为特异响应MeJA诱导的基因,推测RgMYB10基因可能是响应MeJA参与地黄毛蕊花糖苷生物合成的关键转录因子。研究表明,地黄MYB10基因可能参与地黄毛蕊花糖苷的生物合成,为进一步研究MYB10基因在地黄毛蕊花糖苷合成中的功能奠定了基础。     

Effects of acetylsalicylic acid and UV-B on gene expression and tropane alkaloid biosynthesis in hairy root cultures of Anisodus luridus

Anisodus luridus hairy root cultures were established to test biological effects of acetylsalicylic acid (ASA) and ultraviolet ray-B (UV-B) on gene expression, tropane alkaloid (TA) biosynthesis and efflux. The TAs-pathway gene expression was ASA dosage dependant. The expression of PMT, TRI and CYP80F1 showed no significant difference in hairy root cultures in treatment of 0.01 and 0.1 mM ASA, compared with those without ASA treatment; while 0.01 or 0.1 mM ASA slightly upregulated H6H expression. All the four genes including PMT, TRI, CYP80F1 and H6H had a dramatic increase in 1 mM ASA-treated hairy root cultures compared with control. The expressing levels of all the four genes were much significantly higher in 1 mM ASA-treated hairy root cultures than those in 0.01 and 0.1 mM ASA-treated ones. As expected, hairy root cultures treated with 1 mM ASA had the highest capacity of TAs biosynthesis, in which the content of scopolamine and hyoscyamine reached respectively 57.2 and 14.7 μg g^?1 DW. Surprisingly, it was found that 1 mM ASA dramatically induced the efflux of scopolamine. In the liquid medium with 1 mM ASA, the content of scopolamine was 153.4 μg flask^?1, about 6.2 folds compared with that of control. At the same time, hyoscyamine was detected at trace levels in liquid medium. In the UV-B stressed hairy root cultures, all the four genes had a very strong increase of gene expression that led to more accumulation of scopolamine and lower accumulation of hyoscyamine. Only trace amounts of hyoscyamine and scopolamine were detected in the liquid medium when hairy root cultures were stressed under UV-B, and this suggested that UV-B did not affect TAs efflux.     

Multi-modular engineering of Saccharomyces cerevisiae for high-titre production of tyrosol and salidroside

Tyrosol and its glycosylated product salidroside are important ingredients in pharmaceuticals, nutraceuticals and cosmetics. Despite the ability of Saccharomyces cerevisiae to naturally synthesize tyrosol, high yield from de novo synthesis remains a challenge. Here, we used metabolic engineering strategies to construct S. cerevisiae strains for high-level production of tyrosol and salidroside from glucose. First, tyrosol production was unlocked from feedback inhibition. Then, transketolase and ribose-5-phosphate ketol-isomerase were overexpressed to balance the supply of precursors. Next, chorismate synthase and chorismate mutase were overexpressed to maximize the aromatic amino acid flux towards tyrosol synthesis. Finally, the competing pathway was knocked out to further direct the carbon flux into tyrosol synthesis. Through a combination of these interventions, tyrosol titres reached 702.30 ± 0.41 mg l⁻¹ in shake flasks, which were approximately 26-fold greater than that of the WT strain. RrU8GT33 from Rhodiola rosea was also applied to cells and maximized salidroside production from tyrosol in S. cerevisiae. Salidroside titres of 1575.45 ± 19.35 mg l⁻¹ were accomplished in shake flasks. Furthermore, titres of 9.90 ± 0.06 g l⁻¹ of tyrosol and 26.55 ± 0.43 g l⁻¹ of salidroside were achieved in 5 l bioreactors, both are the highest titres reported to date. The synergistic engineering strategies presented in this study could be further applied to increase the production of high value-added aromatic compounds derived from the aromatic amino acid biosynthesis pathway in S. cerevisiae.     

Isolation and genotype-dependent, organ-specific expression analysis of a Rhodiola rosea cDNA encoding tyrosine decarboxylase

Tyrosine decarboxylase (TyrDC) is an important enzyme in the secondary metabolism of several plant species, and was hypothesized to play a key role in the biosynthesis of salidroside, a pharmacologically valuable compound of roseroot. A 1520 bp cDNA was cloned and sequenced, and turned out to contain an ORF of 963 bp, which encodes a protein of 320 amino acids.The expression of the gene was studied by real-time PCR from leaves and roots of both high and low salidroside producer genotype of roseroot. The gene expression analysis showed the gene to be expressed in leaves as well as in roots; however, the expression was significantly higher in roots, which coincides with the fact that salidroside accumulates preferentially in the underground parts of the plant. The expression was also higher in the line accumulating high levels of salidroside, compared to the line with lower salidroside content. The difference in the expression intensity suggests a decisive role for this enzyme in the salidroside biosynthesis.     

Selection of reliable reference genes for RT-qPCR during methyl jasmonate,salicylic acid and hydrogen peroxide treatments in <Emphasis Type="Italic">Ganoderma lucidum</Emphasis>

This study aimed to identify suitable reference genes under three chemical inducers, methyl jasmonate (MeJA), salicylic acid (SA) and hydrogen peroxide (H₂O₂) in Ganoderma lucidum. In this study, expression stabilities of 14 candidate reference genes had been validated. Four algorithms were used: geNorm, NormFinder, BestKeeper, and RefFinder. Our results showed that, in short time, UCE2 (ubiquitin conjugating enzyme) was the most stable gene both in MeJA and H₂O₂ treatments, ACTIN (beta-actin) was the most suitable reference gene for SA treatment. ACTIN/UCE2 were considered the most suitable genes to normalize in MeJA, SA and H₂O₂ conditions. In long time, PP2A (protein phosphatase 2A regulatory subunit) was the most stable gene in MeJA and SA treatments, UCE2 was the most suitable reference gene for H₂O₂ treatment. PP2A/UBQ1 (polyubiquitin 1) were considered the most suitable genes to normalize in MeJA, SA and H₂O₂ conditions. Furthermore, target gene, oxidosqualene cyclase (osc), was selected to validate the most and least stable reference genes under different treatments. Our work provided a better support to study the regulatory mechanism of MeJA, SA and H₂O₂ on biological functions.