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。     

高山红景天细胞悬浮培养中红景天甙生物合成代谢的调控 Ⅰ:前体的影响

探索了高山红景天(Rhodiola sachalinensis A.Bor)细胞培养中红景天甙生物合成的途径,认为甙元酪醇是经由莽草酸途径生成的。在此基础上研究了酪醇、L-酪氨酸与L-苯丙氨酸三种前体加入对红景天甙生物合成的调控作用。结果表明,酪醇、酪氨酸等前体易被多酚氧化酶氧化成褐色,用与前体浓度为1:1的V。来防止褐化效果显著;浓度为0.5mmol/L的酪醇,酪氨酸及苯丙氨酸在细胞培养15d时添加,使红景天甙含量由0.336％分别提高到1.43％、1.11％、0.85％。     

Engineering Salidroside Biosynthetic Pathway in Hairy Root Cultures of Rhodiola crenulata Based on Metabolic Characterization of Tyrosine Decarboxylase

Tyrosine decarboxylase initializes salidroside biosynthesis. Metabolic characterization of tyrosine decarboxylase gene from Rhodiola crenulata (RcTYDC) revealed that it played an important role in salidroside biosynthesis. Recombinant 53 kDa RcTYDC converted tyrosine into tyramine. RcTYDC gene expression was induced coordinately with the expression of RcUDPGT (the last gene involved in salidroside biosynthesis) in SA/MeJA treatment; the expression of RcTYDC and RcUDPGT was dramatically upregulated by SA, respectively 49 folds and 36 folds compared with control. MeJA also significantly increased the expression of RcTYDC and RcUDPGT in hairy root cultures. The tissue profile of RcTYDC and RcUDPGT was highly similar: highest expression levels found in stems, higher expression levels in leaves than in flowers and roots. The gene expressing levels were consistent with the salidroside accumulation levels. This strongly suggested that RcTYDC played an important role in salidroside biosynthesis in R. crenulata. Finally, RcTYDC was used to engineering salidroside biosynthetic pathway in R. crenulata hairy roots via metabolic engineering strategy of overexpression. All the transgenic lines showed much higher expression levels of RcTYDC than non-transgenic one. The transgenic lines produced tyramine, tyrosol and salidroside at higher levels, which were respectively 3.21–6.84, 1.50–2.19 and 1.27–3.47 folds compared with the corresponding compound in non-transgenic lines. In conclusion, RcTYDC overexpression promoted tyramine biosynthesis that facilitated more metabolic flux flowing toward the downstream pathway and as a result, the intermediate tyrosol was accumulated more that led to the increased production of the end-product salidroside.     

Maize phenylalanine ammonia-lyase has tyrosine ammonia-lyase activity.

A full-length cDNA encoding phenylalanine ammonia-lyase (PAL) from Zea mays L. was isolated and the coding region was expressed in Escherichia coli as a C-terminal fusion to glutathione S-transferase. After purification by glutathione-Sepharose chromatography, the glutathione S-transferase moiety was cleaved off and the resulting PAL enzyme analyzed. In contrast to PAL from dicots, this maize PAL isozyme catalyzed the deamination of both L-phenylalanine (PAL activity) and L-tyrosine (tyrosine ammonia-lyase activity). These results provide unequivocal proof that PAL and tyrosine ammonia-lyase activities reside in the same polypeptide. In spite of large differences in the Michaelis constant and turnover number of the two activities, their catalytic efficiencies are very similar. Also, both activities have the same pH and temperature optima. These results imply that maize can produce p-coumaric acid from both phenylalanine and tyrosine.     

Production of Salidroside Through Biotransformation of Exogenous Tyrosol in Rhodiola sachalinensis Cell Suspension Cultures

In Rhodiola sachalinensis A. Bot. cell cultures, low yields of salidroside was supposed to be associated with the low efficiency of glucosylation reaction at the stationary phase of cell growth, when large amounts of the substrate, aglycon tyrosol, were accumulated. Considering the activity of tyrosol glucosyhransferase being the highest at the exponential growth phase, the author added exogenous tyrosol into the cultures at this time so as to produce salidroside through biotransformation. The effects of tyrosol concentration, the way of tyrosol addition as well as the cell density on the transformation rate and salidroside yield were investigated. It was found that the transformation rate attained 95 % after cells were incubated in the medium containing 1 mmol/L tyrosol for 24 h. Excess high concentrations of tyrosol in medium ( > 3 mmol/L) caused inhibition of transformation rate and cell growth. By 3 repeated additions of tyrosol in low concentrations, the salidroside yields of 1 320 mg/L, 1 740 mg/L and 1 980 mg/L to the cell densities of 6 g DW/L, 12 g DW/L and 18 g DW/L were obtained respectively.     

不同来源高山红景天材料中有效成分的HPLC分析

本文用HPLC测定、比较了5种不同来源的高山红景天材料中有效成分—红景天甙及其甙无酪醇的含量,并建立了高山红景天有效成分分析的样品制备方法,结果表明:固体培养、液体培养及反应器培养的高山红景天组织中均含有上述两种有效成分.其中固体培养的材料中有效成分的含量最高.三种提取方法比较表明,用索氏提取器连续回流提取明显优于冷浸提取和批式回流提取.     

利用高山红景天培养细胞生物转化外源酪醇生产红景天甙的研究

高山红景天（ＲｈｏｄｉｏｌａｓａｃｈａｌｉｎｅｎｓｉｓＡ．Ｂｏｒ．）培养细胞中,甙元酪醇在细胞生长静止期大量积累,而此时糖基化反应的效率很低,因而红景天甙（ｓａｌｉｄｒｏｓｉｄｅ）产量较低。考虑到培养细胞中酪醇葡萄糖基转移酶的活性在指数生长期达到最高,考察了在指数生长期添加外源酪醇生物转化生产红景天甙的可能性,并探讨了酪醇添加浓度、添加方法及细胞密度对酪醇转化率及红景天甙产量的影响。结果表明,细胞在酪醇浓度为１ｍｍｏｌ／Ｌ的培养基中培养２４ｈ后可使酪醇转化率达到９５％;过高的酪醇浓度（＞３ｍｍｏｌ／Ｌ）对细胞生长及酪醇转化率都有明显抑制作用;通过较低浓度酪醇的３次重复添加,可使细胞密度为６ｇＤＷ／Ｌ、１２ｇＤＷ／Ｌ及１８ｇＤＷ／Ｌ的培养物中的红景天甙产量分别达到１３２０ｍｇ／Ｌ、１７４０ｍｇ／Ｌ和１９８０ｍｇ／Ｌ。     

High yield production of salidroside in the suspension culture of Rhodiola sachalinensis

Salidroside has been identified as the most potent ingredient of the Chinese medicine herb, Rhodiola sachalinensis. Since the natural supply of this herb is rapidly decreasing, we established a compact callus aggregate (CCA) strain and culturing system for high yield salidroside production. Several callus strains induced from the explants originated from root, stem, leaf and cotyledon of R. sachalinensis were established and screened for rapid growth rate, high salidroside content and easy propagation in suspension culture condition. The CCA strain was established from a callus strain initiated from the cotyledon. The kinetics of dry weight accumulation and cellular salidroside content in various culture conditions for the strain was determined. For high salidroside production, the optimal inoculum amount was 10% and the optimal concentration for 6-benzylaminopurine and indole-3-butyric acid added in the liquid medium was 5 and 2.5 mg l-1, respectively. The acidic culture medium and a faster shaking speed favored the salidroside accumulation. The addition of 2,4-D, in the liquid MS medium and the utilization of L-tyrosol for chemical feeding enhanced salidroside production. Using a proper combination of culture condition and treatment, salidroside accumulation could reach 57.72 mg g-1 dry weight, that was 5-10-fold higher than that detected in field-grown plants. The corresponding salidroside yield was 555.13 mg l-1, a level suitable for cost effective commercial production to compensate the natural resource shortage of R. sachalinensis.     

Maize <Emphasis Type="Italic">Lc</Emphasis> transcription factor enhances biosynthesis of anthocyanins,distinct proanthocyanidins and phenylpropanoids in apple (<Emphasis Type="Italic">Malus domestica</Emphasis> Borkh.)

Flavonoids are a large family of polyphenolic compounds with manifold functions in plants. Present in a wide range of vegetables and fruits, flavonoids form an integral part of the human diet and confer multiple health benefits. Here, we report on metabolic engineering of the flavonoid biosynthetic pathways in apple (Malus domestica Borkh.) by overexpression of the maize (Zea mays L.) leaf colour (Lc) regulatory gene. The Lc gene was transferred into the M. domestica cultivar Holsteiner Cox via Agrobacterium tumefaciens-mediated transformation which resulted in enhanced anthocyanin accumulation in regenerated shoots. Five independent Lc lines were investigated for integration of Lc into the plant genome by Southern blot and PCR analyses. The Lc-transgenic lines contained one or two Lc gene copies and showed increased mRNA levels for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), flavanone 3 beta-hydroxylase (FHT), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin reductases (LAR), anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR). HPLC-DAD and LC-MS analyses revealed higher levels of the anthocyanin idaein (12-fold), the flavan 3-ol epicatechin (14-fold), and especially the isomeric catechin (41-fold), and some distinct dimeric proanthocyanidins (7 to 134-fold) in leaf tissues of Lc-transgenic lines. The levels of phenylpropanoids and their derivatives were only slightly increased. Thus, Lc overexpression in Malus domestica resulted in enhanced biosynthesis of specific flavonoid classes, which play important roles in both phytopathology and human health.     

Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae

Flavonoids are valuable natural products derived from the phenylpropanoid pathway. The objective of this study was to create a host for the biosynthesis of naringenin, the central precursor of many flavonoids. This was accomplished by introducing the phenylpropanoid pathway with the genes for phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides, 4-coumarate:coenzyme A (CoA) ligase (4CL) from Arabidopsis thaliana, and chalcone synthase (CHS) from Hypericum androsaemum into two Saccharomyces cerevisiae strains, namely, AH22 and a pad1 knockout mutant. Each gene was cloned and inserted into an expression vector under the control of a separate individual GAL10 promoter. Besides its PAL activity, the recombinant PAL enzyme showed tyrosine ammonia lyase activity, which enabled the biosynthesis of naringenin without introducing cinnamate 4-hydroxylase (C4H). 4CL catalyzed the conversion of both trans-cinnamic acid and p-coumaric acid to their corresponding CoA products, which were further converted to pinocembrin chalcone and naringenin chalcone by CHS. These chalcones were cyclized to pinocembrin and naringenin. The yeast AH22 strain coexpressing PAL, 4CL, and CHS produced approximately 7 mg liter(-1) of naringenin and 0.8 mg liter(-1) of pinocembrin. Several by-products, such as 2',4',6'-trihydroxydihydrochalcone and phloretin, were also identified. Precursor feeding studies indicated that metabolic flux to the engineered flavonoid pathway was limited by the flux to the precursor l-tyrosine.     

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.     

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.     

Sequence and structure of a phenylalanine ammonia-lyase gene from Glycine max.

The gene encoding a key enzyme in anthocyanin biosynthesis, phenylalanine ammonia-lyase (PAL), was cloned from soybean (Glycine max). The purpose was to obtain a molecular probe to study the organization of this gene family in soybean and to examine novel regulatory mechanisms present in the anthocyanin biosynthetic pathway of this system. A soybean genomic library was constructed in the bacteriophage vector lambda Charon 35. A PAL cDNA clone from Phaseolus vulgaris was used in screening the library, and two PAL genes were isolated. One gene was sequenced entirely and analyzed by sequence homology to the PAL2 gene of Phaseolus vulgaris. Genomic analysis indicates that PAL sequences of Glycine max exist as a small gene family consisting of only two to three members. The representative PAL gene sequenced (PAL1) has a coding region of 2142 basepairs divided among two exons. The single intron is 1519 basepairs and splits the 131st codon.     

Overexpression of the wheat salt tolerance-related gene TaSC enhances salt tolerance in Arabidopsis

A novel gene named TaSC was cloned from salt-tolerant wheat. Northern blot showed that the expression of TaSC in salt-tolerant wheat was up-regulated after salt stress. Real-time quantitative PCR analyses showed that TaSC expression was induced by salt and ABA in wheat. Localization analysis showed that TaSC proteins were localized to the plasma membrane in transgenic Arabidopsis thaliana. The overexpression of TaSC in Col-0 and atsc (SALK_072220) Arabidopsis strains resulted in increased salt tolerance of the transgenic plants. TaSC overexpression in Col-0 and atsc signi?cantly up-regulated the expression of AtFRY1, AtSAD1, and AtCDPK2. AtCDPK2 overexpression in atsc rescued the salt-sensitive phenotype of atsc. The TaSC gene may improve plant salt tolerance by acting via the CDPK pathway.