高效液相色谱法测定红景天根中红景天甙的含量

建立了一种高效液相色谱法测定红景天根中红景天甙的含量。色谱柱为Hypersil—C18（5μm,150mm×4．6mm）,甲醇-水为流动相,梯度洗脱,柱温25℃,在250nm波长处检测。研究结果表明：红景天甙的检测限为50μg/L,线性范围为4—40mg／L,回归方程为Y=5．6348X-4．0931,r=0．9996,加样回收率为98．05％。方法操作简便、快速、准确。     

原花青素的生物合成途径、功能基因和代谢工程

原花青素（PA）广泛分布于高等植物中,与农作物的多种品质性状密切相关。虽长期受到关注,但其生物合成途径和主要功能基因的解析则是近年来随着拟南芥等植物突变体研究的深入才取得突破的。PA经公共苯丙烷。核心类黄酮一原花青素复合途径而合成,先后涉及12个关键酶（PAL、C4H、4CL、CHS、CHI、F3H、F3’H、DFR、LDOX／ANS、LAR、ANR、LAC）的催化反应和3种转运蛋白（GST、MATE、ATPase）的胞内转运,并有6种转录因子（WhD-ZF、MYB、bHLH、WD40、WRKY、MADS）参与调控PA的合成与积累。这些基因在拷贝数、表达特征、蛋白亚细胞定位、蛋白互作、突变体表型等方面具有显著特点。PA的代谢工程在牧草品质改良、农产品脱涩、油菜黄籽材料创新、葡萄和葡萄酒品质改良、茶多酚分子育种、作物抗病虫性提高、新型作物拓展等方向具有重要的应用前景,目前仅在少数方向有所启动,更待广泛关注和深入研究。     

Discovery and Characterization of BlsE,a Radical S-Adenosyl-L-methionine Decarboxylase Involved in the Blasticidin S Biosynthetic Pathway

BlsE, a predicted radical S-adenosyl-L-methionine (SAM) protein, was anaerobically purified and reconstituted in vitro to study its function in the blasticidin S biosynthetic pathway. The putative role of BlsE was elucidated based on bioinformatics analysis, genetic inactivation and biochemical characterization. Biochemical results showed that BlsE is a SAM-dependent radical enzyme that utilizes cytosylglucuronic acid, the accumulated intermediate metabolite in blsE mutant, as substrate and catalyzes decarboxylation at the C5 position of the glucoside residue to yield cytosylarabinopyranose. Additionally, we report the purification and reconstitution of BlsE, characterization of its [4Fe–4S] cluster using UV-vis and electron paramagnetic resonance (EPR) spectroscopic analysis, and investigation of the ability of flavodoxin (Fld), flavodoxin reductase (Fpr) and NADPH to reduce the [4Fe–4S]²⁺ cluster. Mutagenesis studies demonstrated that Cys₃₁, Cys_35, Cys₃₈ in the C×××C×MC motif and Gly₇₃, Gly₇₄, Glu₇₅, Pro₇₆ in the GGEP motif were crucial amino acids for BlsE activity while mutation of Met₃₇ had little effect on its function. Our results indicate that BlsE represents a typical [4Fe–4S]-containing radical SAM enzyme and it catalyzes decarboxylation in blasticidin S biosynthesis.     

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.     

植物内源茉莉酸类物质的生物合成途径及其生物学意义

茉莉酸类物质（JAs）是新确认的一类广泛存在于植物体内的内源激素,在植物的生长发育、应激反应和次生代谢过程中起着重要的调控作用。该文主要概述了植物中茉莉酸类物质的生物合成途径、各关键酶的生理作用及其在植物次生代谢工程等方面的研究进展,并探讨了茉莉酸类物质的潜在应用价值。     

植物内源茉莉酸类物质的生物合成途径及其生物学意义

茉莉酸类物质(JAs)是新确认的一类广泛存在于植物体内的内源激素, 在植物的生长发育、应激反应和次生代谢过程中起着重要的调控作用。该文主要概述了植物中茉莉酸类物质的生物合成途径、各关键酶的生理作用及其在植物次生代谢工程等方面的研究进展, 并探讨了茉莉酸类物质的潜在应用价值。     

Metabolic Engineering of the Carotenoid Biosynthetic Pathway in the Yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a β-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the β-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via β-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3′-4′-didehydro-β-ψ-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.     

不同理化因子对雪莲毛状根生长和总黄酮生物合成的影响

在 1 2MS液体培养基上研究了不同理化因子对水母雪莲毛状根生长和总黄酮生物合成的影响。实验结果表明 :氮源总浓度 (包括NH+4和NO-3)为 30mmol L ;NH⁺₄ NO^-₃比例为 5∶2 5 ;2 %蔗糖和 3%葡萄糖组合 ;0.5mg LGA₃和 0.5mg LIBA ;pH5.8;18h d的光照 (光强为 35.0.0lx) ;2.4℃ ;摇床转速为 100rmin有利于毛状根生长及总黄酮的生物合成。在此培养条件下 ,经过21d的培养毛状根生长量达到 12.8g L(DW) ,总黄酮合成量为 192.2mg L ,即总黄酮含量占毛状根干重的 15 % ,约为干重野生水母雪莲植株总黄酮含量的 2.5倍。     

Polyamine Biosynthetic Enzymes in Chlorella: Characterization of Ornithine and Arginine Decarboxylase

In a Chlorella culture grown asynchronously under autotrophicconditions, two biosynthetic enzymes of putrescine—ornithinedecarboxylase (ODC) and arginine decarboxylase (ADC)—weredetected. Both enzymes require pyridoxal phosphate and dithiothreitolfor their activity but differ in their optimal pH, the ionicstrength of their buffer, temperature of inactivation, and Km.In addition, L-canaline was found to inhibit the activity ofODC but not that of ADC. During the logarithmic phase of growth,ODC activity increased sharply, then decreased before the onsetof the stationary phase. ADC activity changed only slightlyduring growth. ³The work was performed in partial fulfillment of the requirementsfor the Ph.D. Thesis of E.C. (Received September 25, 1982; Accepted June 1, 1983)     

Metabolic Engineering of Corynebacterium glutamicum for Trehalose Overproduction: Role of the TreYZ Trehalose Biosynthetic Pathway

Trehalose has many potential applications in biotechnology and the food industry due to its protective effect against environmental stress. Our work explores microbiological production methods based on the capacity of Corynebacterium glutamicum to excrete trehalose. We address here raising trehalose productivity through homologous overexpression of maltooligosyltrehalose synthase and the maltooligosyltrehalose trehalohydrolase genes. In addition, heterologous expression of the UDP-glucose pyrophosphorylase gene from Escherichia coli improved the supply of glycogen. Gene expression effects were tested on enzymatic activities and intracellular glycogen content, as well as on accumulated and excreted trehalose. Overexpression of the treY gene and the treY/treZ synthetic operon significantly increased maltooligosyltrehalose synthase activity, the rate-limiting step, and improved the specific productivity and the final titer of trehalose. Furthermore, a strong decrease was noted in glycogen accumulation. Expression of galU/treY and galU/treYZ synthetic operons showed a partial recovery in the intracellular glycogen levels and a significant improvement in both intra- and extracellular trehalose content.     

Characterization of the CDP-2-Glycerol Biosynthetic Pathway in Streptococcus pneumoniae

Capsule polysaccharide (CPS) plays an important role in the virulence of Streptococcus pneumoniae and is usually used as the pneumococcal vaccine target. Glycerol-2-phosphate is found in the CPS of S. pneumoniae types 15A and 23F and is rarely found in the polysaccharides of other bacteria. The biosynthetic pathway of the nucleotide-activated form of glycerol-2-phosphate (NDP-2-glycerol) has never been identified. In this study, three genes (gtp1, gtp2, and gtp3) from S. pneumoniae 23F that have been proposed to be involved in the synthesis of NDP-2-glycerol were cloned and the enzyme products were expressed, purified, and assayed for their respective activities. Capillary electrophoresis was used to detect novel products from the enzyme-substrate reactions, and the structure of the product was elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Gtp1 was identified as a reductase that catalyzes the conversion of 1,3-dihydroxyacetone to glycerol, Gtp3 was identified as a glycerol-2-phosphotransferase that catalyzes the conversion of glycerol to glycerol-2-phosphate, and Gtp2 was identified as a cytidylyltransferase that transfers CTP to glycerol-2-phosphate to form CDP-2-glycerol as the final product. The kinetic parameters of Gtp1 and Gtp2 were characterized in depth, and the effects of temperature, pH, and cations on these two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-2-glycerol has been identified biochemically; this pathway provides a method to enzymatically synthesize this compound.Capsule polysaccharide (CPS) of Gram-positive bacteria, external to the cell wall, provides resistance to phagocytosis. CPS in Streptococcus pneumoniae is the most important virulence factor and the target of pneumococcal vaccines (2). Ninety individual CPS serotypes have been recognized so far by immunological and chemical techniques (9). Each has a structurally distinct CPS, composed of repeating oligosaccharide units joined by glycosidic linkages. The components of the repeat units are transferred from nucleoside diphosphate (NDP) derivatives. Among the 54 identified CPS structures, several sugars and related compounds have been found. Seven NDP-monosaccharide precursors (glucopyranose, N-acetylglucosamine, galactopyranose, N-acetylgalactosamine, 2-acetamido-4-amino-2,4,6-trideoxy-d-galactopyranose, ribitol-phosphate, and phosphorylcholine) are available from housekeeping metabolic pathways, and the biosynthetic genes for 14 NDP-monosaccharide precursors were found in the pneumococcal cps loci. Among the 14 components, the pathways of five (NDP-d-mannitol, NDP-d-arabinitol, NDP-ribofuranose [Rib], CDP-glycerol [CDP-Gro], and NDP-2-glycerol) are putative and have not yet been identified (1, 4).Glycerol-2-phosphate is rarely present in bacteria and has been found in S. pneumoniae types 15A and 23F. The NDP-2-glycerol biosynthetic pathway has been proposed to include three enzymes: Gtp1, Gtp2, and Gtp3. Gtp3 has been proposed to be a glyceraldehyde-2-phosphotransferase and to be involved in the synthesis of glyceraldehyde-2-phosphate from glyceraldehyde. Gtp1, a putative dehydrogenase, has been proposed to be responsible for the conversion of glyceraldehyde-2-phosphate to glycerol-2-phosphate. The last step of the synthesis of CDP-2-glycerol is catalyzed by the putative glycerol-2-phosphate cytidyltransferase Gtp2 (14). The three genes, gtp1, gtp2, and gtp3, have also been found to be present in the cps loci of S. pneumoniae serotypes 15B, 15C, 15F, 23A, 23B, 28A, and 28F (4). However, the biosynthetic pathway for NDP-2-glycerol has never been identified by molecular and biochemical methods.In this study, we found that the enzymes were not reactive by the previously proposed CDP-2-glycerol biosynthetic pathway. Therefore, a new pathway was proposed, and the three enzymes, Gtp1, Gtp2, and Gtp3, were identified and confirmed biochemically as 1,3-dihydroxyacetone/glyceraldehyde reductase, glycerol-2-phosphate cytidylyltransferase, and glycerol-2-phosphotransferase, respectively. This is the first report on the characterization of the CDP-2-glycerol biosynthetic pathway.     

Transformation of Gynostermma pentaphyllum by Agrobacterium rhizogenes and Saponin Production in Hairy Root Cultures

Authors report here the establishment of an efficient transformation system for Gynosternrna pentaphyllum (Thunb.) Makino using Agrobacteriurn rhizogenes R1600. Hairy roots appeared on leaf explants 10 days after inoculation with the bacteria . Frequency of the explants transformed by R1600 was up to 94%. Transformation was confirmed by Southern analysis. Biomass of hairy root cultures suspended in hormone-free MS medium increased 9 times after 20 days of incubation. There was no callus formation on the hairy roots during suspension culture. Saponin content in the hairy root cultures was about 2 times as much as in the natural roots, saponins of the hairy root cultures were also released into growth medium as well.     

Functional Characterization of Proanthocyanidin Pathway Enzymes from Tea and Their Application for Metabolic Engineering

A reevaluation of flux data for Arabidopsis mutants reveals that nitrate uptake through AtNRT1.1 conforms to a single low-affinity transport system that makes virtually no contribution to high-affinity nitrate uptake.In papers by Wang et al. (1998), Liu et al. (1999), and Liu and Tsay (2003), it was proposed that Arabidopsis thaliana Nitrate Transporter1.1 (AtNRT1.1; CHL1) encodes a dual-affinity nitrate transporter that “plays a major role in high-affinity nitrate uptake.” Here, we evaluate this concept by reexamining the uptake kinetics of Arabidopsis (Arabidopsis thaliana) mutant lines defective in NRT1.1 or other nitrate transporters.The uptake of inorganic ions by plant roots conforms to a pattern of biphasic kinetics. At low external ion concentration, ions are absorbed by saturable high-affinity transport systems (HATS), while at high concentrations, nonsaturating low-affinity transport systems (LATS) operate. Such is the case for K⁺, NH₄⁺, NO₃⁻, and ClO₃⁻ (a NO₃⁻ analog; Kochian and Lucas, 1982; Ullrich et al., 1984; Pace and McClure, 1986; Guy et al., 1988; Siddiqi et al., 1990; Aslam et al., 1992). The LATS for ³⁶ClO₃⁻ uptake was linear at [ClO₃⁻] down to 200 μm in tobacco (Nicotiana tabacum; Guy et al., 1988) and for nitrate uptake by barley (Hordeum vulgare) down to 100 μm NO₃⁻ (Aslam et al., 1992). These concentrations were the lowest examined by the latter authors. In the studies by Pace and McClure (1986), Guy et al. (1988), Siddiqi et al. (1990), and Aslam et al. (1992), LATS fluxes were extremely small at low external [NO₃⁻] and linear at both low and high [NO₃⁻].In barley, both constitutive HATS (CHATS) and inducible HATS (IHATS) were demonstrated at low [NO₃⁻], while a constitutive LATS (CLATS) failed to saturate even at 50 mm NO₃⁻ (Siddiqi et al., 1990). Likewise, CHATS and IHATS for nitrate have been demonstrated in Arabidopsis, as well as CLATS and inducible LATS (ILATS; Tsay et al., 1993; Huang et al., 1999).Doddema and Telkamp (1979) isolated an Arabidopsis B1 mutant that was defective in the LATS for nitrate (but not the HATS) by screening for survival on ClO₃⁻. Tsay et al. (1993) isolated the nitrate-inducible AtNRT1.1 gene that encodes the ILATS. Interestingly, Touraine and Glass (1997) were unable to detect reduced LATS or HATS influxes in AtNRT1.1 mutants grown on KNO₃, while Muños et al. (2004) reported increased HATS influx in AtNRT1.1 mutants. Likewise, Remans et al. (2006) failed to detect reduced uptake rates at low (0.5 mm) or high (10 mm) nitrate in AtNRT1.1 mutants.Among eukaryotes, genes encoding IHATS for nitrate were first isolated from Aspergillus nidulans (Unkles et al., 1991) and subsequently from Chlamydomonas reinhardtii (Quesada et al., 1994) and several higher plants (Glass, 2009), and based on the correlations between AtNRT2.1 expression and IHATS influx, it became accepted that IHATS was encoded by AtNRT2.1. This conclusion was supported by the demonstration that transfer DNA mutants disrupted in both AtNRT2.1 and AtNRT2.2 exhibited 67% reduction of HATS but no reduction in LATS function (Filleur et al., 2001). A gene encoding CHATS has not yet been identified, although a mutant with defective CHATS has been isolated (Wang and Crawford, 1996). In summary, it was held that in Arabidopsis, AtNRT2.1 was responsible for IHATS, while AtNRT1.1 and AtNRT1.2 encoded ILATS and CLATS, respectively (Forde, 2000; Li et al., 2007).In papers by Wang et al. (1998), Liu et al. (1999), and Liu and Tsay (2003), it was demonstrated that AtNRT1.1 mutants of Arabidopsis exhibited reduced nitrate uptake even at 10 μm nitrate. The authors concluded that AtNRT1.1 fluxes exhibited saturation kinetics in planta and in Xenopus laevis oocytes and proposed that NRT1.1 encodes a dual-affinity nitrate transporter that “plays a major role in high-affinity nitrate uptake” (Wang et al., 1998). Liu and Tsay (2003) demonstrated that the AtNRT1.1 protein was capable of switching between high- and low-affinity states by phosphorylation of Thr residue 101; under low-nitrogen (N) conditions, phosphorylation mediated via the activation of protein kinase CIPK23 generated a high-affinity transporter (Ho et al., 2009), whereas high-N favored the dephosphorylated low-affinity configuration.     

Effects of Fungal Elicitors on Cell Growth and Artemisinin Accumulation in Hairy Root Cultures of Artemisia annua

The artemisinin accumulation in the hairy root cultures of Artemisia annua L. was enhanced via a treatment of three fungal elicitors separately ( Verticillium dahliae Kleb., Rhizopus stolonifer (Ehrenb. ex Fr. ) Vuill and Colletotrichum dematium (Pers.) Grove). Among these three elicitors, V. dahliae had the highest inducing efficiency, but none of them manifests any noticeable effects on the cell growth of the hairy root cultures. The artemisinin content of the hairy root cultures treated with V. dahliae elicitor was 1.12 mg/g DW, which was 45% higher than the control (0.77 mg/g DW). The results showed that elicitation was dependent on the elicitor concentration, the incubation period and the physiological stage at which the hairy root cultures were treated. In addition, the authors found that for V. dahliae, the optimum concentration was 0.4 mg carbohydrate per millilitre medium, the strongest response of A. annua hairy root cultures to the elicitation was at the late exponential growth stage, and the highest artemisinin content of the hairy root cultures was on the 4th day post treatment.     

真菌诱导子对青蒿发根细胞生长和青蒿素积累的影响

３种真菌诱导子（大菌丽花轮枝孢（Ｖｅｒｔｉｃｉｌｌｉｕｍ ｄａｈｉａｅ Ｋｌｅｂ．）、葡枝根霉（Ｒｈｉｚｏｐｕｓ ｓｔｏｌｏｎｉｆｅｒ（Ｅｈｒｅｎｂ．ｅｘＦｒ．）Ｖｕｉｌｌ）和束状刺盘孢（Ｃｏｌｌｅｔｏｒｉｃｈｕｍ ｄｅｍａｔｉｕｍ（Ｐｅｒｓ．）Ｇｒｏｖｅ）处理青蒿（Ａｒｔｅｍｉｓｉａ ａｎｎｕａＬ．）的发根,均能促进发根中青蒿素的积累,其中以大丽花轮枝孢的诱导效果最好;对细胞生长均没有明显影响,     

酵母葡聚糖对丹参毛状根过氧化物酶和苯丙氨酸解氨酶的影响

为利用转化的丹参毛状根生产丹参中的药理活性物质,从啤酒酵母细胞壁中应用碱处理方法制备β-1,3-葡聚糖。利用全酵母细胞壁和酵母葡聚糖的水解产物分别诱导悬浮培养的丹参毛状根,比较它们对丹参毛状根的形态和根组织过氧化物酶、苯丙氨酸解氨酶的影响。结果表明,酵母葡聚糖比全酵母细胞壁水解产物更显著促进丹参毛状根组织的过氧化物酶和苯丙氨酸解氨酶的总活性,酵母葡聚糖的诱导效应具有浓度依赖性和时效性。酵母葡聚糖显著促进丹参毛状根的生长和根端膨大。葡聚糖是有潜力的丹参生长和次生代谢调节剂。     

Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum

The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vertès, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7:182-196, 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.     

HPLC法测定红景天居群中红景天苷的分布式样

本文以HPLC法作为测定红景天苷的方法,调查红景天苷在红景天不同种、不同居群中的分布式样。实验中采集不同种、同种不同居群、同居群不同个体的红景天样品,进行红景天苷的含量测定。结果表明红景天苷在不同红景天中分布差异较大,同种不同居群间多型性、居群内及个体间多态性显著。HPLC比法简便、快速、准确、重复性好,适用于研究红景天苷在药用红景天居群中的分布。