Isoflavonoids as systematic markers

A taxonomic procedure, applicable to biogenetic groups (BG) of secondary metabolities, involves initially the determination of the relative probability of occurrence (RPO) with respect to skeleton and substitution for each compound of the BG. The means of the RPO-values of the constituents represent the evolutionary advancement parameters (EAP) of the species. Consideration of the EAPs and of the chemical composition leads to the classification of species along chemical lines. This procedure, which is independent of morphological evidence, applied to isoflavonoids, suggests the sequence of structural changes which accompany the evolution of this BG in nature, and, used as an auxiliary criterion to the conventional morphological classification, provides the basis for understanding of the evolutionary development in the Lotoideae.     

Isoflavonoids of Dalbergia ecastophyllum

In addition to the known isoflavonoids formononetin and (±)-mucronulatol, the vine wood of Dalbergia ecastophyllum (L.) Taub. contains (R)-8-demethylduartin. The assigned structure (II) has been confirmed by synthesis. Its absolute stereochemistry is deduced from examination of the CD spectrum of its acetate.     

Isoflavonoids from Myroxylon peruiferum

Considerable differences in flavonoid composition of the trunkwood characterize different specimens of Myroxylon balsamum (L.) Harms. Only calycosin among the 11 flavonoids found in M. peruiferum L.f., presently considered synonymous with M. balsamum, had previously been located in the latter species. Two of these flavonoids, 2′-hydroxy-7,3′,4′-trimethoxyisoflavanone and 2′-hydroxy-7,3′,4′-trimethoxyisoflavone are new natural products.     

Isoflavonoids of Mildbraedeodendron excelsa

The heartwood of Mildbraedeodendron excelsa has yielded five isoflavones and an ( ± )-isoflavanone. The structure of 7,4′-dihydroxy-6-methoxyisoflavone (glycetein) was confirmed by synthesis.     

黄花木的异黄酮类成分研究(英文)

黄花木(Piptanthus concolor Harrow)茎枝85%乙醇提取物采用多种色谱技术分离纯化,从中分离到11个异黄酮类化合物,经波谱分析鉴定为美迪紫檀素(1),山槐素(2),阿弗洛莫生(3),11b-羟基-11b,1-二氢美迪紫檀素(4),多花紫藤苷(5),trifolirhizin(6),wighteone(7),3’-甲氧基-大豆苷元(8),红车轴草素(9),毛蕊异黄酮(10),erythrinin C(11)。其中,化合物1～9和11为首次从该植物中分离得到。     

产丁二酸工程菌的构建及其厌氧发酵

为了考察过量表达苹果酸酶对于E.coli NZN111(ldhA::Kan pfl::Cam)厌氧发酵产丁二酸的影响, 将连接有苹果酸酶基因sfcA的表达载体pTrc99a-sfcA转化进NZN111中, 构建了重组NZN111(pTrc99a-sfcA)。0.5 mmol/L IPTG诱导8 h后, 测定的苹果酸酶比酶活为30.67 u/mg, 比受体菌提高了140倍。采用两阶段发酵模式, 结果表明: 过量表达的苹果酸酶在NZN111体内催化了从丙酮酸到苹果酸的逆向反应, 丁二酸是发酵过程中积累的主要有机酸, 且当加入0.7 mmol/L IPTG诱导, 初始葡萄糖糖浓度为18.5 g/L时, 选择对数生长期后期的菌种以10%的接种量转入厌氧发酵, 发酵结束时发酵液中丁二酸的浓度为12.84 g/L, 对葡萄糖的收率为69.43%, 乙酸为0.58 g/L, 二者浓度比为22:1, 没有检测到甲酸和乳酸。构建的菌种具有高产丁二酸和副产物极少的优点, 在同类菌种中处于先进水平。     

崖豆藤属植物中异黄酮类化学成分及其药理活性的研究进展

本文对豆科崖豆藤属植物中的异黄酮类化学成分及此类成分药理活性的国内外研究进行综述。从崖豆藤属植物中分离得到异黄酮类化合物159个,包括异黄酮及其苷类、鱼藤酮类、异黄烷类、紫檀烷类、二氢异黄酮类,它们具有抗肿瘤、抗炎、抗雌激素等作用。     

Isoflavonoids and flavone glycosides from rhizomes of Iris carthaliniae

Two new isoflavonoid biosides, tectorigenin 4′-glucosyl (1→6)glucoside and iristectorigenin B 7-glucosyl (1→6)glucoside, a new isoflavonoid monoside, 4′-methyltectorigenin 7-glucoside and a new flavone glucoside, 6,4′-dimethoxy-5-hydroxyflavone 7-glucoside, together with tectoridin and tectorigenin 4′-glucoside were isolated from rhizomes of Iris carthaliniae. The structures of the isolated compounds were determined by NMR spectral analysis.     

葛根异黄酮的快速分离检测方法研究

以SDS正丁醇-正庚烷-水组成的微乳体系作为展开剂,通过聚酰胺薄层层析,探讨不同类型微乳液对葛根异黄酮分离检测效果的影响。结果表明,选择含水量为70％的微乳液作为展开剂,分离效果明显,一次检测出12个黄酮化合物;与以三氯甲烷-甲醇-水(7:2．5:0．25)为展开剂的硅胶薄层相比,微乳薄层色谱对葛根异黄酮的检测效果和灵敏度显著提高,为实验研究中常规检测提供了简便快捷的方法。     

Engineered biosynthesis of medium-chain esters in Escherichia coli

Medium-chain esters such as isobutyl acetate (IBAc) and isoamyl acetate (IAAc) are high-volume solvents, flavors and fragrances. In this work, we engineered synthetic metabolic pathways in Escherichia coli for the total biosynthesis of IBAc and IAAc directly from glucose. Our pathways harnessed the power of natural amino acid biosynthesis. In particular, the native valine and leucine pathways in E. coli were utilized to supply the precursors. Then alcohol acyltransferases from various organisms were investigated on their capability to catalyze esterification reactions. It was discovered that ATF1 from Saccharomyces cerevisiae was the best enzyme for the formation of both IBAc and IAAc in E. coli. In vitro biochemical characterization of ATF1 confirmed the fermentation results and provided rational guidance for future enzyme engineering. We also performed strain improvement by removing byproduct pathways (Δldh, ΔpoxB, Δpta) and increased the production of both target chemicals. Then the best IBAc producing strain was used for scale-up fermentation in a 1.3-L benchtop bioreactor. 36 g/L of IBAc was produced after 72 h fermentation. This work demonstrates the feasibility of total biosynthesis of medium-chain esters as renewable chemicals.     

The ADP-glucose binding site of the Escherichia coli glycogen synthase

Bacterial glycogen/starch synthases are retaining GT-B glycosyltransferases that transfer glucosyl units from ADP-Glc to the non-reducing end of glycogen or starch. We modeled the Escherichia coli glycogen synthase based on the coordinates of the inactive form of the Agrobacterium tumefaciens glycogen synthase and the active form of the maltodextrin phosphorylase, a retaining GT-B glycosyltransferase belonging to a different family. In this model, we identified a set of conserved residues surrounding the sugar nucleotide substrate, and we replaced them with different amino acids by means of site-directed mutagenesis. Kinetic analysis of the mutants revealed the involvement of these residues in ADP-Glc binding. Replacement of Asp21, Asn246 or Tyr355 for Ala decreased the apparent affinity for ADP-Glc 18-, 45-, and 31-fold, respectively. Comparison with other crystallized retaining GT-B glycosyltransferases confirmed the striking similarities among this group of enzymes even though they use different substrates.     

Novel insight into the secretory expression of recombinant enzymes in Escherichia coli

The secretory expression of recombinant enzymes in Escherichia coli has generally been a challenging task. In the present study, we investigated the expression of the extracellular enzyme cyclodextrin glycosyltransferase in E. coli. Our results indicated that when the overexpressed pre-proteins were not translocated across the inner membrane in a timely manner, they aggregated near the inner side of the E. coli inner membrane, resulting in the formation of insoluble inclusion bodies, which eventually blocked the pre-protein translocation channels and subsequently impeded further protein secretion. This mechanism suggests that for the efficient production of extracellular enzymes in E. coli, it is very important to maintain a balance between the rate of pre-protein synthesis and translocation, which can be achieved by altering the cultivation process. Our findings provide novel insight into the secretory expression of extracellular enzymes and may shed light on the further development of new strategies for extracellular protein production in E. coli.     

Engineered polyketide biosynthesis and biocatalysis in Escherichia coli

Polyketides are important bioactive natural products biosynthesized by bacteria, fungi, and plants. The enzymes that synthesize polyketides are collectively referred to as polyketide synthases (PKSs). Because many of the natural hosts that produce polyketides are difficult to culture or manipulate, establishing a universal heterologous host that is genetically tractable has become an important goal toward the engineered biosynthesis of polyketides and analogues. Here, we summarize the recent progresses in engineering Escherichia coli as a heterologous host for reconstituting PKSs of different types. Our increased understanding of PKS enzymology and structural biology, combined with new tools in protein engineering, metabolic engineering, and synthetic biology, has firmly established E. coli as a powerful host for producing polyketides.     

Isoflavonoids from Phaseolus coccineus

After treatment with CuCl₂, the following isoflavonoids have been isolated from the runner bean, Phaseolus coccineus: daidzein, genistein, isoprunetin, 2′-hydroxygenistein, phaseoluteone, 2′-hydroxydihydrodaidzein, isoferreirin, kievitone, cyclokievitone, glycinol, phaseollidin, phaseollin, demethylvestitol, phaseollinisoflavan, 2′-hydroxyisoprunetin and 7,4′-dihydroxy-5,2′-dimethoxyisoflavanone. The latter two compounds are novel natural products.     

Engineered citrate synthase alters Acetate Accumulation in Escherichia coli

Metabolic engineering is used to improve titers, yields and generation rates for biochemical products in host microbes such as Escherichia coli. A wide range of biochemicals are derived from the central carbon metabolite acetyl-CoA, and the largest native drain of acetyl-CoA in most microbes including E. coli is entry into the tricarboxylic acid (TCA) cycle via citrate synthase (coded by the gltA gene). Since the pathway to any biochemical derived from acetyl-CoA must ultimately compete with citrate synthase, a reduction in citrate synthase activity should facilitate the increased formation of products derived from acetyl-CoA. To test this hypothesis, we integrated into E. coli C ΔpoxB twenty-eight citrate synthase variants having specific point mutations that were anticipated to reduce citrate synthase activity. These variants were assessed in shake flasks for growth and the production of acetate, a model product derived from acetyl-CoA. Mutations in citrate synthase at residues W260, A267 and V361 resulted in the greatest acetate yields (approximately 0.24 g/g glucose) compared to the native citrate synthase (0.05 g/g). These variants were further examined in controlled batch and continuous processes. The results provide important insights on improving the production of compounds derived from acetyl-CoA.     

Engineered isoprenoid pathway enhances astaxanthin production in Escherichia coli

The isoprenoid pathway is a versatile biosynthetic network leading to over 23,000 compounds. Similar to other biosynthetic pathways, the production of isoprenoids in microorganisms is controlled by the supply of precursors, among other factors. To engineer a host that has the capability to supply geranylgeranyl diphosphate (GGPP), a common precursor of isoprenoids, we cloned and overexpressed isopentenyl diphosphate (IPP) isomerase (encoded by idi) from Escherichia coli and GGPP synthase (encoded by gps) from the archaebacterium Archaeoglobus fulgidus. The latter was shown to be a multifunctional enzyme converting dimethylallyl diphosphate (DMAPP) to GGPP. These two genes and the gene cluster (crtBIYZW) of the marine bacterium Agrobacterium aurantiacum were introduced into E. coli to produce astaxanthin, an orange pigment and antioxidant. This metabolically engineered strain produces astaxanthin 50 times higher than values reported before. To determine the rate-controlling steps in GGPP production, the IDI-GPS pathway was compared with another construct containing idi, ispA (encoding farnesyl diphosphate (FPP) synthase in E. coli), and crtE (encoding GGPP synthase from Erwinia uredovora). Results show that the conversion from FPP to GGPP is the first bottleneck, followed sequentially by IPP isomerization and FPP synthesis. Removal of these bottlenecks results in an E. coli strain providing sufficient precursors for in vivo synthesis of isoprenoids.     

Engineered Synthetic Pathway for Isopropanol Production in Escherichia coli

A synthetic pathway was engineered in Escherichia coli to produce isopropanol by expressing various combinations of genes from Clostridium acetobutylicum ATCC 824, E. coli K-12 MG1655, Clostridium beijerinckii NRRL B593, and Thermoanaerobacter brockii HTD4. The strain with the combination of C. acetobutylicum thl (acetyl-coenzyme A [CoA] acetyltransferase), E. coli atoAD (acetoacetyl-CoA transferase), C. acetobutylicum adc (acetoacetate decarboxylase), and C. beijerinckii adh (secondary alcohol dehydrogenase) achieved the highest titer. This strain produced 81.6 mM isopropanol in shake flasks with a yield of 43.5% (mol/mol) in the production phase. To our knowledge, this work is the first to produce isopropanol in E. coli, and the titer exceeded that from the native producers.     

Homolactate Fermentation by Metabolically Engineered Escherichia coli Strains

We report the homofermentative production of lactate in Escherichia coli strains containing mutations in the aceEF, pfl, poxB, and pps genes, which encode the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, and phosphoenolpyruvate synthase, respectively. The process uses a defined medium and two distinct fermentation phases: aerobic growth to an optical density of about 30, followed by nongrowth, anaerobic production. Strain YYC202 (aceEF pfl poxB pps) generated 90 g/liter lactate in 16 h during the anaerobic phase (with a yield of 0.95 g/g and a productivity of 5.6 g/liter · h). Ca(OH)₂ was found to be superior to NaOH for pH control, and interestingly, significant succinate also accumulated (over 7 g/liter) despite the use of N₂ for maintaining anaerobic conditions. Strain ALS961 (YYC202 ppc) prevented succinate accumulation, but growth was very poor. Strain ALS974 (YYC202 frdABCD) reduced succinate formation by 70% to less than 3 g/liter. ¹³C nuclear magnetic resonance analysis using uniformly labeled acetate demonstrated that succinate formation by ALS974 was biochemically derived from acetate in the medium. The absence of uniformly labeled succinate, however, demonstrated that glyoxylate did not reenter the tricarboxylic acid cycle via oxaloacetate. By minimizing the residual acetate at the time that the production phase commenced, the process with ALS974 achieved 138 g/liter lactate (1.55 M, 97% of the carbon products), with a yield of 0.99 g/g and a productivity of 6.3 g/liter · h during the anaerobic phase.     

纤维素酶解-超声偶联法提取葛根中总异黄酮的工艺优化

采用酶解-超声偶联新工艺对葛根中总异黄酮提取进行优化,以提高其收率.在单因素试验的基础上,利用Box-Behnken响应面试验设计,以蒸馏水作为提取溶剂,对温度、酶浓度、时间进行三因素三水平的试验设计优化.结果表明最佳提取工艺条件为:温度34℃、酶浓度0.63 mg/mL、时间62 min.在此条件下,葛根总异黄酮提取率为7.10％,与预测值的相对误差为0.34％.酶解-超声偶联工艺是一种新型、快速、有效的总黄酮提取方法.