黄酮6位羟基化酶催化机制的理论研究与应用

灯盏乙素发酵生产过程中,黄酮6位羟基化酶催化效率不足,导致产生至少约18%的副产物。本研究以2种黄酮6位羟基化酶CYP82D4与CYP706X为研究目标,通过分子动力学模拟与量子化学计算,对两种黄酮6位羟基化酶的催化机制进行解析。结果表明,CYP82D4与CYP706X在反应决速步的能垒几乎相同,应当具有相似的反应速率,而CYP82D4相对较小的底物结合能可能有利于产物释放,是其具有更高催化效率的直接原因。最后,基于对底物进出过程的研究,CYP82D4的L540A突变将催化效率提高了1.37倍,证明了理论计算指导黄酮6位羟基化酶改造优化的可行性。本研究揭示了黄酮6位羟化酶的催化机制,为对其进行改造优化以提高灯盏乙素的发酵生产效率提供了参考。     

柚皮素合成关键基因表达水平对目标产物积累水平的量化影响

柚皮素是一种天然黄酮类化合物,具有抗炎、抗氧化、抗病毒、预防动脉粥样硬化等多种药理活性,也是其他黄酮类化合物合成的重要前体,具有重要的应用价值。目前,微生物法生产柚皮素等黄酮类化合物由于代谢通路不平衡等原因导致产量较低,在很大程度上限制了其工业应用。文中以一株产柚皮素的酿酒酵母菌株Y-01为研究对象,利用启动子和拷贝数控制柚皮素合成代谢途径关键酶4-香豆酸:CoA连接酶(4CL)、查尔酮合成酶(CHS)和查尔酮异构酶(CHI)编码基因的表达水平,考察这些基因的表达水平对目标产物积累水平的量化影响。结果表明,柚皮素产量与4CL或CHI编码基因的表达量之间关联性较低,而与chs基因的表达量存在显著的正相关性。通过调控chs基因的表达水平,获得一株高产柚皮素的酿酒酵母工程菌株Y-04,产量较出发菌株Y-01提高了4.1倍。研究结果表明,CHS是柚皮素合成过程的关键调控靶点,合理调控CHS表达可以显著促进酿酒酵母积累柚皮素。相关结果为采用代谢工程强化微生物合成柚皮素等重要黄酮类化合物提供了重要的理论参考。     

操纵茶树类黄酮3′-羟基化酶生物合成B环-3′,4′-二羟基黄酮类化合物

【目的】操纵茶树类黄酮3′-羟基化酶,生物合成B环-3′,4′-二羟基黄酮类化合物圣草酚、二氢槲皮素和槲皮素。【方法】构建了4个茶树类黄酮3′-羟基化酶基因(CsF3′H)和拟南芥的P450还原酶基因(ATR)融合表达质粒:SUMO-CsF3'H[7-517]::ATR1[49-688]3 AA、SUMO-CsF3'H[28-517]::ATR1[49-688]3 AA、SUMO-CsF3'H[7-517]::ATR2[75-711]3 AA和SUMO-CsF3'H[28-517]::ATR2[75-711]3 AA,分别转化大肠杆菌菌株TOP10、DH5α和BL21,获得12个转化菌株S1–S12;构建了茶树类黄酮3′-羟基化酶基因CsF3′H表达质粒p YES-Dest52-CsF3′H,转化酵母菌株WAT11,得到转化菌株S13;构建了茶树类黄酮3′-羟基化酶基因CsF3′H表达质粒pES-URA-CsF3′H,及茶树黄烷酮3-羟基化酶基因CsF3H与拟南芥黄酮醇合成酶基因At FLS的融合表达质粒pES-HIS-CsF3H::At FLS 9AA,二者共转化酵母菌株WAT11,获得转化菌株S14。【结果】转化SUMO-CsF3'H[28-517]::ATR1[49-688]3 AA质粒的TOP10菌株S6在25°C条件下发酵,转化效率最高,能将1000μmol/L柚皮素、二氢山奈酚和山奈酚,分别转化生成287.93μmol/L圣草酚、131.76μmol/L二氢槲皮素和188.62μmol/L槲皮素。发酵菌株S13能分别将1000μmol/L柚皮素、二氢山奈酚和山奈酚,最多能转化生成734.32μmol/L圣草酚、446.07μmol/L二氢槲皮素和594.64μmol/L槲皮素。喂食S14发酵菌株5 mmol/L的底物柚皮素,在发酵36–48 h中,最多能生成1412.16μmol/L圣草酚、490.25μmol/L山奈酚、445.75μmol/L槲皮素、66.75μmol/L二氢槲皮素和73.50μmol/L二氢山奈酚。【结论】本研究首次将茶树类黄酮3′-羟基化酶基因应用于B环-3′,4′-二羟基黄酮类化合物圣草酚、二氢槲皮素和槲皮素的生物合成。     

柚皮素-7-O-乙酸酯和柚皮素-7-O-丙酸酯的制备及抗血小板聚集活性

为改善柚皮素的水溶性而不降低其抗血小板聚集活性,本文以柚皮苷为原料,经"4'位羟基苄基化-酸水解苷键-酰化-加氢脱苄基"四步反应,合成出柚皮素-7-O-乙酸酯和柚皮素-7-O-丙酸酯。两种衍生物在水中的溶解度分别为637.34±53.23μg/m L和59.74±4.81μg/m L,均高于柚皮素的溶解度。两种衍生物均对二磷酸腺苷诱导的兔体外和大鼠体内血小板聚集有显著的抑制活性,且抑聚率均高于柚皮素。实验结果表明,通过选择酰化柚皮素的7位羟基,引入含1~2个碳的短脂肪烃基链,能显著改善水溶性,提高抗血小板聚集活性。     

黄酮类化合物清除羟自由基(HO·)功能及其构效关系研究

本文采用体外实验的方法,系统地研究了八种黄酮类植物化学物槲皮素、异槲皮素、芦丁、橙皮素、橙皮苷、柚皮素、柚皮苷和淫羊藿苷对羟自由基(HO.)的清除作用及构效关系。结果表明,八种黄酮类化合物中,只有槲皮素、芦丁和异槲皮素对HO·有明显的清除作用,在浓度为260μM时其对HO·清除率分别为30.69%±0.06、13.70%±0.04和10.23%±0.03。构效比较结果表明:黄酮类化合物C环C3位羟基和B环3’-4’邻位二羟基是其清除HO.的重要功能基团。本研究为选择有效的能清除HO·的膳食黄酮类化合物用于预防和治疗相关疾病提供了一定实验基础和理论依据。     

合成8二甲基异戊烯基柚皮素的人工酿酒酵母菌株构建

8二甲基异戊烯基柚皮素(8DN)作为生产黄酮类药物淫羊藿苷的重要前体,在医药合成领域具有重大应用潜力。由于其合成路径及相关基因的复杂性,目前主要通过饲喂8DN的直接前体(柚皮素、异黄腐酚等)的方式合成8DN,而在生物体内全合成8DN的研究工作还未见报道。为了实现8DN在酿酒酵母体内的生物全合成,通过组合筛选8DN前体物柚皮素合成所需的多种外源基因(TAL、4CL、CHS、CHI),获得30株柚皮素生产菌,发现不同来源的基因组合使柚皮素产量的具有明显差异(0.37~22.33mg/L)。并且利用Delta位点将较优的基因组合整合至酵母基因组,实现了稳定的柚皮素高产菌株(Sy BE_Sc02050031)构建。在此基础上进一步导入带有苦参来源的异戊烯基转移酶基因(N8DT)多拷贝质粒,实现8DN合成的完整反应过程,8DN的摇瓶发酵产量达到36.7μg/L。另外,通过关键限速酶N8DT的序列优化策略,发现截断定位信号肽序列的N8DT明显提高了从柚皮素到8DN这一关键反应的催化效果,8DN的产量提高到52.6μg/L(144.2%)。首次在酿酒酵母中成功构建高产8DN的生物全合成路径,为在微生物体内合成其他黄酮类天然产物提供了参考,具有重要的指导意义。     

酸豆外果皮黄酮类成分及其抗氧化活性评价

为了研究酸豆外果皮成分及其抗氧化活性,采用现代色谱分离技术从酸豆(Tamarindus indica Linn.)外果皮丙酮提取物中分离和纯化得到18个黄酮类化合物,通过核磁共振波谱数据对它们进行鉴定,分别为木犀草素(1)、7,3′,4′-三羟基黄酮(2)、芹菜素(3)、7,4′-二羟基黄酮(4)、7,4′-二羟基-3′-甲氧基黄酮(5)、紫云英苷(6)、槲皮素(7)、山柰酚(8)、金合欢素(9)、圣草酚(10)、柚皮素(11)、紫铆素(12)、二氢山柰酚(13)、5,7,3′,5′-四羟基二氢黄酮(14)、5,6,7,4′-四羟基二氢黄酮(15)、3,5,7,3′,5′-五羟基二氢黄酮醇(16)、儿茶素(17)、表儿茶素(18)。10个化合物(2、4～6、9、11、13～16)为首次从该属植物中分离得到;采用ORAC法体外抗氧化活性测定,发现酸豆外果皮的4份萃取物及9个化合物(1、2、7～10、12、13、17)有较好的抗氧化活性。     

牛奶菜中黄酮类成分的研究

从牛奶菜(Marsdenia sinensis Hemsl)根中分离得到6个黄酮类化合物,经光谱分析确定其结构为:芹菜素(1),(2S)-柚皮素(2),木犀草素(3),异牡荆素(4),8-C-β-D-Glucopyranosyl apigeninidin(5),5,7-二羟基色原酮(6)。所有化合物均为首次从该植物中分离得到,同时对分离得到的单体化合物进行细胞毒活性测试,化合物1～5均表现出较强的细胞毒活性。     

华中冬青化学成分的研究

从华中冬青（Ｉｌｅｘ ｃｅｎｔｒｏｃｈｉｎｅｎｓｉｓ Ｓ．Ｙ．Ｈｕ）的叶片分离到９ 种黄酮类化合物：１ 种新化合物鉴定为３,５,５,７－四羟基二氢黄酮,命名为华中冬青黄酮（Ⅰ）;８ 种化合物鉴定为柚皮素（Ⅱ）、橙皮素（Ⅲ）、异樱花素（Ⅳ）、枸柑甙（Ⅴ）、橙皮甙（Ⅵ）、洋芹素（Ⅶ）、紫云英甙（Ⅷ）和野漆树甙（Ⅸ）。它们皆首次从该植物分得     

水团花黄酮类成分及其体外抗病毒活性

从水团花乙醇提物的乙酸乙酯部位分离到6个黄酮类化合物,根据光谱数据分别鉴定为柚皮素（1）、圣草酚（2）、槲皮素（3）、柚皮素-7-O-β-D-葡萄糖苷（4）、圣草酚-7-O-β-D-葡萄糖苷（5）、槲皮素-3-O-β-D-葡萄糖苷（6）,化合物1、2,4和5为首次从该植物中分离得到。采用细胞病变抑制法（CPEr eductionassay）和MTT法测定化合物的体外抗病毒活性,结果显示,三个黄酮苷元1、2和3均具有不同程度的体外抑制呼吸道合胞病毒（RSV）和柯萨奇B3型病毒（CVB3）活性,反之,3个黄酮苷均不显示活性。     

Biochemical and genetic characterization of Arabidopsis flavanone 3beta-hydroxylase

Flavanone 3beta-hydroxylase (F3H; EC 1.14.11.9) is a 2-oxoglutarate dependent dioxygenase that catalyzes the synthesis of dihydrokaempferol, the common precursor for three major classes of 3-hydroxy flavonoids, the flavonols, anthocyanins, and proanthocyanidins. This enzyme also competes for flux into the 3-deoxy flavonoid branch pathway in some species. F3H genes are increasingly being used, often together with genes encoding other enzymes, to engineer flavonoid synthesis in microbes and plants. Although putative F3H genes have been cloned in a large number of plant species, only a handful have been functionally characterized. Here we describe the biochemical properties of the Arabidopsis thaliana F3H (AtF3H) enzyme and confirm the activities of gene products from four other plant species previously identified as having high homology to F3H. We have also investigated the surprising "leaky" phenotype of AtF3H mutant alleles, uncovering evidence that two related flavonoid enzymes, flavonol synthase (EC 1.14.11.23) and anthocyanidin synthase (EC 1.14.11.19), can partially compensate for F3H in vivo. These experiments further indicate that the absence of F3H in these lines enables the synthesis of uncommon 3-deoxy flavonoids in the Arabidopsis seed coat.     

高选择性不对称还原N,N-二甲基-3-酮-3-(2-噻吩)-1-丙胺的重组氧化还原酶催化性质及其酶促转化

【目的】通过表达多种重组立体选择性氧化还原酶,分析其催化不对称还原N,N-二甲基-3-酮-3-(2-噻吩)-1-丙胺(DKTP)的性质,从而构建酶促合成(S)-N,N-二甲基-3-羟基-3-(2-噻吩)-1-丙胺(DHTP)的反应体系。【方法】基于已有立体选择性氧化还原酶重组大肠杆菌,通过Ni离子亲和层析法纯化得到重组氧化还原酶,以DKTP为底物,考察不同重组氧化还原酶对DKTP的催化活性和选择性,进一步对高选择性酶促合成(S)-DHTP的重组酶CR2进行性质分析,并考察其在最适条件下不对称还原DKTP的过程。【结果】筛选获得产物构型为(S)-型的催化活性最高的酶为CR2,该酶米氏常数Km为0.135 mmol/L,kcat/Km为3.689 L/(mmol·s),最适p H 8.4(0.1 mol/L三乙醇胺缓冲液),最适反应温度为35°C,在10-45°C条件下和p H 7.5-8.5较为稳定,Zn2+离子对酶活有促进作用。CR2催化DKTP不对称还原反应6 h后,DHTP的产率达92.1%、光学纯度达99.9%。【结论】基于活性和选择性分析,获得不对称还原DKTP的目标酶CR2,其催化特性有利于高立体选择性还原DKTP生成度洛西汀中间体(S)-DHTP,从而为进一步提高酶促不对称还原DKTP的转化效率提供研究基础。     

Flavanone 3beta-hydroxylases from rice: key enzymes for favonol and anthocyanin biosynthesis

Flavanone 3beta-hydroxylases (F3H) are key enzymes in the synthesis of flavonol and anthocyanin. In this study, three F3H cDNAs from Oryza sativa (OsF3H-1 approximately 3) were cloned by RT-PCR and expressed in E. coli as gluthatione S-transferase (GST) fusion proteins. The purified recombinant OsF3Hs used flavanone, naringenin and eriodictyol as substrates. The reaction products with naringen and eriodictyol were determined by nuclear magnetic resonance spectroscopy to be dihydrokaempferol and taxifolin, respectively. OsF3H-1 had the highest enzymatic activity whereas the overall expression of OsF3H-2 was highest in all tissues except seeds. Flavanone 3beta-hydroxylase could be a useful target for flavonoid metabolic engineering in rice.     

蓝亚麻花瓣中类黄酮化合物及代谢途径分析

花色是观赏植物重要的观赏性状之一,而类黄酮是其主要的呈色物质。该研究以蓝亚麻花瓣为研究对象,将蓝亚麻开花过程分为5个阶段,并用高效液相色谱—光电二极管阵列检测技术(HPLC-PAD)和高效液相色谱—电喷雾离子化—质谱连用技术(HPLC-ESI-MS)分析不同开花阶段花瓣中类黄酮化合物的成分和含量。结果表明:蓝亚麻花瓣中积累飞燕草素苷、矢车菊素苷和锦葵素苷,未检测到天竺葵素苷,其中以酰基化的飞燕草素苷为主要呈色物质;而总花青素苷含量在第2阶段达到最高。根据花青素苷终产物和类黄酮中间代谢产物推定了蓝亚麻花瓣中类黄酮代谢途径,其中以F3'5'H所引导的分支途径占优势,其主要原因可能是F3'5'H酶活高于F3'H。     

Directed evolution of mammalian cytochrome P450 2B1: mutations outside of the active site enhance the metabolism of several substrates, including the anticancer prodrugs cyclophosphamide and ifosfamide

Cytochrome P450 2B1 has been subjected to directed evolution to investigate the role of amino acid residues outside of the active site and to engineer novel, more active P450 catalysts. A high throughput screening system was developed to measure H(2)O(2)-supported oxidation of the marker fluorogenic substrate 7-ethoxy-4-trifluoromethylcoumarin (7-EFC). Random mutagenesis by error-prone polymerase chain reaction and activity screening were optimized using the L209A mutant of P450 2B1 in an N-terminally modified construct with a C-terminal His tag (P450 2B1dH). Two rounds of mutagenesis and screening and one subcloning step yielded the P450 2B1 quadruple mutant V183L/F202L/L209A/S334P, which demonstrated a 6-fold higher k(cat) than L209A. Further random or site-directed mutagenesis did not improve the activity. When assayed in an NADPH-supported reconstituted system, V183L/L209A demonstrated lower 7-EFC oxidation than L209A. Therefore, F202L/L209A/S334P was generated, which showed a 2.5-fold higher k(cat)/K(m) for NADPH-dependent 7-EFC oxidation than L209A. F202L/L209A/S334P also showed enhanced catalytic efficiency with 7-benzyloxyresorufin, benzphetamine, and testosterone, and a 10-fold increase in stereoselectivity for testosterone 16alpha-versus 16beta-hydroxylation compared with 2B1dH. Enhanced catalytic efficiency of F202L/L209A/S334P was also retained in the full-length P450 2B1 background with 7-EFC and testosterone as substrates. Finally, the individual mutants were tested for metabolism of the anti-cancer prodrugs cyclophosphamide and ifosfamide. Several of the mutants showed increased metabolism via the therapeutically beneficial 4-hydroxylation pathway, with L209A/S334P showing 2.8-fold enhancement of k(cat)/K(m) with cyclophosphamide and V183L/L209A showing 3.5-fold enhancement with ifosfamide. Directed evolution can thus be used to enhance P450 2B1 catalytic efficiency across a panel of substrates and to identify functionally important residues distant from the active site.     

酵母还原乙酰乙酸乙酯制备(S)-3-羟基丁酸乙酯的研究

研究了五种酵母催化乙酰乙酸乙酯生成(S)-3-羟基丁酸乙酯的能力,筛选出催化性能较好的菌株酿酒酵母,并以该酵母为出发菌株进行紫外诱变筛选出催化性能更优良的菌株LY7;另外还对菌株LY7催化乙酰乙酸乙酯生成(S)-3-羟基丁酸乙酯的反应特性进行了研究。研究表明:采用200g/L蔗糖作为碳源,初始乙酰乙酸乙酯浓度为0.25mol/L,初始细胞浓度为150g/L,反应温度为36℃时所得产物得率和对映体过剩值最高。     

Structural studies of zoospore attractants from Trachelospermum jasminoides var. pubescens: taxifolin 3-O-glycosides

(2S, 3S)-Taxifolin 3-O-arabinoside, a new dihydroflavonol glycoside, together with the known substances, (2R 3R)-taxifolin 3-O-arabinoside, astragalin, isoquercitrin, and cosmosiin, were isolated from the leaves of Trachelospermum jasminoides var. pubescens. Structural elucidation was carried out by spectroscopic methods, and the absolute configurations of C-2 and C-3 in taxifolin 3-O-arabinosides were determined on the basis of circular dichroism. Unlike the dihydroflavonol glycosides, (2R 3R)-taxifolin 3-O-glucoside and (2R, 3R-taxifolin 3-O-arabinoside, the novel (2S, 3S)-taxifolin 3-O-arabinoside is not effective as a zoospore attractant of the plant pathogenic fungus Aphanomyces cochlioides.     

Mitochondrial ATP synthase. Crystal structure of the catalytic F1 unit in a vanadate-induced transition-like state and implications for mechanism

ATP synthesis from ADP, P(i), and Mg2+ takes place in mitochondria on the catalytic F1 unit (alpha3beta3gammedeltaepsilon) of the ATP synthase complex (F0F1), a remarkable nanomachine that interconverts electrochemical and mechanical energy, producing the high energy terminal bond of ATP. In currently available structural models of F1, the P-loop (amino acid residues 156GGAGVGKT163) contributes to substrate binding at the subunit catalytic sites. Here, we report the first transition state-like structure of F1 (ADP.V(i).Mg.F1) from rat liver that was crystallized with the phosphate (P(i)) analog vanadate (VO(3-)4 or V(i)). Compared with earlier "ground state" structures, this new F1 structure reveals that the active site region has undergone significant remodeling. P-loop residue alanine 158 is located much closer to V(i) than it is to P(i) in a previous structural model. No significant movements of P-loop residues of the subunit were observed at its analogous but noncatalytic sites. Under physiological conditions, such active site remodeling involving the small hydrophobic alanine residue may promote ATP synthesis by lowering the local dielectric constant, thus facilitating the dehydration of ADP and P(i). This new crystallographic study provides strong support for the catalytic mechanism of ATP synthesis deduced from earlier biochemical studies of liver F1 conducted in the presence of V(i) (Ko, Y. H., Bianchet, M., Amzel, L. M., and Pedersen, P. L. (1997) J. Biol. Chem. 272, 18875-18881; Ko, Y. H., Hong, S., and Pedersen, P. L. (1999) J. Biol. Chem. 274, 28853-28856).     

Modular Optimization of Heterologous Pathways for De Novo Synthesis of (2S)-Naringenin in Escherichia coli

Due to increasing concerns about food safety and environmental issues, bio-based production of flavonoids from safe, inexpensive, and renewable substrates is increasingly attracting attention. Here, the complete biosynthetic pathway, consisting of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS), chorismate mutase/prephenate dehydrogenase (CM/PDH), tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), malonate synthetase, and malonate carrier protein, was constructed using pre-made modules to overproduce (2S)-naringenin from D-glucose. Modular pathway engineering strategies were applied to the production of the flavonoid precursor (2S)-naringenin from L-tyrosine to investigate the metabolic space for efficient conversion. Modular expression was combinatorially tuned by modifying plasmid gene copy numbers and promoter strengths to identify an optimally balanced pathway. Furthermore, a new modular pathway from D-glucose to L-tyrosine was assembled and re-optimized with the identified optimal modules to enable de novo synthesis of (2S)-naringenin. Once this metabolic balance was achieved, the optimum strain was capable of producing 100.64 mg/L (2S)-naringenin directly from D-glucose, which is the highest production titer from D-glucose in Escherichia coli. The fermentation system described here paves the way for the development of an economical process for microbial production of flavonoids.     

二氢黄酮醇 4 还原酶在花青素合成中的功能及调控研究进展

植物色素主要有花青素、类胡萝卜素和生物碱类色素三大类,其中花青素是决定大部分被子植物组织或器官颜色的重要色素。花青素通过类黄酮途径合成,该途径是生物学上研究较多且较为清楚的代谢途径之一。近年来的研究表明,在该途径中除了查尔酮合成酶(chalcone synthase,CHS)、查尔酮异构酶(chalcone isomerase,CHI)和黄烷酮-3-羟化酶(flavanone-3-hydrolase,F3H)起着关键作用外,二氢黄酮醇-4-还原酶(dihydroflavonol 4-reductase,DFR)对花青素的合成也至关重要。DFR可催化3种二氢黄酮醇和2种黄烷酮生成5种不同的花青素前体,且DFR基因家族不同成员对各个底物的催化效率不同,因此它在一定程度上决定着植物中花青素的种类和含量,从而影响植物组织或器官的颜色。该文对近年来国内外有关DFR在花青素合成过程中的生物学功能与调控,包括DFR的特征、作用机制和系统进化以及环境、转录因子和一些结构基因与DFR的关系等方面的研究进展进行了综述,以期为DFR今后的研究和利用基因工程改变植物组织或器官的颜色提供理论依据。