高通量筛选高产酪氨酸的酿酒酵母菌株

酪氨酸是重要的芳香族氨基酸,自身不仅具有重要的营养价值,也是合成香豆素类化合物和黄酮类化合物的重要前体。文中以实验室前期构建的一株解除了酪氨酸反馈抑制的酿酒酵母Saccharomyces cerevisiae LTH0 (ARO4K229L,ARO7G141S,Δaro10,Δzwf1,Δura3) 为出发菌株,异源表达甜菜黄素合成基因DOD和CYP76AD1,使酿酒酵母产生黄色荧光。然后利用紫外诱变和常压室温等离子体 (Atmospheric and room temperature plasma,ARTP) 诱变相结合的方法对上述菌株进行随机诱变,并通过流式细胞仪筛选荧光强度显著提高的突变株。其中突变株LTH2-5-DOD-CYP76AD1在激发波长485 nm、发射波长505 nm处荧光强度为 (5 941±435) AU/OD,比诱变前提高了8.37倍。对诱变后荧光强度提高较多的14株突变株进行发酵生产酪氨酸,胞外酪氨酸产量最高为26.8 mg/L,比出发菌株提高了3.96倍。进一步异源表达约翰逊黄杆菌Flavobacterium johnsoniae来源的酪氨酸解氨酶FjTAL,对香豆酸产量达到119.8 mg/L,比出发菌株LTH0-FjTAL提高了1.02倍。     

产对香豆酸酿酒酵母工程菌株的构建与优化

对香豆酸是黄酮类、芪类等天然活性化合物的重要前体,在生物医药、食品等行业应用广泛。与传统植物提取和化学合成相比,微生物合成对香豆酸因其具有生产周期短、转化效率高等优势而得到广泛关注。为构建高产对香豆酸酵母工程菌株,以酿酒酵母为出发菌,通过敲除酪氨酸合成竞争路径基因ARO10和PDC5,突变芳香族氨基酸合成调控基因ARO4^K229L与ARO7^G141S、解除酪氨酸负反馈抑制、并整合酪氨酸解氨酶FjTAL,获得的工程菌C001对香豆酸产量为296.73 mg/L。为进一步提高对香豆酸合成前体积累,分别敲除8个与氨基酸、糖类等转运相关基因并强化糖异生途径,分析其对对香豆酸积累的影响。结果表明,敲除GAL2及过表达EcppsA,对香豆酸产量提高至475.11 mg/L。最后,分析了FjTAL蛋白锚定至酵母液泡对产物积累的影响,结果表明其定位液泡后对香豆酸产量明显提升,达到593.04mg/L。通过强化前体物供应,阻断竞争旁路途径,利用亚细胞定位等策略有效提高对香豆酸产量,为后续黄酮类及芪类化合物的合成提供高效平台菌株,具有重要的应用前景。     

类芽胞杆菌碱性果胶裂解酶的纯化与酶学性质表征

从类芽胞杆菌Paenibacillus sp.WZ008的发酵上清液中纯化得到一个高活力碱性果胶裂解酶,经SDS-PAGE电泳估算其亚基相对分子质量为4.5×104。通过对该酶进行酶学性质研究发现：该酶能催化裂解果胶酸、低酯果胶和高酯果胶;酶催化反应最适温度范围为55～60℃,最适pH为9.6,在最适条件下以低酯果胶为底物酶的比酶活达3 021.6 U/mg;Ca2＋能增强该酶的活力,而Mn2＋,Ba2＋和EDTA强烈抑制该酶活力;当没有Ca2＋存在时,高度酯化的果胶是该酶的最适底物,在4 mmol/L Ca2＋存在时,该酶以果胶酸为底物比酶活最高（25 467 U/mg）。该酶N端序列比对分析发现与类芽胞杆菌Paenibacillus amylolyticus strain 27c64果胶裂解酶高度同源。     

毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸*

目的:改造毕赤酵母使其异源合成类黄酮生物合成途径的重要中间体肉桂酸、对香豆酸,并优化前体芳香族氨基酸生物合成途径以提高毕赤酵母的生产能力。方法:在毕赤酵母GS115中利用乙醇诱导型人工转录系统表达Rhodotorula glutinis来源的苯丙氨酸解氨酶,并在该重组菌株中分别过表达胞内芳香族氨基酸生物合成途径中的关键酶或其突变体以进行优化。结果:异源表达苯丙氨酸解氨酶可使毕赤酵母将自身产生的L-苯丙氨酸、L-酪氨酸转化为肉桂酸(38.8 mg/L)、对香豆酸(34.2 mg/L),而通过过表达相关酶进行优化,最终肉桂酸和对香豆酸的产量分别达到124.1 mg/L和302.0 mg/L。结论:利用新的异源宿主毕赤酵母成功合成了肉桂酸、对香豆酸,并对胞内的芳香族氨基酸生物合成途径进行了优化,表明毕赤酵母具有生产黄酮类化合物的应用潜力,也为其他芳香族氨基酸衍生物或植物化合物在毕赤酵母中的异源合成奠定了基础。     

大肠杆菌中从头合成白藜芦醇途径的设计及优化

白藜芦醇是一种极具药用价值的植物源芪类化合物。为了在E.coli实现白藜芦醇的从头合成,构建了由酪氨酸解氨酶(TAL),香豆酸-CoA合成酶(4CL)和白藜芦醇合成酶(STS)组成的非天然合成途径。经3天发酵后,白藜芦醇产量仅为2.67 mg/L,而其中间体香豆酸的积累达到了95.64 mg/L。为了进一步改善异源途径的效率,对4CL和STS模块采取融合表达、高拷贝表达及启动子工程改造的策略,最终使白藜芦醇产量提高到了9.6倍,达到了25.76 mg/L,同时香豆酸的积累减少到了20.38 mg/L。这些研究结果为更高效白藜芦醇从头合成工程菌的构建及最终实现白藜芦醇的微生物大规模生产奠定了基础。     

3-(4-羟基苯基)丙酸在酿酒酵母中的生物合成

3-(4-羟基苯基)丙酸(HPPA)是一种芳香类化合物,作为中间体主要应用于医药、食品、化学领域,具有重要的经济价值。旨在实现HPPA在酿酒酵母中的从头合成,通过在酿酒酵母中过表达约氏黄杆菌来源的酪氨酸转移酶(Fj TAL)、拟南芥来源的对香豆酰辅酶A连接酶(At4CL),同时利用酿酒酵母BY4742自身来源的超长链烯酰辅酶A还原酶(Sc TSC13)、硫酯水解酶,实现了在酿酒酵母中从头合成HPPA,产量约为70 mg/L左右。在以4 mmol/L酪氨酸为前体、半乳糖诱导Fj TAL过表达、30℃发酵培养72 h的条件下,约36%的酪氨酸转化生成对香豆酸;在以4 mmol/L对香豆酸或咖啡酸为前体,半乳糖诱导At4CL过表达,同时利用酵母内源Sc TSC13、硫酯水解酶,30℃发酵培养72 h的条件下,约94%的对香豆酸被还原成HPPA,约91%的咖啡酸被还原成3,4-二羟基苯丙酸(DHPPA),为进一步生物合成HPPA及其衍生物奠定了基础。     

Flavobacterium johnsoniae昆布多糖酶的分离纯化及其催化性质

约氏黄杆菌Flavobacterium johnsoniae具有分泌裂解酵母细胞壁酶系的能力,经初步分析发现其发酵液中具有葡聚糖酶、几丁质酶和蛋白酶等活性。通过离子交换层析、疏水层析和凝胶过滤层析,从该菌发酵液中分离纯化到一种昆布多糖酶。该酶分子量为35 kD左右,其最适反应温度为50°C,最适反应pH为5.0。以昆布多糖和昆布寡糖为底物的反应表明,该酶以内切酶作用模式进行催化水解。     

产对香豆酸酿酒酵母菌株的构建及优化

对香豆酸(p-coumaric acid)作为苯丙素类物质、芪类物质及黄酮类物质的重要前体化合物,在生物医药、化妆品及食品工业中均有广泛的应用价值。以酿酒酵母作为底盘菌株,利用合成生物学原理构建一株高产对香豆酸的人工酵母细胞。通过对比不同拷贝数的酪氨酸解氨酶(tyrosine ammonia lyase)合成的对香豆酸产量,发现随着基因拷贝数的增加对香豆酸的产量也相应提高;同时对酪氨酸的负反馈调控相关的蛋白质进行氨基酸定点突变得到Aro4pK229L和Aro7pG141S,利用delta位点将突变后的基因整合至酵母基因组,并挑取24株构建成功的酵母细胞进行发酵验证,发现菌株最高产量与最低产量相差28.87mg/L;为了进一步增加对香豆酸的代谢通量,对生成芳香醇类物质的旁路基因ARO10和PDC5进行敲除,发现同时敲除两个基因的菌株对香豆酸的产量最高,是敲除前产量的2.05倍(从42.71mg/L到87.56mg/L)。此外,通过设计前体酪氨酸的梯度添加实验,发现当添加1mmol/L的酪氨酸时,对香豆酸产量达到峰值(174.57±0.30)mg/L,相较于未添加时提高了将近1倍。通过运用合成生物学原理在酿酒酵母中实现了对香豆酸的高产,为后续的芪类化合物和黄酮类化合物生物合成奠定了基础。     

定向进化改造酪氨酸解氨酶提高大肠杆菌合成对香豆酸产量

对香豆酸是一种具有多种药理活性的天然酚类化合物,也是多种天然药用产物生物合成的前体物质,广泛应用于食品、化妆品、医药等领域。通过微生物合成对香豆酸相对于化学合成和植物提取工艺具有节能减排等优势。但是,目前微生物合成对香豆酸产量较低,难以满足大规模工业发酵生产的要求。为了进一步提高对香豆酸产量,对粘红酵母酪氨酸解氨酶(Tyrosineammonia-lyase,TAL)进行定向进化改造,利用高通量筛选方法从随机突变体文库中筛选TAL催化活性提高的突变体。通过初筛和复筛两轮筛选,从大约10 000个突变体中获得1个TAL催化活性提高1倍的突变体。该突变体包含3个氨基酸突变位点,分别为S9Y、A11N、E518A。进一步通过单点氨基酸饱和突变验证,当S9位点突变为Y、I、N和A11位点突变为N、T、Y时,TAL的催化活性提高1倍以上。通过对S9和A11位点3种类型突变进行组合突变验证,S9Y/A11N和S9N/A11Y突变体的TAL催化活力显著高于其他组合。将S9N/A11Y突变体质粒转入酪氨酸高产菌株CP032。通过摇瓶发酵,该菌株在48 h时的对香豆酸产量达到394.2 mg/L,比对照...     

嗜热子囊菌JCM12803来源的双功能木聚糖/纤维素酶

纤维素和木聚糖的充分利用对于生物燃料的生产是非常重要的。文中利用PCR的方法从嗜热子囊菌Thermoascus crustaceus JCM12803中克隆到一个新颖的双功能木聚糖/纤维素酶基因Tcxyn10a,并将其在毕赤酵母Pichia pastoris GS115中实现高效异源表达。经过蛋白纯化和酶学性质研究分析,TcXyn10A的最适pH值和最适温度分别为5.0和65-70℃,能够在酸性至碱性(pH 3.0-11.0)条件下和60℃下保持稳定;对榉木木聚糖、小麦阿拉伯木聚糖、羧甲基纤维素钠和地衣多糖均有降解活性,比活分别为(1 480±26)U/mg、(2 055±28)U/mg、(7.4±0.2)U/mg和(10.9±0.4)U/mg;同源建模结构以及分子对接试验表明,双功能酶TcXyn10A只含有单一催化结构域,且木聚糖底物与纤维素底物共用一条催化通道。文中为探索双功能酶结构与其功能的关系提供了很好的素材。     

Systematic significance of mature embryo of bamboos

The mature embryo of seven species belonging to five genera of Indian bamboos is described. In all these the basic pattern of embryo organisation is same: the scutellar and coleoptilar bundles are not separated by an internode, the epiblast is absent, the lower portion of the scutellum and the coleorhiza are separated by a cleft and the margins of embryonic leaves overlap. The features unique to fleshy fruited bamboos are: presence of a massive scutellum, the juxtaposition of plumule and radicle and the occurrence of a bud in the axil of the coleoptile. The fleshy fruit bearing bamboos should be classified into one group, the tribeMelocanneae. Evidence is provided to recognise additional groups in the subfamilyBambusoideae.     

Taxonomic status of the species of the green algal genusNeochloris

Komárek has recently reviewed the various species assigned to the green algal genusNeochloris Starr (Chlorococcales, Chlorococcaceae) and removed those with uninucleate vegetative cells to a new genus,Ettlia. Watanabe & Floyd, unaware ofKomárek's work, also reviewed the species ofNeochloris and distributed them among three genera—Neochloris, Chlorococcopsis gen. nov., andParietochloris gen. nov.—on the basis of details of the covering of the zoospore and the arrangement of the basal bodies of the flagellar apparatus. This paper reconciles these two treatments and makes additional recommendations at the ranks of genus, family, order, and class.     

Phylogeny of cardinalfishes (Teleostei: Gobiiformes: Apogonidae) and the evolution of visceral bioluminescence

The cardinalfishes (Apogonidae) are a diverse clade of small, mostly reef-dwelling fishes, for which a variety of morphological data have not yielded a consistent phylogeny. We use DNA sequence to hypothesize phylogenetic relationships within Apogonidae and among apogonids and other acanthomorph families, to examine patterns of evolution including the distribution of a visceral bioluminescence system. In conformance with previous studies, Apogonidae is placed in a clade with Pempheridae, Kurtidae, Leiognathidae, and Gobioidei. The apogonid genus Pseudamia is recovered outside the remainder of the family, not as sister to the superficially similar genus Gymnapogon. Species sampled from the Caribbean and Western Atlantic (Phaeoptyx, Astrapogon, and some Apogon species) form a clade, as do the larger-bodied Glossamia and Cheilodipterus. Incidence of visceral bioluminescence is found scattered throughout the phylogeny, independently for each group in which it is present. Examination of the fine structure of the visceral bioluminescence system through histology shows that light organs exhibit a range of morphologies, with some composed of complex masses of tubules (Siphamia, Pempheris, Parapriacanthus) and others lacking tubules but containing chambers formed by folds of the visceral epithelium (Acropoma, Archamia, Jaydia, and Rhabdamia). Light organs in Siphamia, Acropoma, Pempheris and Parapriacanthus are distinct from but connected to the gut; those in Archamia, Jaydia, and Rhabdamia are simply portions of the intestinal tract, and are little differentiated from the surrounding tissues. The presence or absence of symbiotic luminescent bacteria does not correlate with light organ structure; the tubular light organs of Siphamia and chambered tubes of Acropoma house bacteria, those in Pempheridae and the other Apogonidae do not.     

Host plants of someRhinanthoideae (Scrophulariaceae) of eastern North America

Twenty three species in 11 genera were examined in the field to determine hosts. OnlyStriga asiatica andSeymeria cassioides have a narrow host range being restricted to grasses and pines, respectively. These are the only species which cause pronounced and sometimes serious host damage. The other species attach to a great diversity of hosts.     

Apoptotic Regulation of Caspase Activity

Intracellular cysteine aspartate-specific proteases (caspases) play both signaling and effector roles in realizing the program of cell death. Caspases function as proteolytic cascades unique for each cell type and signal triggering apoptosis. All parts of the proteolytic cascades are duplicated and controlled by feedback signals. Amplification cycles between pairs of caspases (the third and the sixth, the ninth and the third, the twelfth and the sixth, and others) help multiply the initial apoptotic signal. The presence of physiological inhibitors of apoptosis that directly interact with caspases creates a multilevel regulatory network of apoptosis in cell. The caspase proteolytic cascades are also regulated by sphingolipid secondary messengers, among them ceramide, sphingosine, and their phosphates. Moreover, an association of the caspase signaling with ubiquitin-dependent proteolysis is shown in cells. In particular, the use of extracellular activators and inhibitors of caspases allows irreversible activation of apoptosis in tumor cells or the prevention of apoptosis in cortical neurons under neurodegenerative diseases.     

A molecular phylogeny of Hebeloma species from Europe

In order to widen the scope of existing phylogenies of the ectomycorrhizal agaric genus Hebeloma a total of 53 new rDNA ITS sequences from that genus was generated, augmented by sequences retrieved from GenBank, and analysed using Bayesian, strict consensus and neighbour joining methods. The lignicolous Hebelomina neerlandica, Gymnopilus penetrans, and two species of Galerina served as outgroup taxa. Anamika indica, as well as representatives of the genera Hymenogaster and Naucoria, were included to test the monophyly of Hebeloma, which is confirmed by the results. Hebeloma, Naucoria, Hymenogaster and Anamika indica cluster in a strongly supported monophyletic hebelomatoid clade. All trees largely reflect the current infrageneric classification within Hebeloma, and divide the genus into mostly well-supported monophyletic groups surrounding H. crustuliniforme, H. velutipes, H. sacchariolens, H. sinapizans, and H. radicosum, with H. sarcophyllum being shown at an independent position; however this is not well supported. The section Indusiata divides with strong support into three groups, the position of the pleurocystidiate Hebeloma cistophilum suggests the possible existence of a third subsection within sect. Indusiata. Subsection Sacchariolentia is raised to the rank of section.     

Analysis of twin of eyeless regulation during early embryogenesis in Drosophila melanogaster

The Drosophila Pax6 homolog twin of eyeless (toy) is so far the first zygotically expressed gene involved in eye morphogenesis in Drosophila. The study of its expression during embryogenesis is therefore informative of the initial events of eye development in Drosophila. We have analyzed how the initial expression domain of toy at cellular blastoderm is regulated. We show that the three maternal patterning systems active in the cephalic region (the anterior, terminal and dorsal-ventral systems) cooperate with zygotically activated gap genes to shape the initial expression domain of toy. Whereas Bicoid, Dorsal and Torso signaling synergistically act as activators, Hunchback, Knirps and Decapentaplegic act as repressors.     

New species of Peruvian Orchidaceae III

Sixteen new species are proposed in the generaAckermania, Dressleria, Epidendrum, Maxillaria, Oncidium, Rodriguezia, Sigmatostalix, andTrigonidium. All new species are illustrated.Maxillaria vittariifolia L. O. Williams is newly recorded for Peru. A key is provided forTrigonidium of Peru.Trigonidium loretoense Schltr. andT. peruvianum Schltr. are lectotypified.     

Interaction of Synthetic Analogues of Natural Chalcones and Flavones with Guanidine

The reaction of chalcones and flavones with guanidine in ethanol results in 2-(2-aminopyrimidinyl-4)-phenols.     

Demographic characteristics of an intertidal bay goby (Lepidogobius lepidus)

Synopsis In a fourteen month study (May 1976 – June 1977) I examined the following characteristics of an intertidal bay goby (Lepidogobius lepidus) in Morro Bay, California, U.S.A.: annual and seasonal patterns of abundance, age composition and growth rates, survivorship and mortality patterns, and the reproductive cycle for female gobies. Fishes were collected with the aid of quinaldine and otoliths and ovaries removed. Age and growth rates were estimated from otolith annuli using a back calculation formula and a Brody-Bertalanffy growth curve. Mortality rates were derived using the methods of Heincke (1913), Robson & Chapman (1960), mean age, and a catch curve (Ricker 1975). A gonad index was used to describe the annual reproductive cycle. Results indicated that abundance fluctuated seasonally and that these fluctuations appeared to be caused by reproductive emigrations. Bay gobies reached an age of 7+ and a standard length of 87 mm. Growth was relatively constant (6 mm yr⁻¹) until age 5, at which point it began to decline. The mean rates of survivorship, mortality, and instantaneous mortality were 0.75, 0.25, and 0.29 respectively. Mortality rates for individual age classes ranged from 0.13 to 0.51 and increased with age. This stock appears to reproduce mainly during the winter.