大肠杆菌代谢途径改造策略与应用研究进展

大肠杆菌由于具有稳定性强和易于操作的特点成为基因改造常用的宿主微生物。利用基因工程手段改造大肠杆菌的代谢途径,可用于生物燃料、手性药物及其衍生物的合成。利用代谢组学和合成生物学能够高效地以大肠杆菌作为生物催化剂生产目标物。综述了大肠杆菌中丙酮酸、乙酰辅酶A、甲羟戊酸和莽草酸代谢途径的改造策略,以及大肠杆菌代谢途径的改造在合成生物燃料、砌块化合物中的应用,为研究者以大肠杆菌合成目标化合物的研究提供一个整体的思路和方法。     

代谢工程改造大肠杆菌生产L-高丝氨酸

L-高丝氨酸是一种非必需氨基酸,在工业生产中常被用作重要的平台化合物和添加剂.为提高产物合成效率,本文以实验室构建的L-高丝氨酸高产工程菌大肠杆菌Escherichia coli H0-0作为底盘菌株进行代谢改造.首先对ppc和pyccgP458S基因进行过表达来优化柠檬酸循环,然后通过thrAC1034T和lysCc...     

基于代谢组学分析的O-琥珀酰-L-高丝氨酸发酵调控

【目的】O-琥珀酰-L-高丝氨酸(O-succinyL-L-homoserine, OSH)是合成L-蛋氨酸、L-草铵膦等重要前体，在医药、农药、食品等领域具有重要的应用前景，其绿色高效制造受到广泛关注。本研究通过解析OSH发酵过程代谢途径和代谢产物变化规律，建立OSH发酵调控策略，提升其产量和糖酸转化率。【方法】运用代谢组学技术，系统考察OSH生产菌在发酵不同时间段的代谢物变化情况，探究与OSH合成显著关联的代谢途径，通过在不同时间外源添加关键代谢物，平衡关键代谢物及其前体通量，减少旁路途径对前体的竞争性利用。【结果】在5 L发酵罐中产量达70.1 g/L，糖酸转化率达0.52 g/g (葡萄糖)。【结论】研究结果表明，基于代谢组学分析技术的OSH发酵体系优化和发酵过程调控显著提升了目标产物生产效率，奠定了OSH的产业化基础。     

假单胞菌株M18gacA基因对BHL和HHL合成正调控及对PCA合成负调控无相关性

经初步鉴定,假单胞菌株(Pseudomonassp.)M18至少能产生5种N-酰基高丝氨酸内酯类(N-acyl-homoserinelactones,AHLs)信号分子,它们是:N-丁酰高丝氨酸内酯(N-butyryl-L-homoserine lactone,C4-HSL,BHL)、N-己酰高丝氨酸内酯(N-hexanoyl-L-homoserine lactone,C6-HSL,HHL)、N-3-氧-己酰高丝氨酸内酯[N-(3-oxohexanoyl)-L-homoserinelactone,3-Oxo-C6-HSL,OHHL]、N-3-氧-辛酰高丝氨酸内酯[N-(3-oxooctanoyl)-L-homoserine lactone,3-Oxo-C8-HSL,OOHL]和N-3-氧-癸酰高丝氨酸内酯[N-(3-oxodecanoyl)-L-homoserine lactone,3-Oxo-C10-HSL,ODHL)。在gacA突变菌株M18G中,信号分子的积累量明显减少,且只能检测出其中的4种;同时,吩嗪-1-羧酸(Phenazine-1-carboxylic acid,PCA)的合成量比野生株M18提高了2倍左右。在M18菌株中,基因rhlⅠ的编码产物参与BHL和HHL的合成。构建rhlI’-’lacZ翻译融合表达质粒pMEIZ,分别导入野生株M18和突变株M18G,突变株M18G的半乳糖苷酶活性比野生株M18下降约40%,表明GacA对基因rhlI的表达具有正调控作用。但是,在野生株M18和突变株M18G的发酵液中,分别或同时添加过量的外源BHL和HHL,对PCA合成的影响不显著,表明在突变株M18G中,PCA合成量的增加与BHL和HHL合成量的减少没有明显的相关性。     

N-琥珀酰低聚壳聚糖的抗氧化性能研究(英文)

低聚壳聚糖经化学改性得到三种取代度不同的N-琥珀酰低聚壳聚糖(NSCOSA、NSCOSB和NSCOSC,取代度(DS)分别是为0.25、0.57和0.70。对其结构进行红外表征,并且并考察了其对超氧阴离子自由基(O-·2)、羟基自由基(·OH)、DPPH的清除活性以及还原能力。结果表明:N-琥珀酰低聚壳聚糖衍生物的抗氧化活性与低聚壳聚糖相比有所降低。随着DS的升高,琥珀酰衍生物对·OH的清除能力下降;对O-·2的清除能力大小顺序为NSCBNSCANSCC;对DPPH的清除能力以及还原能力大小顺序均为:NSCANSCCNSCB。这可能是由于N-琥珀酰低聚壳聚糖的取代度、引进基团的性质以及对自由基的清除机理不同所致。     

高效表达N-乙酰高丝氨酸内酯酶-木聚糖酶融合蛋白及其酶学性质

摘要: 【目的】构建N-乙酰高丝氨酸内酯酶-木聚糖酶双酶活性表达毕氏酵母重组菌株,并对经纯化的重组蛋白SL2B 进行N-乙酰高丝氨酸内酯酶及木聚糖酶酶学性质的研究。【方法】利用PCR 拼接技术得到N-乙酰高丝氨酸内酯酶基因aiiA-B546 和木聚糖酶基因xynAS27cd 融合而成的基因Sl2b。构建重组表达载体pPIC9 / Sl2b 转化毕氏酵母,筛选得到同时具有木聚糖酶和N-乙酰高丝氨酸内酯酶活性的重组子,随后对经硫酸铵沉淀、分子筛纯化后得到的重组蛋白SL2B 进行N-乙酰高丝氨酸内酯酶及木聚糖酶     

大肠杆菌O11 O-抗原基因簇序列的破译及特异分子标识的鉴定

大肠杆菌O11是一种可在人畜间交叉传染的强致病菌,具有潜在流行性爆发的危险。现完成了O11 O-抗原基因簇的破译,筛选和鉴定了多种特异分子标识,并实现了对大肠杆菌O11的快速、灵敏和准确的分子分型检测。利用鸟枪法测定大肠杆菌O11 O-抗原基因簇的序列全长为14180bp,生物信息学方法分析序列结构,共发现12个基因:GDP-L型岩藻糖合成途径基因(gmd,fcl,gmm,manC,manB)、UDP-N乙酰葡萄糖C4异构酶基因(gne)、O-抗原转运酶基因(wzx)、O-抗原聚合酶基因(wzy)和4个糖基转移酶基因;用PCR方法筛选出2个针对大肠杆菌O11的特异基因和4对特异引物,并进行环境样品检测实验鉴定了该PCR检测方法的灵敏度;设计并筛选出8条针对大肠杆菌O11的特异探针。     

植物蛋白质O-糖基化修饰的研究进展

糖基化是最主要的蛋白质翻译后修饰方式之一,主要有N-糖基化、O-糖基化和糖基磷脂酰肌醇锚定修饰三种类型。在植物细胞中, O-糖基化修饰广泛发生,它不仅参与蛋白质转录调节、信号转导,还与细胞壁合成等生物学过程紧密相关。在多种O-糖基化修饰类型中, O-N-乙酰氨基葡萄糖(O-GlcNAc)糖基化修饰结构独特、易于检测和表征,因此已经有许多相关技术实现了对其的表征。然而,其他类型O-糖基化修饰蛋白的结构和功能仍有待更全面的研究。该文综述了植物蛋白中不同类型O-糖基化修饰的相关研究进展,总结了植物O-糖基化修饰蛋白检测技术的优缺点,最后展望了这些技术在植物蛋白质O-糖基化修饰研究中的应用前景。     

基于组学技术的维生素C二步混菌发酵研究进展

二步发酵法是工业化生产维生素C (vitamin C, Vc)的主要方法,其中第二步由伴生菌与产酸菌(普通生酮基古龙酸菌)组合进行混菌发酵产生Vc前体2-酮基-l-古龙酸(2-keto-l-gluonic acid, 2-KLG)的机制,一直是科研人员研究的重要科学问题。通过高通量基因组学、转录组学、蛋白组学、代谢组学等组学技术揭示生物系统中各个组分相互作用关系已经成为主要的研究手段。本文对近年来利用组学技术解析Vc混菌发酵中两菌互作关系、解除发酵系统的氧化胁迫、伴生活性物质、产酸菌群体感应、外源添加物、基因工程改造产酸菌促进产2-KLG等方面的研究进行综述,并为进一步的探索和深入研究提供思路。     

生物酶法合成L-精氨酸衍生物的研究进展

L-精氨酸是一种碱性氨基酸,具有多样化的官能团,是合成多种有用化合物的前体,其衍生物广泛应用于医疗、食品和化妆品等领域。L-精氨酸衍生物的合成方法有化学法、发酵法和酶法。在当前绿色经济和可持续发展的背景下,对比各种生产方法,生物酶法合成L-精氨酸衍生物具有明显优势。因此本文重点介绍了L-精氨酸衍生化的典型产品和合成技术,并介绍了生物酶法合成L-精氨酸衍生物未来可能的发展方向。     

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.