代谢工程改造大肠杆菌合成D-1,2,4-丁三醇

【目的】D-1,2,4-丁三醇是一种四碳的多元醇,在军事和医药领域具有广泛的应用。为实现生物法一步转化生产D-1,2,4-丁三醇,对Escherichia coli W3100的木糖代谢途径进行改造。【方法】将来源于柄杆菌的D-木糖脱氢酶基因xylB和恶臭假单胞菌的苯甲酰甲酸脱羧酶基因mdlC克隆至E.coli W3100,得到重组菌E.coli(pEtac-mdlC-tac-xylB)。在此基础上对重组菌代谢木糖合成D-1,2,4-丁三醇的能力进行考察。【结果】在30°C下,以30 g/L D-木糖为底物,重组菌E.coli(pEtac-mdlC-tac-xylB)的D-1,2,4-丁三醇产量达到了0.9 g/L,摩尔转化率为4%。【结论】实现了D-1,2,4-丁三醇的一步法发酵生产,为国内开展相关研究奠定了坚实的基础。     

副产物途径的缺失对大肠杆菌合成D-1,2,4-丁三醇的影响

【目的】敲除副产物途径,提高重组大肠杆菌D-1,2,4-丁三醇(D-1,2,4-Butanetriol,BT)产量。【方法】利用Red重组技术敲除木糖途径xyl AB基因及2-酮-3-脱氧木糖酸代谢途径的yag E及yjh H基因,考察其对重组菌生长、BT生产及副产物积累的影响。【结果】敲除xyl AB基因后,重组菌生物量降低57%,BT产量降低20%,单位菌体产量提高84%,木糖酸积累量提高52%。yag E或yjh H基因单独缺失重组菌生物量分别提高10%和5%,BT产量提高36%和14%。基因共同缺失后重组菌生物量降低了21%,BT产量提高184%,达到2.44 g/L,单位菌体产量提高258%。而共同敲除两途径,生物量降低了72%,虽然单位菌体产量提高了约4倍,但BT产量仅提高43%。p H调控下,重组菌木糖酸积累量下降,BT产量进一步提高,最高达3.11 g/L。【结论】xyl AB基因缺失后,虽有利于提高BT途径的效率,但由于木糖无法进入PPP途径及木糖酸积累,造成生物量降低,不利于BT合成。单独敲除yag E或yjh H后BT产量略有提高,而共同敲除这两基因更为有效地调整碳流向BT合成偏转。两途径共同敲除利于BT的合成,但由于菌体量的减少,无法大量获得BT。     

代谢木糖和葡萄糖的重组酿酒酵母的构建*

为使酿酒酵母(Saccharomyces cerevisiae)YS58代谢木糖产乙醇,采用PCR方法克隆得到树干毕赤酵母(Pichia stipitis)木糖醇脱氢酶基因xyl2,并将该基因和克隆得到的休哈塔假丝酵母(Candida shehatae)缺终止子的木糖还原酶基因xyt1一起连接到酵母表达载体pYES2的强启动子GAL下,得到融合表达载体pYES2-P12。通过醋酸锂转化的方法将pY- ES2-P12转入S．Cerevisiae YS5     

新的热带假丝酵母载体-宿主系统的建立

热带假丝酵母(Candida tropicalis)是一种可以利用多种非糖碳源代谢的微生物,在石油发酵、石油化工生产领域已得到长期应用^[1]。随着分子生物学研究技术的发展。通过代谢工程技术改变热带假丝酵母的代谢途径和流向．发展出能够把石油中的烃类物质发酵转化成各种重要化工原料、中间体的新型生产菌株,已普遍受到国内外研究机构的重视。另一方面,热带假丝酵母具有细胞生长密度高,分泌蛋白能力强等特点,可以发展成一个重要的异源蛋白表达体系。建立稳定高效的热带假丝酵母载体一宿主系统是实现上述构想的前提和关键。迄今为止,在热带假丝酵母细胞内尚未发现能游离于染色体外自主复制的天然质粒存在。目前已有20余种热带假丝酵母基因被克隆和鉴定。大部分与热带假丝酵母的氧化代谢过程有关,其中5个过氧物酶体蛋白基因的结构和2个细胞色素P450系统基因的表达调控规律巳被阐明^[2-5],与DNA复制过程有关的基因或功能序列均未见报道。本文研究和探索Candida属其他微生物基因元件在热带假酵母中的功能作用,选用热带假丝酵母细胞色素P450单加氧酶基因的启动子和侧翼调控序列^[3],构建了一套新的热带假丝酵母载体一宿主系统,并成功地表达了小鼠CYPlAl基因。     

代谢改造克雷伯氏菌合成D-1,2,4-丁三醇

【背景】D-1,2,4-丁三醇(D-1,2,4-butanetriol,BT)是一种重要的四碳多元醇,应用范围广,以木糖为底物的四步生化反应是目前最高效的BT生物合成路线。但大肠杆菌宿主存在严重的碳代谢抑制,限制了工程菌在木糖葡萄糖混合糖下的生长和BT合成。然而克雷伯氏菌具有生长速度更快、葡萄糖木糖混合糖利用效果好等优点。【目的】在碳代谢抑制效应较弱的克雷伯氏菌中构建以木糖为底物的BT合成途径,以提高混合糖下BT合成能力。【方法】将来源于Clostridium crescenti的木糖脱氢酶基因xdh和来源于Lactococcus lactis的2-酮异戊酸脱羧酶基因kivD及来源于Escherichia coli W3110的木糖酸脱水酶基因yjhG克隆至KlebsiellapneumoniaeZG25,得到重组菌K.pneumoniae ZG25-BT,对重组菌进行培养条件和培养基优化,进一步敲除xylA以提高BT产量。【结果】在37°C、200 r/min、接种量1%、诱导时间2 h、添加10.0 g/L CaCO3控制pH条件下,敲除xylA的重组菌在1.5倍LB培养基中以30.0 g/L木糖和10.0 g/L葡萄糖为底物,BT的产量达到4.52 g/L,摩尔转化率为0.21mol/mol,收率为15%,较优化前分别提高150%、62%和67%。【结论】实现了BT在K.pneumoniaeZG25中的发酵生产,同时通过培养条件和培养基的优化及xylA的敲除提高了BT合成能力,为进一步实验奠定了基础。     

异源表达木糖异构酶基因对克雷伯氏菌合成1,3-丙二醇的影响

【目的】提高克雷伯氏菌胞内还原力以强化1,3-丙二醇合成。【方法】将来源于大肠杆菌的木糖异构酶基因在克雷伯氏菌中异源表达,构建重组菌。研究重组菌添加不同浓度木糖为辅底物与甘油共发酵过程中代谢产物和NADH的变化规律。【结果】与对照菌相比,重组菌细胞内还原力NADH提高了0.1?0.3倍,1,3-丙二醇产量达到23.31 g/L,提高20%,1,3-丙二醇转化率从0.60 mol/mol提高到0.73 mol/mol。【结论】木糖异构酶基因的表达强化了木糖代谢途径,经磷酸戊糖途径积累大量还原力,促进了1,3-丙二醇的生成。     

木糖醇脱氢酶基因高拷贝表达载体构建及在酿酒酵母中的表达

木糖醇脱氢酶（xylitol dehydrogenase, XDH）可以氧化木糖醇生成木酮糖,处于木糖代谢的节点位置。利用PCR方法克隆得到了休哈塔假丝酵母（Candida shehatae） 20335的木糖醇脱氢酶基因、质粒pKT0150的ADH1终止子序列和G418抗性基因（KanR）,以及酿酒酵母(Saccharomyces cerevisiae) W5特定的2.2 kb的rDNA片段。以酿酒酵母整合载体p406ADH1为骨架,利用基因工程手段构建一个多拷贝整合表达载体pLX-AGRX。将重组载体pLX-AGRX线性化转入到酿酒酵母W5后,通过高浓度G418筛选和PCR双重鉴定,证实重组载体pLX-AGRX已整合到酿酒酵母W5基因组上,测定木糖醇脱氢酶酶活可达65.957 4 U/mg。     

木酮糖激酶表达水平对酿酒酵母木糖代谢产物流向的影响

在酿酒酵母中分别引入真菌和细菌的木糖代谢关键酶,木糖还原酶基因XYL1、木糖醇脱氢酶基因XYL2和木糖异构酶基因xylA. 并在此基础上以共转化策略超表达下游关键酶木酮糖激酶基因XKS1. 与亲本菌株相比,用pMA91和YEp24质粒表达XKS1的重组菌株,木酮糖激酶（xylulokinase,XK）活性分别提高了14和6.7倍. 在限氧条件下,重组菌株对木糖和葡萄糖的共发酵结果显示,表达XYL1,XYL2以及XKS1的重组菌株HSXY-251木糖消耗为12.4 g/L,提高了120.9%,乙醇产量达到9.4 g/L,提高了36%,副产物木糖醇产量为0.7 g/L,下降了84.9%.     

在无天然游离质粒产甘油假丝酵母中非整合表达合成咖啡酸

【背景】咖啡酸（3,4-二羟基肉桂酸）是一种有多种生物活性和药用价值的天然酚类化合物,产甘油假丝酵母（Candida glycerinogenes）具有咖啡酸前体代谢途径,高耐酸且生长与发酵速率快,是潜在高产咖啡酸的底盘细胞,但无游离载体将影响咖啡酸合成的深入研究。【目的】探索在无天然游离质粒的C. glycerinogenes中构建操作更简便、表达能力更强的游离载体合成咖啡酸的可行性。【方法】筛选自主复制序列（autonomously replicating sequence,ARS）,构建适用于C. glycerinogenes合成咖啡酸的游离载体,并通过改造其ARS位置、标记基因URA5启动子长度、基因表达元件和利用Kozak序列优化表达并合成咖啡酸。【结果】构建的5个分别含不同ARS的载体中,pTGAPU-CA-AOX1t-KLARS在C. glycerinogenes中能自我复制并表达合成咖啡酸的基因,而且当ARS位于目的基因表达元件上游、URA5启动子截短250 bp,或分别采用Kozak序列与终止子URA5t后,咖啡酸产量较改造前均有明显提升,最高产量为初始产量的3.73倍,达29.1 mg/L,高于前期整合表达产量。【结论】在C. glycerinogenes中非整合表达合成咖啡酸且优于整合表达,为今后利用游离载体改造咖啡酸合成代谢途径提供了新工具,同时为其他无游离质粒菌株构建非整合表达体系提供参考。     

大肠杆菌合成1,2,4-丁三醇的途径优化

1,2,4-丁三醇(BT)是一种在工业中有多种用途的重要的非天然化合物。文中通过将外源基因xdh和mdlC导入大肠杆菌BW25113表达,并敲除了xylA、xylB、yagE、yjhH、yiaE和ycdW等木糖和中间产物代谢旁路基因,构建了能够将D-木糖转化为BT的重组菌株。为优化BT合成途径,针对BT合成途径中的限速步骤——3-脱氧-D-甘油-戊酮糖酸的脱羧反应,进行了新酶的筛选和评价,获得了可显著提高反应效率的新的2-酮酸脱羧酶——KivD,并构建了表达该酶的重组菌株BW-025。在此基础上,通过初步条件优化,将BT产量提高至2.38g/L;进一步调节途径中各个酶的表达量,探究了它们对BW-025合成BT的影响,最终获得了BT产量较BW-025提高了48.62%的重组菌株BW-074。     

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.