工业分枝杆菌甾醇代谢基因组学的研究进展

甾类化合物具有重要的生理医药作用,市场需求巨大。分枝杆菌对降解甾类中关键甾类药物中间体有很高的转化效率。为了更深入地了解甾体降解途径,本文从植物甾醇结构种类、分枝杆菌特点、不同分枝杆菌菌株基因组学、代谢组等方面的研究进展进行了归纳。     

高效转化植物甾醇为9α-OH-AD的分枝杆菌诱变选育及工艺优化

以实验室前期筛选得到的分枝杆菌(Mycobacterium sp.LY-1)为出发菌株,采用等离子诱变(ARTP)技术选育出能高效转化植物甾醇为重要甾体药物中间体9α-羟基雄甾-4-烯-3,17-二酮(9α-OH-AD)的菌株,并对其转化工艺进行优化。经过ARTP诱变选育得到遗传稳定性较好的分枝杆菌突变株Mycobacterium sp.C33,当底物植物甾醇投料浓度为15 g/L时,转化生成9α-OH-AD的摩尔得率达到15.5%,较原始菌株提高34.8%。继而采用正交实验设计方法,对突变菌的发酵培养基组分进行优化,并建立了油水双相转化体系,进一步提高了突变菌株C33的产物摩尔得率,最高达到47.0%,较优化前(15.5%)提高了2倍。     

植物甾醇酯合成研究进展

植物甾醇广泛存在于植物的根、茎、叶、果实和种子中,易受光、热、氧化等作用而被分解,具有生物利用率低、水溶性和脂溶性较差等特点。植物甾醇与脂肪酸酯化生成的植物甾醇酯可以有效降低植物甾醇的氧化率,具有更好的脂溶性和降血清、胆固醇的能力,更易被人体吸收。作为一类新型的功能食品添加剂,植物甾醇酯的市场需求量逐年增长。对植物甾醇酯的原料植物甾醇和脂肪酸来源进行了简单介绍,就近年来国内外植物甾醇酯的合成方法进行了系统阐述,重点介绍了酸/碱化学催化法和脂肪酶催化合成方法,对这两类方法的优缺点分别进行了评价,并对未来研究的重点内容和待突破的核心技术进行了展望。     

植物甾醇与心血管疾病关系的研究进展

对植物甾醇与心血管疾病关系的研究进展及其应用现状进行综述,以期为植物甾醇对心血管疾病的防治提供科学依据。     

植物甾醇保护胃粘膜及抗消化道肿瘤的研究进展

天然产物是新药发现的重要来源,具有多途径、多层次、多靶点和多种生物活性的特点。植物甾醇作为其中一类重要的甾体类化合物,种类繁多,分布广泛,药理活性显著,是一种多功能活性因子,具有保护胃黏膜、抗炎、抗癌、降低胆固醇、抗氧化等功效。胃黏膜损伤是世界范围内的常见病和多发病,几乎所有人在一生中都罹患此疾病,寻找具有保护胃黏膜功能的生物活性物质,已成为人类关注的热点话题之一。本研究概述植物甾醇的理化性质、药理活性及其安全性,并阐述植物甾醇保护胃粘膜及抗消化道肿瘤的活性,为植物甾醇功能性食品、药品地进一步开发提供理论依据。     

植物中甾醇糖基转移酶的研究进展

甾醇(sterol)是植物细胞膜结构和天然植物激素的重要组成成分。甾醇糖基转移酶(sterol glycosyltransferases,SGTs)作为糖基转移酶一号家族(GT family 1)较为保守的一支,是一类参与甾醇下游修饰的酶,具有调控植物初期生长发育、信号转导、次生代谢产物合成以及响应生物、非生物胁迫等生物学功能。本文主要综述了SGTs在植物生长调控、生物合成、早期发育研究的进展,最后讨论了甾醇糖基转移酶在工业生产药用活性分子方面的前景和主要限制,旨在为更深入开展甾醇糖基转移酶的研究和应用提供参考。     

植物天冬氨酸代谢途径关键酶基因研究进展

谷类作物和豆类作物的营养价值主要体现在以天冬氨酸为前体的必需氨基酸的含量上。植物中这些氨基酸的含量受天冬氨酸代谢途径中关键酶基因的影响。本文综述了这些酶基因的克隆、在大肠杆菌和不同植物中表达的研究进展。     

植物甾醇在植物逆境胁迫中的研究进展

植物甾醇是一类重要的生理活性物质,对植物的生长发育具有重要作用,对响应植物逆境胁迫也具有重要功能.植物甾醇是细胞膜和脂质筏的重要组分,与膜的稳定性密切相关,主要通过甾醇含量的相对变化维持膜的稳定性及影响脂质筏的生物功能响应逆境胁迫.植物甾醇作为信号分子参与逆境胁迫中的信号传导,油菜素内酯类(BRs)是植物甾醇合成途径的重要产物,作为一种重要的信号分子调控植物甾醇合成酶基因的表达以响应逆境胁迫.     

基于转录组测序的铁皮石斛植物甾醇生物合成相关基因分析

为了解铁皮石斛(Dendrobium officinale)植物甾醇的生物合成途径,利用Illumina HiSeq 4000高通量测序技术对茎和叶进行转录组测序,比较植物甾醇生物合成关键酶基因的表达。结果表明,共获得43 085条Unigenes,其中24 459条在Nr、Swiss-prot、KOG和KEGG数据库获得注释,7 333条获得共同注释。KEGG代谢途径分析表明,铁皮石斛植物甾醇生物合成分为3个阶段,共有50个Unigenes (30种酶)参与。表达分析表明,DXR和HMED的表达量明显高于MK和MVD;成熟期茎、叶SMT1的表达量比生长期高,SMT2则生长期高于成熟期;同一时期,SMT1和SMT2在叶的表达量都比茎高。这为铁皮石斛植物甾醇的开发利用和调控植物甾醇生物合成研究提供了科学依据。     

甾醇在调节植物生长发育中的研究进展

甾醇是一种异戊二烯类化合物,在生物的生长发育中起着重要作用。甾醇不仅是真核细胞膜的结构成分,而且也是甾醇激素生物合成的前体,在植物细胞分裂、胚胎发生和发育、参与逆境胁迫中起着关键作用。在植物中,甾醇衍生的油菜素内酯(brassinosteroids,BRs)在生长发育中的多种功能已被广泛研究,BRs作为一类植物激素,协同其他激素在植物生长发育中发挥多种功能,从细胞分裂、细胞扩张、气孔导度和根系发育,BRs在植物生命周期的各个方面都发挥着重要的作用。除了这些功能外,BRs作为植物甾醇合成途径的重要产物,作为一种重要的信号分子响应逆境胁迫及调控植物的形态建成。本文对BRs合成途径中的相关基因的研究进展进行了概述,并且综述了油菜素内酯的生物合成及其在调节植物生长发育中的研究进展,最后对油菜素内酯的研究前景进行了讨论和展望。     

结核杆菌的药物外排泵

外排泵基因过度表达促使细胞内的药物转移到细胞外,胞内药物浓度下降,致使药物临床疗效降低或无效。外排泵上有多个与识别底物结构无关的阴离子凹穴(底物结合位点),通过静电引力非特异性结合各种含阳离子的药物,在能量协助下将药物排到细胞外,进而导致细菌抗药性的产生。研究表明,结核杆菌基因组中存在外排泵基因,但仅分离到几个有功效的外排泵。本文就相关研究进展进行综述。     

Structural studies of shikimate 5-dehydrogenase from Mycobacterium tuberculosis

Tuberculosis (TB) remains the leading cause of mortality due to a single bacterial pathogen, Mycobacterium tuberculosis. The reemergence of TB as a potential public health threat, the high susceptibility of human immunodeficiency virus-infected persons to the disease, the proliferation of multi-drug-resistant strains (MDR-TB) and, more recently, of extensively drug resistant isolates (XDR-TB) have created a need for the development of new antimycobacterial agents. Amongst the several proteins and/or enzymes to be studied as potential targets to develop novel drugs against M. tuberculosis, the enzymes of the shikimate pathway are attractive targets because they are essential in algae, higher plants, bacteria, and fungi, but absent from mammals. The mycobacterial shikimate pathway leads to the biosynthesis of chorismate, which is a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Here we report the structural studies by homology modeling and circular dichroism spectroscopy of the shikimate dehydrogenase from M. tuberculosis (MtSDH), which catalyses the fourth step of the shikimate pathway. Our structural models show that the MtSDH has similar structure to other shikimate dehydrogenase structures previously reported either in presence or absence of NADP, despite the low amino acid sequence identity. The circular dichroism spectra corroborate the secondary structure content observed in the MtSDH models developed. The enzyme was stable up to 50 degrees C presenting a cooperative unfolding profile with the midpoint of the unfolding temperature value of approximately 63-64 degrees C, as observed in the unfolding experiment followed by circular dichroism. Our MtSDH structural models and circular dichroism data showed small conformational changes induced by NADP binding. We hope that the data presented here will assist the rational design of antitubercular agents.     

花粉管通道法在植物遗传转化中的应用

本文综述了花粉管通道法的创立与发展,应用此方法所取得的最新研究成果,对遗传转化中所涉及的机理进行了探讨,最后比较了该方法较其它常用的转化法的优势与局限性。     

Superoxide dismutase from Mycobacterium species,strain Takeo

Superoxide dismutase from Mycobacterium species, strain Takeo, has been purified to homogeneity as judged by disc gel electrophoresis and ultracentrifugation. The enzyme was found to have a molecular weight of approximately 61 500 by sedimentation equilibrium and to contain manganese by atomic absorption and electron spin resonance spectra. The amino acid composition was also determined. The enzyme was considerably stable to the treatment with sodium dodecyl sulfate; unless incubating at 80°C for 2 min, it was not completely dissociated into the subunits. The molecular weight of the subunit was found to be approximately 21 000. Antibodies against the superoxide dismutase were produced by immunization of rabbits with the enzyme, and the -globulin fraction was purified. Superoxide dismutase preparations obtained from various species of mycobacteria and nocardia cross-reacted to different degrees with these antibodies on the Ouchterlony double diffusion plates. Comparative immunological studies indicated that strain Takeo might be most closely related to Myobacterium smegmatis among species of mycobacteria and nocardia tested. The antibodies against superoxide dismutase may be used as a valuable tool for the classification of mycobacteria.     

恩拉霉素生产菌株的遗传改造

【目的】提高杀真菌素链霉菌发酵生产恩拉霉素的产量。【方法】利用定点突变技术,对恩拉霉素生产菌株杀真菌素链霉菌F1中影响细胞次级代谢及抗生素合成的核糖体S12蛋白的编码基因rps L进行改造,将第43位的赖氨酸(Lys)分别替换为天冬酰胺(Asn)和精氨酸(Arg),并对改造菌株L-M1(Asn43)和L-M2(Arg43)的生长特性、抗生素合成以及摇瓶发酵性能进行研究。【结果】与野生型菌株相比,改造菌株的生长特性及生理生化特性均发生了明显的改变:产孢周期明显缩短,野生型菌株在MS培养基中,28°C下需要培养5-7 d后才能产生孢子,而在相同条件下,改造菌株3 d后就能产生大量的孢子;恩拉霉素产量相对提高,摇瓶发酵条件下,改造菌株L-M1(Asn43)和L-M2(Arg43)的恩拉霉素产量分别可达到1 334 U/m L和1 456 U/m L,与野生型菌株F1相比分别提高了11.9%和22.1%。【结论】通过遗传改造,恩拉霉素的产量得到了提高,为其他位点的遗传改造提供了可行性。     

Metabolic characterization and transformation of the non‐dairy Lactococcus lactis strain KF147, for production of ethanol from xylose

The non‐dairy lactic acid bacterium Lactococcus lactis KF147 can utilize xylose as the sole energy source. To assess whether KF147 could serve as a platform organism for converting second generation sugars into useful chemicals, the authors characterized growth and product formation for KF147 when grown on xylose. In a defined medium KF147 was found to co‐metabolize xylose and arginine, resulting in bi‐phasic growth. Especially at low xylose concentrations, arginine significantly improved growth rate. To facilitate further studies of the xylose metabolism, the authors eliminated arginine catabolism by deleting the arcA gene encoding the arginine deiminase. The fermentation product profile suggested two routes for xylose degradation, the phosphoketolase pathway and the pentose phosphate pathway. Inactivation of the phosphoketolase pathway redirected the entire flux through the pentose phosphate pathway whereas over‐expression of phosphoketolase increased the flux through the phosphoketolase pathway. In general, significant amounts of the mixed‐acid products, including lactate, formate, acetate and ethanol, were formed irrespective of xylose concentrations. To demonstrate the potential of KF147 for converting xylose into useful chemicals the authors chose to redirect metabolism towards ethanol production. A synthetic promoter library was used to drive the expression of codon‐optimized versions of the Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase, and the outcome was a strain producing ethanol as the sole fermentation product with a high yield corresponding to 83% of the theoretical maximum. The results clearly indicate the great potential of using the more metabolically diverse non‐dairy L. lactis strains for bio‐production based on xylose containing feedstocks.     

Degradation of isooctane by Mycobacterium austroafricanum IFP 2173: growth and catabolic pathway

AIMS: Isooctane (2,2,4-trimethylpentane), a major component of gasoline formulations, is recalcitrant to biodegradation probably because of the quaternary carbon group it contains. Information on the biodegradability of this hydrocarbon is essential to evaluate its fate in the environment. For these reasons, the degradation kinetics and the catabolic pathway of isooctane were investigated in Mycobacterium austroafricanum IFP 2173, the only strain characterized to use it as sole carbon and energy source. METHODS AND RESULTS: The selected strain exhibited a rather moderate maximum growth rate (micromax = 0.053 h(-1)) but degraded isooctane up to 99% with a mineralization yield of 45%, indicating attack of the quaternary carbon group. The GC/MS identification of metabolites, 2,4,4-trimethylpentanoic and dimethylpropanoic (pivalic) acids, which transiently accumulated in the cultures indicated that degradation started from the isopropyl extremity of the molecule and subsequently proceeded by catabolism of the tert-butyl moiety. The degradation of putative metabolic intermediates was investigated. The initial isooctane oxidation system was tentatively characterized. CONCLUSIONS: The isooctane-degrading strain harboured two candidate systems for initial alkane oxidation. Although a cytochrome P450 was induced by isooctane degradation, the functional oxidation system was probably a nonheme alkane monooxygenase as indicated by PCR amplification and RT-PCR expression of an alkB gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Isooctane is a recalcitrant branched alkane. A plausible pathway of its degradation by Myco. austroafricanum was put forward.     

线性泛素化修饰研究进展

蛋白质泛素化修饰过程在调节各种细胞生物学功能的过程中发挥了非常重要的作用,如细胞周期进程、DNA损伤修复、信号转导和各种蛋白质膜定位等。泛素化修饰可分为多聚泛素化修饰和单泛素化修饰。多聚泛素化修饰系统可以通过对底物连接不同类型的多泛素化链调节蛋白质的功能。多聚泛素化修饰中已知7种泛素链连接方式均为泛素内赖氨酸连接方式。近几年发现了第8种类型的泛素链连接形式即线性泛素化,其泛素链的连接方式是由泛素甲硫氨酸的氨基基团与另一泛素甘氨酸的羧基基团相连形成泛素链标记。目前研究表明线性泛素化修饰在先天性免疫和炎症反应等多个过程中发挥着非常重要的作用。募集线性泛素链的泛素连接酶E3被称为LUBAC复合体,其组成底物以及其活性调控机制和功能所知甚少。本文综述了募集线性泛素化链的泛素连接酶、去泛素化酶、底物等活性调控机制及其在先天性免疫等多个领域中的功能,分析了后续研究方向,以期为相关研究提供参考。     

白三叶转基因及其生态适应性研究进展

牧草基因工程是近年来国内国际研究的热点之一 ,白三叶作为温带地区优良的豆科牧草 ,是人工草地建植的首选草种 ,也是各类观赏性草坪和绿地的主要组分。但白三叶的生态幅较窄 ,适宜在温暖 (适宜生长温度为 19～ 2 4℃ )湿润、年降雨量 70 0～10 0 0 mm的地区生长 ,对土壤酸碱度的要求比较严 ,适宜的土壤 p H为 6～ 7,耐酸性和耐盐碱能力都比较差 ,从而限制了其在许多地区的应用。利用转基因手段提高其抗逆性和生态适应性 ,培育抗性强、适应性广的白三叶新品种 ,对我国尤其是北方畜牧业、草坪业和生态建设都具有重要意义。在提高其对生物胁迫和非生物胁迫的抗性和生态适应性方面 ,白三叶的转基因研究已经取得了较大进展。结合国内外白三叶基因工程及生态适应性研究动态 ,系统介绍了白三叶转基因研究的主要内容和方法 ,在对已有成果进行综合分析的基础上 ,就目前白三叶转基因和生态适应性研究中亟待解决的一些问题进行了探讨。