酿酒酵母对数生长后期代谢重构的全基因组表达谱芯片分析

为了探讨酵母进入对数生长后期以后酒精生产速度降低的原因, 我们利用酵母表达谱芯片技术对酿酒酵母细胞从对数生长中期进入对数生长后期时的全基因组表达谱进行了分析, 发现酵母在对数生长中期的表达谱非常稳定, 而一旦进入对数生长后期, 则出现明显的代谢重构现象。许多氨基酸合成和代谢相关的基因、离子转移以及与能量的生成和储存等功能相关的基因出现了不同程度的上调; 而许多涉及酵母转座和DNA重组的基因则表达下调; 一些中心代谢途径也发生了代谢重构, 包括: 琥珀酸和a-酮戊二酸生成途径基因的一致上调, 都与氨基酸合成和代谢相关基因表达的结果相吻合。结果表明: 由于氨基酸合成的需求量增加, 进入对数生长后期酵母的代谢转向TCA循环和乙醛酸循环, 导致酒精的生产速率降低。     

低产高级醇酿酒酵母突变菌株的差异蛋白组分析及

【目的】高级醇是白酒微量香气成分中的重要组成部分,但是高级醇含量过高会对酒的品质产生不利影响,而白酒中的高级醇主要是在酒精发酵过程由酵母产生的,因此酿酒酵母高级醇合成相关蛋白的研究对于控制高级醇产生具有重要意义。【方法】以诱变得到的低产高级醇酿酒酵母菌株ARTP5和原始酿酒酵母菌株CF4为研究对象,比较两株酵母的胞内蛋白组差异,寻找高级醇合成相关蛋白。【结果】与原始菌株CF4相比,诱变菌株ARTP5高级醇产量降低了20%,有45个胞内蛋白表达量差异2倍以上,通过MALDITOF-MS质谱鉴定出29个,主要包括碳源和能量代谢、胁迫反应过程、蛋白翻译和折叠过程、氨基酸代谢和高级醇代谢等途径的蛋白,其中ARTP5菌株表达上调的支链氨基酸代谢合成途径的ILV5蛋白和表达下调的高级醇合成途径中的ADH1蛋白与诱变菌株ARTP5高级醇降低具有一定相关性。【结论】与高级醇合成具有一定相关性蛋白的发现对于酿酒酵母酒精发酵过程高级醇的合成机制的解析以及白酒酿造过程高级醇产量的控制有重要意义。     

碱性条件下苏云金芽胞杆菌基础代谢分析

【目的】探索苏云金芽胞杆菌(Bacillus thuringiensis)形成转录差异的碱性条件,明确B.thuringiensis在该条件下的基础代谢途径变化。【方法】采用半定量RT-PCR技术及实时荧光定量PCR技术,确定碱刺激下参考基因psp A存在表达差异的碱性处理条件。在该条件下提取RNA进行Agilent定制B.thuringiensis表达谱芯片杂交,对芯片数据进行差异表达分析、GO富集分析及生物途径富集分析等。【结果】通过检测psp A表达变化,将对数生长中期的菌体加入终浓度为28 mmol/L的Na OH并诱导培养10min,作为B.thuringiensis响应碱刺激的研究条件。富集分析表明碳代谢、脂肪酸合成代谢、氨基酸合成代谢途径变化明显。细胞糖酵解途径至少19个酶促基因上调表达,三羧酸循环中催化α-酮戊二酸转化为苹果酸的大部分酶蛋白编码基因上调2倍以上。【结论】本研究发现在碱性条件下B.thuringiensis基础代谢明显增强,细胞可能通过大量合成酸性物质如乳酸、苹果酸等来提高细胞对于碱性环境的适应能力。     

基于转录组分析的金针菇冷诱导原基形成的调控网络

金针菇是低温结实型菌类,原基形成需要低温诱导,子实体发育也需要较低的温度,因此在工厂化生产过程中能源消耗大,生产成本高。本研究利用转录组测序对金针菇菌丝体和原基进行RNA-Seq分析,筛选得到7 935个差异表达基因,在原基形成后,有4 025个基因上调表达以及3 910个基因下调表达。通过GO注释和KEGG通路注释等生物信息学手段对代谢途径进行分析,可推测冷诱导形成原基的代谢调控为：当金针菇菌丝细胞接受到冷信号后,糖分转运相关的基因表达量下降,导致碳源摄取效率下降,因而糖酵解途径大部分相关基因表达量下调,进而导致三羧酸循环的底物乙酰辅酶A（乙酰CoA）合成量减少,整个细胞能量产出下降。此负反馈信号使细胞内储存的脂质进行氧化代谢的基因表达上调,产出乙酰CoA以供三羧酸循环产能。此负反馈导致不饱和脂肪酸的合成基因表达上调,以调节细胞流动性;同时磷脂和鞘脂代谢通路的相关基因表达大多上调,合成增多,细胞膜的组分因此改变,因此细胞进行重构进入另一种状态。与DNA复制、RNA转录和蛋白质合成的相关基因表达均大部分上调,表明了原基形成时细胞正处于增殖旺盛时期。本研究结果从分子水平上揭示了金针菇原基的形成伴随着能量来源的转变,各个代谢途径的相互调控以及相关基因的表达影响,为有目的培育高温型金针菇新品种,减少栽培能耗提供理论依据。     

酿酒酵母GAL基因的表达调控

酵母菌一直是人们进行遗传分析的理想材料,对许多酵母基因的表达调节途径人们也已有了较深人的了解。酵母中研究最多的基因表达调节途径是GAL基因表达调节过程,它不仅为比较研究原核和真核生物的协同表达调节机制提供了可能性,而且由于酵母GAL基因表达可以被生长条件所控制,所以GAL基因区域对外源基因在酵母中的克隆表达也具有潜在的应用价值I‘,‘］。现在就酵母GAL基因的表达调节做一概述。IGAL基因组成及转录调节模型半乳糖是通过转化为6一磷酸葡萄糖后进人糖解途径而被酵母利用的。其中至少有五种基因产物参与从半乳糖的运…     

双孢蘑菇子实体不同发育时期的转录组分析

双孢蘑菇是世界第一大宗栽培食用菌,具有重要经济价值。为探讨双孢蘑菇子实体不同发育时期基因表达变化,利用高通量测序技术对双孢蘑菇原基期、采收期和开伞后期等不同发育时期进行RNA‐Seq分析,共筛选到6 328个差异表达基因,其中3 941个上调基因,2 387个下调基因。Gene Ontology(GO)功能聚类分析表明,差异表达基因主要富集在结合、催化分子功能组和代谢过程生物学通路中,且发育过程和有性繁殖相关的基因全部为上调表达,以利于细胞分化发育形成成熟子实体进入生殖生长阶段。KEGG功能富集分析结果表明,差异基因参与了氨基酸代谢、碳水化合物代谢、核苷酸代谢、脂类代谢和能量代谢这五大代谢通路,其中差异基因主要富集在氨基酸代谢通路中,氨基酸合成相关的多数基因上调表达,表明双孢蘑菇子实体发育形成需要一系列代谢反应协同调控,氨基酸代谢相关基因可能在双孢蘑菇子实体发育过程中起重要作用。本文通过全面分析双孢蘑菇子实体发育时期基因表达变化,获得了大量转录本信息,为深入了解双孢蘑菇子实体发育调控分子机理和相关功能基因提供了重要的基因数据资源。     

牛磺胆酸作用下副溶血弧菌全基因组比较转录谱的表达分析

目的利用DNA芯片技术研究副溶血弧菌对牛磺胆酸刺激反应的全局性基因转录变化概况,找出其中的表达调控变化规律,为副溶血弧菌基因转录调控网络的构建提供实验和理论依据。方法副溶血弧菌分别在正常和添加了50mmol／L牛磺胆酸的培养基中孵育至对数中期,收集菌体,提取RNA,利用全基因组DNA芯片分析比较两者基因转录变化。并应用聚类分析比较其中的变化规律。结果比较转录谱分析证实一共有255个基因的转录表达发生显著性变化,和对照组相比,上调的基因明显占主导优势。而在这些变化的基因中,关于蛋白合成和硫代谢以及谷氨酸合成相关的基因均呈现明显的转录上调变化。结论我们利用DNA芯片技术描绘出了副溶血弧菌在添加牛磺胆酸后全部基因转录水平变化的概图,并发现了蛋白合成,硫代谢和谷氨酸合成相关的基因的变化规律,这给我们下一步的转录调控网络研究提供了良好的靶标。     

乙醇胁迫抑制毕赤酵母表达外源蛋白的转录组学分析

在5 L发酵罐中进行毕赤酵母发酵表达猪?干扰素的实验,发现甘油培养末期乙醇的积累会抑制外源蛋白的表达。从转录组学角度系统分析不同浓度乙醇胁迫条件下,毕赤酵母甘油培养期和甲醇诱导期细胞的生理状态变化。研究结果表明,在甘油培养期,乙醇胁迫使得毕赤酵母细胞中的545个基因发生了显著差异表达(265个基因表达上调,280个基因表达下调),这些差异表达基因的功能主要涉及蛋白质合成、能量代谢、细胞周期和过氧化物酶代谢。乙醇胁迫增加了蛋白质错误折叠的情况,降低了核糖体和线粒体的结构完整性,使得甘油培养末期无法得到大量具有健全功能的酵母细胞。在甲醇诱导期,与甲醇代谢、蛋白质加工合成、氨基酸代谢等途径相关的294个基因发生了显著差异表达(171个基因表达上调,123个基因表达下调),导致内质网胁迫不能被及时解除,破坏了细胞内的氨基酸正常代谢。     

酿酒酵母表达系统

酿酒酵母是单细胞真核微生物,人们以酿酒酵母为宿主菌,用载体表达外源基因的过程中,积累了丰富的经验,掌握了酿酒酵母表达的许多优缺点,如它繁殖速度快,可以大规模发酵生产,但在发酵过程中会产生乙醇,而乙醇在培养基中积累会影响酵母的生长代谢和基因产物的表达,尤其是进行高密度发酵时该效应更明显;针对这些缺点,可以采取积极的应对措施.主要就酿酒酵母表达系统的组成、优缺点及高效表达的策略等作一综述.     

施芝麻饼肥对烟草叶片基因差异表达的影响

施用芝麻饼可提高烟叶油份和质量,但对其机理了解很少.为了解施用芝麻饼肥条件下烟草基因表达的影响,采用拟南芥基因芯片检测了施用芝麻饼肥后的烟草叶片的基因谱.在22810个基因微矩阵点中,有效差异表达的基因有54个,上调32个,下调22个.其中包括一些与烟叶质量形成关系密切的基因,如与促进脂类分解的类黄酮合成有关的4-二氢黄酮醇还原酶基因表达上调,与糖类物质合成有关的糖基转移酶基因表达下调,与根毛形成代谢有关的CslD3基因表达上调,推动各种离子和小分子代谢产物进行跨膜运输的ATPase subunit 6基因表达上调,促进氨基酸合成的γ - 谷氨酰转肽酶基因上调,还检测到与光合系统有关的基因如psaK、PsbI以及多个参与复制、转录和翻译过程基因的差异表达,如SWIM锌指家族蛋白、H4组蛋白、mTERF-related等 .此外检测到19个未知基因,它们的差异表达可能和施用饼肥有关.通过分析这些特异表达基因,了解芝麻饼肥促进烟草生长及品质形成的有关机理,揭示出一些潜在的生物学规律,为研究烟草栽培生理提供有价值的信息.     

Metabolome remodeling during the acidogenic-solventogenic transition in Clostridium acetobutylicum

The fermentation carried out by the biofuel producer Clostridium acetobutylicum is characterized by two distinct phases. Acidogenesis occurs during exponential growth and involves the rapid production of acids (acetate and butyrate). Solventogenesis initiates as cell growth slows down and involves the production of solvents (butanol, acetone, and ethanol). Using metabolomics, isotope tracers, and quantitative flux modeling, we have mapped the metabolic changes associated with the acidogenic-solventogenic transition. We observed a remarkably ordered series of metabolite concentration changes, involving almost all of the 114 measured metabolites, as the fermentation progresses from acidogenesis to solventogenesis. The intracellular levels of highly abundant amino acids and upper glycolytic intermediates decrease sharply during this transition. NAD(P)H and nucleotide triphosphates levels also decrease during solventogenesis, while low-energy nucleotides accumulate. These changes in metabolite concentrations are accompanied by large changes in intracellular metabolic fluxes. During solventogenesis, carbon flux into amino acids, as well as flux from pyruvate (the last metabolite in glycolysis) into oxaloacetate, decreases by more than 10-fold. This redirects carbon into acetyl coenzyme A, which cascades into solventogenesis. In addition, the electron-consuming reductive tricarboxylic acid (TCA) cycle is shutdown, while the electron-producing oxidative (clockwise) right side of the TCA cycle remains active. Thus, the solventogenic transition involves global remodeling of metabolism to redirect resources (carbon and reducing power) from biomass production into solvent production.     

Metabolic analysis of the synthesis of high levels of intracellular human SOD in Saccharomyces cerevisiae rhSOD 2060 411 SGA122

The synthesis of human superoxide dismutase (SOD) in batch cultures of a Saccharomyces cerevisiae strain using a glucose-limited minimal medium was studied through metabolic flux analysis. A stoichiometric model was built, which included 78 reactions, according to metabolic pathways operative in these strains during respirofermentative and oxidative metabolism. It allowed calculation of the distribution of metabolic fluxes during diauxic growth on glucose and ethanol. Fermentation profiles and metabolic fluxes were analyzed at different phases of diauxic growth for the recombinant strain (P+) and for its wild type (P-). The synthesis of SOD by the strain P+ resulted in a decrease in specific growth rate of 34 and 54% (growth on glucose and ethanol respectively) in comparison to the wild type. Both strains exhibited similar flux of glucose consumption and ethanol synthesis but important differences in carbon distribution with biomass/substrate yields and ATP production 50% higher in P-. A higher contribution of fermentative metabolism, with 64% of the energy produced at the phosphorylation level, was observed during SOD production. The flux of precursors to amino acids and nucleotides was higher in the recombinant strain, in agreement with the higher total RNA and protein levels. Lower specific growth rates in strain P+ appear to be related to the decrease in the rate of synthesis of nonrecombinant protein, as well as a decrease in the activities of the pentose phosphate (PP) pathway and TCA cycle. A very different way of entry into the stationary phase was observed for each strain: in the wild-type strain most metabolic fluxes decreased and fluxes related to energy reserve synthesis increased, while in the P+ strain the flux of 22 reactions (including PP pathway and amino acids biosynthesis) related to SOD production increased their fluxes. Changes in SOD production rates at different physiological states appear to be related to the differences in building blocks availability between respirofermentative and oxidative metabolism. Using the present expression system, ideal conditions for SOD synthesis are represented by either active growth during respirofermentative metabolism or transition from a growing to a nongrowing state. An increase in SOD flux could be achieved using an expression system nonassociated to growth and potentially eliminating part of the metabolic burden.     

Accumulation of cAMP in the cells of Candida tropicalis at an early stage of ethanol-induced filamentous growth and its prevention by myo-inositol

Phosphatidylinositol metabolism is enhanced in the cells of Candida tropicalis Pk 233 at an early stage of filamentous growth caused by ethanol, and myo-inositol prevents the ethanol-induced changes in the metabolism and morphology [Uejima et al. (1987) FEBS Lett. 214, 127-129]. The accumulation of cAMP and an increase in adenylate cyclase activity were observed in the cells grown with ethanol to the mid-log phase. Myo-inositol abolished these effects of ethanol also. The activity of cAMP phosphodiesterase was affected by neither ethanol nor myo-inositol. These results suggest that the inositol phospholipid-linked and cAMP-linked signaling pathways may be involved in the mechanism of ethanol-induced filamentous growth of this yeast and also that myo-inositol would affect morphogenesis by controlling these pathways.     

Metabolic flux analysis of hepatocyte function in hormone- and amino acid-supplemented plasma

Understanding the metabolic and regulatory pathways of hepatocytes is important for biotechnological applications involving liver cells. Previous attempts to culture hepatocytes in plasma yielded poor functional results. Recently we reported that hormone (insulin and hydrocortisone) and amino acid supplementation reduces intracellular lipid accumulation and restores liver-specific function in hepatocytes exposed to heparinized human plasma. In the current study, we performed metabolic flux analysis (MFA) using a simplified metabolic network model of cultured hepatocytes to quantitively estimate the changes in lipid metabolism and relevant intracellular pathways in response to hormone and amino acid supplementation. The model accounts for the majority of central carbon and nitrogen metabolism, and assumes pseudo-steady-state with no metabolic futile cycles. We found that beta-oxidation and tricarboxylic acid (TCA) cycle fluxes were upregulated by both hormone and amino acid supplementation, thus enhancing the rate of lipid oxidation. Concomitantly, hormone and amino acid supplementation increased gluconeogenic fluxes. This, together with an increased rate of glucose clearance, caused an increase in predicted glycogen synthesis. Urea synthesis was primarily derived from ammonia and aspartate generated through transamination reactions, while exogenous ammonia removal accounted for only 3-6% of the urea nitrogen. Amino acid supplementation increased the endogenous synthesis of oxaloacetate, and in turn that of aspartate, a necessary substrate for the urea cycle. These findings from MFA provide cues as to which genes/pathways relevant to fatty acid oxidation, urea production, and gluconeogenesis may be upregulated by plasma supplementation, and are consistent with current knowledge of hepatic amino acid metabolism, which provides further credence to this approach for evaluating the metabolic state of hepatocytes under various environmental conditions.