佐夫色绿藻高产虾青素突变体的筛选及虾青素合成机理研究

为了提高佐夫色绿藻(Chromochloris zofingiensis)细胞内虾青素含量,研究通过甲基磺酸乙酯诱变构建了含有20000个单克隆的突变体库,并筛选出一株高产虾青素的突变体12C10。在异养正常培养条件下,当葡萄糖耗完时, 12C10虾青素含量比野生型提高74%;缺氮诱导第4天时,虾青素含量比野生型高25%。利用广泛靶向代谢组学分析在正常培养条件下12C10与野生型在代谢物水平上的差异。与野生型相比, 12C10中除谷氨酸外的氨基酸及脂肪酸的合成普遍下降,但是谷氨酸的水平显著提高。氨基酸和脂肪酸合成减少为虾青素合成提供更多的碳骨架、NADPH和ATP。谷氨酸的积累可能一方面刺激了磷酸戊糖途径中6-磷酸葡萄糖脱氢酶的活性促进NADPH的产生,另一方面导致氧自由基产生促进虾青素合成。代谢物组分析结果还表明12C10中虾青素合成的增强可能与乙烯合成的增强有关。研究为进一步通过代谢调控提高C. zofingiensis虾青素含量奠定了基础,对指导C. zofingiensis虾青素积累新工艺的开发具有重要意义。     

碳氮比和光强对小球藻合成虾青素的影响

本文研究了碳氮比和光强对小球藻Chlorella zofingiensis合成虾青素的影响。结果表明, 随着碳氮比的增加, 虾青素含量提高, 但是过高的碳氮比限制藻细胞的生长。当碳氮比为 133 时, 虾青素的产量最大, 达到 9.19 mg/L。高光诱导在高碳氮比基础上进一步提高虾青素的含量及产量。200 mmol/m2﹒s 光强在没有严重影响小球藻生长的同时, 明显提高虾青素含量, 最大虾青素产量达到 12.52 mg/L。文章对不同诱导条件下虾青素的合成机理以及利用异养自养相结合的方法提高虾青素产量的可能性进行了讨论。     

碳氮比和光强对小球藻合成虾青素的影响

本文研究了碳氮比和光强对小球藻Chlorella zofingiensis合成虾青素的影响。结果表明,随着碳氮比的增加,虾青素含量提高,但是过高的碳氮比限制藻细胞的生长,当碳氮比为133时,虾青素的产量最大,达到9.19mg／L。高光诱导在高碳氮比基础上进一步提高虾青素的含量及产量。200μmol／m^2.S光强在没有严重影响小球藻生长的同时,明显提高虾青素含量,最大虾青素产量达到12.52mg／L。文章对不同诱导条件下虾青素的合成机理以及利用异养自养相结合的方法提高虾青素产量的可能性进行了讨论。     

缺氮胁迫下雨生红球藻虾青素积累过程中的基因组MSAP分析

虾青素具有多种生物学活性,雨生红球藻为天然虾青素的最佳来源,缺氮胁迫会导致雨生红球藻积累虾青素。为了解缺氮条件下雨生红球藻虾青素积累的分子机制,该研究通过对雨生红球藻进行缺氮胁迫,结合MSAP法,研究了雨生红球藻在缺氮胁迫下虾青素积累过程中基因组甲基化水平的变化,结果表明:缺氮胁迫0~72 h期间,雨生红球藻生长速度减慢,而虾青素积累主要发生在缺氮处理12~24 h期间,随后积累速度减慢。同时,对缺氮胁迫0、24、72 h的雨生红球藻基因组DNA进行甲基化敏感扩增多态性分析,共得到了291个甲基化多态性位点,其中发生甲基化变化的位点在0~24 h和24~72 h分别占总位点的29.90%和53.95%。在缺氮胁迫24 h处DNA半甲基化率最大(为12.71%),全甲基化率最低(为26.80%);缺氮胁迫72 h处DNA全甲基化率最高(为28.52%),半甲基化率最低(为1.72%)。这表明DNA甲基化调节方式的改变是虾青素积累过程中的一种重要调控模式。     

乳酸钠促进法夫酵母虾青素合成代谢流作用分析

【目的】研究乳酸钠(一种糖代谢产物)的加入对法夫酵母JMU-VDL668发酵过程中细胞生长和虾青素合成的影响。【方法】分别在摇瓶和7 L发酵罐实验基础上,采用代谢通量分析的方法分析添加乳酸钠对法夫酵母菌株JMU-VDL668合成虾青素代谢流的影响。【结果】在7 L发酵罐实验中添加乳酸钠,虾青素产量最高可达17.70 mg/L,与对照组相比提高26%。代谢通量分析表明,乳酸钠可以调节丙酮酸、乙酰辅酶A节点处的代谢通量分布,乳酸在乳酸脱氢酶的作用下可以直接进入代谢网络的后半程,乙酰辅酶A的通量和进入TCA循环的通量得到了显著加强。【结论】乳酸钠的加入提供了更多的乙酰辅酶A等前体物质和能量供给,因此促进了虾青素的合成。     

氮源对C.vulgaris油脂积累的影响

实验室条件下,考察了在发酵过程中不同氮源对小球藻的生物量和油脂积累的影响,确定了小球藻的最佳氮源;并对比分析了含氮培养与缺氮培养的生物量、油脂含量、氮消耗量、生物量氮消耗比率和油脂氮消耗比率的不同。结果表明:小球藻在1.6 g/L Na NO3时获得最大生物量,为562.2 mg/L,在0.8 g/L Na NO3时获得最大相对油脂含量为12.01%;以油脂含量为考察指标时,培养小球藻的最佳氮源为0.8 g/L Na NO3;缺氮培养时,最大油脂含量为13.49%,比含氮培养高约15%;含氮培养时,最高生物量为626.3 mg/L,比缺氮培养高约1.9倍。氮源对生物量,相对油脂含量,生物量氮消耗比率和油脂氮消耗比率具有明显的影响。藉此,提出了通过改变培养方式,达到调控小球藻细胞内生理代谢组分的可行性。     

高产虾青素红法夫酵母的选育及代谢通量分析

以野生型红法夫酵母As2.1557为出发菌株,依次进行两轮紫外线诱变和亚硝基胍诱变,并以10-4与10-3mol/Lβ-紫罗酮作为筛选剂,得到突变株UV-N2-7,其虾青素产量和含量分别较出发菌株提高81.7%和2.25倍。采用Plackett-Burman设计法及响应面分析法对发酵培养基的组分及发酵条件进行了优化,突变株的虾青素产量由从初始的2.674 mg/L提高到了6.338 mg/L。建立了红法夫酵母的代谢网络,并对比了突变株和野生株间歇培养条件下对数生长期的代谢通量分布。结果表明:突变株与野生株相比,PP途径通量有所减小,而EMP途径和TCA循环有所增强,突变株用于菌体生长的通量有所减小;二者的丙酮酸脱氢酶的活性均较低,分泌至胞外的丙酮酸的代谢通量约为32%。因此预计通过遗传改造和发酵控制提高丙酮酸脱氢酶的活性可能会进一步提高虾青素的产率。     

光诱导雨生红球藻虾青素积累的信号通路转录组分析

雨生红球藻Haematococcuspluvialis在逆境胁迫条件下可大量积累虾青素,被认为自然界最理想的虾青素生产者。高光能有效诱导其虾青素的合成与积累,但藻细胞感知和转导光信号进而调控虾青素积累的机制尚不清楚。文中采用Illumina Hiseq 2000高通量测序技术分别获得正常、高白光及高蓝光处理组4.0 G、3.8 G及3.6 G的原始数据量,经质控与拼接之后获得51 954条长度至少为200 bp的unigenes基因,经过比对分析,共有20 537个unigenes在NR、NT、KO、SwissProt、PfamGO及KOG等数据库中的至少1个数据库中注释成功,达到39.52%。差异表达基因分析显示,高白光vs正常组共获得1 255个DEGs;高蓝光vs正常组共获得1 494个DEGs;高白光与高蓝光vs正常组共同的DEGs有1 008个。KEGG富集分析显示,与对照组相比高白光与高蓝光共同的显著富集通路包括光合作用、类胡萝卜素合成、脂肪酸合成、氧化磷酸化、DNA复制、碳代谢及氮代谢等过程。通过对转录组数据进一步分析,挖掘鉴定了大量光受体及其信号转导通路中的互作蛋白。随机筛选DEGs基因15条,采用荧光定量PCR技术检测其转录水平,结果表明与转录组差异表达数据高度一致。光受体及其信号转导通路中的互作蛋白基因差异表达分析,推测"光信号→光受体→互作蛋白(互作蛋白→转录因子/转录调节子)→功能基因表达→虾青素积累"的信号转导通路可能参与上述调控过程,为深入解析光诱导虾青素合成的转录调控机制奠定了基础。     

黄腐酸对雨生红球藻虾青素的积累和CHY基因表达量影响

实验以雨生红球藻Haematococcus pluvialis LUGU为对象,研究了不同浓度的黄腐酸对微藻细胞生长、虾青素积累以及β-胡萝卜素羟化酶(CHY)基因表达量的影响。结果表明,FA浓度为5 mg/L,藻细胞生物量产率达到了79.39 mg/(L·d),虾青素产量达到了20.82 mg/L,分别比对照组提高了4.25%和86.89%;FA浓度为10 mg/L,藻细胞的生物量产率和虾青素产量分别比对照组提高了5.44%和9.78%。RT-PCR分析显示,虾青素合成的关键基因CHY的表达受FA的诱导,当添加5和10 mg/L的黄腐酸时,CHY基因最大的表达量分别为对照的18.1倍和7.3倍,当添加20 mg/L的黄腐酸时CHY基因的最大的表达量仅为对照的3.2倍,FA诱导下的雨生红球藻虾青素的积累含量和CHY基因表达量呈正相关。实验表明,适当浓度的黄腐酸不仅能够显著提高虾青素合成关键酶基因CHY的表达水平,并且明显促进了藻细胞内虾青素的积累,因此黄腐酸可作为虾青素生产的一种有效诱导子。     

不同培养条件对枝鞘藻生长、虾青素和油脂积累的影响

以丝状绿藻枝鞘藻(Oedocladium sp.)为实验材料,研究在100、300μmol·m-2·s-1和双侧300μmol·m-2·s-13种光强以及1、3、9、18 mmol/L 4种初始氮浓度下,两步法培养(第12 d时实验组分别更换为无氮培养基及加盐培养基)对枝鞘藻生长、油脂和虾青素积累的影响。结果显示:枝鞘藻最大生物量在双侧300μmol·m-2·s-1光强,18 mmol/L初始氮浓度更换为无氮培养基的条件下达到,为9.61 g/L;最高虾青素含量和最高油脂含量在双侧300μmol·m-2·s-1光强,3 mmol/L初始氮浓度更换为加盐培养基条件下达到,分别达到干重的1.62%和51.19%。研究结果表明高光条件有利于枝鞘藻的生长,双侧高光条件下低氮浓度更换为加盐培养基最有利于枝鞘藻虾青素和油脂的积累。     

Quantification of metabolism in Saccharomyces cerevisiae under hyperosmotic conditions using elementary mode analysis

Yeast metabolism under hyperosmotic stress conditions was quantified using elementary mode analysis to obtain insights into the metabolic status of the cell. The fluxes of elementary modes were determined as solutions to a linear program that used the stoichiometry of the elementary modes as constraints. The analysis demonstrated that distinctly different sets of elementary modes operate under normal and hyperosmotic conditions. During the adaptation phase, elementary modes that only produce glycerol are active, while elementary modes that yield biomass, ethanol, and glycerol become active after the adaptive phase. The flux distribution in the metabolic network, calculated using the fluxes in the elementary modes, was employed to obtain the flux ratio at key nodes. At the glucose 6-phosphate (G6P) node, 25% of the carbon influx was diverted towards the pentose phosphate pathway under normal growth conditions, while only 0.3% of the carbon flux was diverted towards the pentose phosphate pathway during growth at 1?M NaCl, indicating that cell growth is arrested under hyperosmotic conditions. Further, objective functions were used in the linear program to obtain optimal solution spaces corresponding to the different accumulation rates. The analysis demonstrated that while biomass formation was optimal under normal growth conditions, glycerol synthesis was closer to optimal during adaptation to osmotic shock.     

Analysis of optimal phenotypic space using elementary modes as applied to Corynebacterium glutamicum

Background  

Quantification of the metabolic network of an organism offers insights into possible ways of developing mutant strain for better productivity of an extracellular metabolite. The first step in this quantification is the enumeration of stoichiometries of all reactions occurring in a metabolic network. The structural details of the network in combination with experimentally observed accumulation rates of external metabolites can yield flux distribution at steady state. One such methodology for quantification is the use of elementary modes, which are minimal set of enzymes connecting external metabolites. Here, we have used a linear objective function subject to elementary modes as constraint to determine the fluxes in the metabolic network of Corynebacterium glutamicum. The feasible phenotypic space was evaluated at various combinations of oxygen and ammonia uptake rates.     

System-level insights into yeast metabolism by thermodynamic analysis of elementary flux modes

One of the most obvious phenotypes of a cell is its metabolic activity, which is defined by the fluxes in the metabolic network. Although experimental methods to determine intracellular fluxes are well established, only a limited number of fluxes can be resolved. Especially in eukaryotes such as yeast, compartmentalization and the existence of many parallel routes render exact flux analysis impossible using current methods. To gain more insight into the metabolic operation of S. cerevisiae we developed a new computational approach where we characterize the flux solution space by determining elementary flux modes (EFMs) that are subsequently classified as thermodynamically feasible or infeasible on the basis of experimental metabolome data. This allows us to provably rule out the contribution of certain EFMs to the in vivo flux distribution. From the 71 million EFMs in a medium size metabolic network of S. cerevisiae, we classified 54% as thermodynamically feasible. By comparing the thermodynamically feasible and infeasible EFMs, we could identify reaction combinations that span the cytosol and mitochondrion and, as a system, cannot operate under the investigated glucose batch conditions. Besides conclusions on single reactions, we found that thermodynamic constraints prevent the import of redox cofactor equivalents into the mitochondrion due to limits on compartmental cofactor concentrations. Our novel approach of incorporating quantitative metabolite concentrations into the analysis of the space of all stoichiometrically feasible flux distributions allows generating new insights into the system-level operation of the intracellular fluxes without making assumptions on metabolic objectives of the cell.     

铜绿微囊藻与小球藻对低温和黑暗的响应与恢复

研究以水华蓝藻铜绿微囊藻(Microcystis aeruginosa PCC 7806)与绿藻小球藻(Chlorella sp. FACHB-31)为研究对象, 探讨低温和黑暗对其生长、色素含量、最大光化学效率(F_v/F_m)、丙二醛(MDA)含量及过氧化氢酶活性的变化。结果表明, 30d的低温和黑暗处理, 显著降低了铜绿微囊藻和小球藻的叶绿素a浓度, 增加了单位细胞类胡萝卜素含量。在低温黑暗条件下, 铜绿微囊藻的MDA含量及CAT活性均显著增加, 而小球藻变化不明显。30d低温黑暗处理, 铜绿微囊藻的存活率为54.6%, 显著高于小球藻的31.3%。当恢复正常温度与光照, 2种藻均迅速生长。这些结果表明低温黑暗影响了微囊藻和小球藻的生理特性。在低温黑暗处理下, 微囊藻的F_v/F_m显著降低, 而小球藻则保持较为恒定的F_v/F_m, 表明微囊藻通过降低自身光合活性来渡过冬季低温黑暗的条件, 而小球藻在低温黑暗条件下仍保持较高的光合活性。     

Quantitative elementary mode analysis of metabolic pathways: the example of yeast glycolysis

Background  

Elementary mode analysis of metabolic pathways has proven to be a valuable tool for assessing the properties and functions of biochemical systems. However, little comprehension of how individual elementary modes are used in real cellular states has been achieved so far. A quantitative measure of fluxes carried by individual elementary modes is of great help to identify dominant metabolic processes, and to understand how these processes are redistributed in biological cells in response to changes in environmental conditions, enzyme kinetics, or chemical concentrations.     

Production and analysis of secondary carotenoids in green algae

The microalgae Neochloris wimmeri, Scenedesmus vacuolatus, Scotiellopsisoocystiformis, Chlorella zofingiensis and Protosiphon botryoides were grown under secondarycarotenoid inductive conditions. The results indicatethat nitrogen deficiency and high light intensity arepotential inducers of astaxanthin formation in thefive microalgae studied. All these microalgaeaccumulate significant quantities of secondarycarotenoids, mainly as astaxanthin esters andcanthaxanthin. They also showed high resistance toenvironmental conditions. All these qualities makethese microalgae good candidates for successfulculture in open ponds.     

Application of hybrid cybernetic model in simulating myeloma cell culture co-consuming glucose and glutamine with mixed consumption patterns

A dynamic model called hybrid cybernetic model (HCM) based on structured metabolic network is established for simulating mammalian cell metabolism featured with partially substitutable and partially complementary consumption patterns of two substrates, glucose and glutamine. Benefiting from the application of elementary mode analysis (EMA), the complicated metabolic network is decomposed into elementary modes (EMs) facilitating the employment of the hybrid cybernetic framework to investigate the external and internal flux distribution and the regulation mechanism among them. According to different substrate combination, two groups of EMs are obtained, i.e., EMs associated with glucose uptake and simultaneous uptake of glucose and glutamine. Uptake fluxes through various EMs are coupled together via cybernetic variables to maximize substrate uptake. External fluxes and internal fluxes could be calculated and estimated respectively, by the combination of the stoichiometrics of metabolic networks and fluxes through regulated EMs. The model performance is well validated via three sets of experimental data. Through parameter identification of limited number of experimental data, other external metabolites are precisely predicted. The obtained kinetic parameters of three experimental cultures have similar values, which indicates the robustness of the model. Furthermore, the prediction performance of the model is successfully validated based on identified parameters.     

The Effect of Salt Stress on the Production of Canthaxanthin and Astaxanthin by Chlorella zofingiensis Grown Under Limited Light Intensity

The fresh water green microalga Chlorella zofingiensisis known to accumulate ketocarotenoids – primarily astaxanthin but also canthaxanthin – when grown under stress conditions of high light irradiance and low nitrogen. We found that salt stress can replace light stress with respect to inducing carotenoid production: cells of C. zofingiensis grown under low light irradiance and subjected to salt and low nitrogen stress accumulated higher amounts of total secondary carotenoids than those growing under high light and low nitrogen stress. Furthermore, C. zofingiensis growing under conditions of salt stress and low light accumulated higher amounts of canthaxanthin than astaxanthin. It is suggested that for canthaxanthin accumulation under salt stress, light is not a limiting factor, but for astaxanthin accumulation high light irradiance is mandatory. These results may be applied in the future for the commercial production of canthaxanthin by C. zofingiensis in systems in which light availability is poor.