不同比生长速率对毕赤酵母发酵生产植酸酶的影响

不同比生长速率控制条件下毕赤酵母代谢流流向不同,从而导致产酶量的不同。为了得到毕赤酵母发酵生产植酸酶的最适比生长速率,对毕赤酵母工程菌50 L发酵生产植酸酶的工艺条件进行了研究。结果表明,通过调节甲醇的流加速度,控制菌体比生长速率为0.02 h^-1,发酵结束时,植酸酶酶活17 445 U/mL,比对照提高69.7 %,该比生长速率对发酵生产植酸酶最有利。     

Expanded flux variability analysis on metabolic network of Escherichia coli

Flux balance analysis, based on the mass conservation law in a cellular organism, has been extensively employed to study the interplay between structures and functions of cellular metabolic networks. Consequently, the phenotypes of the metabolism can be well elucidated. In this paper, we introduce the Expanded Flux Variability Analysis （EFVA） to characterize the intrinsic nature of metabolic reactions, such as flexibility, modularity and essentiality, by exploring the trend of the range, the maximum and the minimum flux of reactions. We took the metabolic network of Escherichia coli as an example and analyzed the variability of reaction fluxes under different growth rate constraints. The average variability of all reactions decreases dramatically when the growth rate increases. Consider the noise effect on the metabolic system, we thus argue that the microorganism may practically grow under a suboptimal state. Besides, under the EFVA framework, the reactions are easily to be grouped into catabolic and anabolic groups. And the anabolic groups can be further assigned to specific biomass constitute. We also discovered the growth rate dependent essentiality of reactions.     

肌苷产生菌枯草芽孢杆菌分批发酵的代谢流分析

为了更好地掌握代谢规律和代谢方式，代谢流作为一个关键而重要的变量需要测定．然而，由于代谢流测定的困难，在许多代谢研究中代谢流并没有被完全应用．在肌苷高产菌枯草芽孢杆菌中，利用得到的实验数据包括葡萄糖的消耗速率和代谢主产物以及副产物的形成速率，利用代谢通量平衡模型，得到糖酵解、三羟酸循环及磷酸戊糖途径的代谢流，并对其进行了分析．     

Scaling metabolism from organisms to ecosystems

Understanding energy and material fluxes through ecosystems is central to many questions in global change biology and ecology. Ecosystem respiration is a critical component of the carbon cycle and might be important in regulating biosphere response to global climate change. Here we derive a general model of ecosystem respiration based on the kinetics of metabolic reactions and the scaling of resource use by individual organisms. The model predicts that fluxes of CO2 and energy are invariant of ecosystem biomass, but are strongly influenced by temperature, variation in cellular metabolism and rates of supply of limiting resources (water and/or nutrients). Variation in ecosystem respiration within sites, as calculated from a network of CO2 flux towers, provides robust support for the model's predictions. However, data indicate that variation in annual flux between sites is not strongly dependent on average site temperature or latitude. This presents an interesting paradox with regard to the expected temperature dependence. Nevertheless, our model provides a basis for quantitatively understanding energy and material flux between the atmosphere and biosphere.     

Metabolite-enabled eradication of bacterial persisters by aminoglycosides

Bacterial persistence is a state in which a sub-population of dormant cells, or 'persisters', tolerates antibiotic treatment. Bacterial persisters have been implicated in biofilms and in chronic and recurrent infections. Despite this clinical relevance, there are currently no viable means for eradicating persisters. Here we show that specific metabolic stimuli enable the killing of both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) persisters with aminoglycosides. This potentiation is aminoglycoside-specific, it does not rely on growth resumption and it is effective in both aerobic and anaerobic conditions. It proceeds by the generation of a proton-motive force which facilitates aminoglycoside uptake. Our results demonstrate that persisters, although dormant, are primed for metabolite uptake, central metabolism and respiration. We show that aminoglycosides can be used in combination with specific metabolites to treat E. coli and S. aureus biofilms. Furthermore, we demonstrate that this approach can improve the treatment of chronic infections in a mouse urinary tract infection model. This work establishes a strategy for eradicating bacterial persisters that is based on metabolism, and highlights the importance of the metabolic environment to antibiotic treatment.     

青藏高原珍稀大型真菌亚东木耳菌丝发酵条件研究

为了更好地开发利用西藏珍稀大型真菌亚东木耳,对亚东木耳菌丝生长和次级代谢条件进行研究。考察了不同培养基成分和培养条件对亚东木耳菌丝生长速度、生物量和次级代谢产物产量的影响。结果表明,可溶性淀粉、酵母浸粉和CaCl₂分别为亚东木耳生长代谢的较佳碳源、氮源和无机盐,维生素B₁、B₂对菌丝生长无明显促进作用。较佳培养温度为26℃,较佳培养时间为63d,菌丝生长较佳初始pH值为7.0,较佳次级代谢初始pH值为6.0。HPLC图谱分析结果表明,次级代谢产物种类多达200余种,其中初始pH值和温度条件对次级代谢多样性的影响较为明显。     

Escherichia coli K-12 undergoes adaptive evolution to achieve in silico predicted optimal growth

Annotated genome sequences can be used to reconstruct whole-cell metabolic networks. These metabolic networks can be modelled and analysed (computed) to study complex biological functions. In particular, constraints-based in silico models have been used to calculate optimal growth rates on common carbon substrates, and the results were found to be consistent with experimental data under many but not all conditions. Optimal biological functions are acquired through an evolutionary process. Thus, incorrect predictions of in silico models based on optimal performance criteria may be due to incomplete adaptive evolution under the conditions examined. Escherichia coli K-12 MG1655 grows sub-optimally on glycerol as the sole carbon source. Here we show that when placed under growth selection pressure, the growth rate of E. coli on glycerol reproducibly evolved over 40 days, or about 700 generations, from a sub-optimal value to the optimal growth rate predicted from a whole-cell in silico model. These results open the possibility of using adaptive evolution of entire metabolic networks to realize metabolic states that have been determined a priori based on in silico analysis.     

强化工业大肠杆菌生长特性的研究

大肠杆菌是制药工业中的重要蛋白质药物生产菌株。然而乙酸积累、中心碳代谢负担过重等不良因素严重制约着大肠杆菌的高密度生长。利用Red同源重组技术,构建icd和ptsG双基因敲除大肠杆菌菌株,以分流碳代谢流,提高碳利用效率,最终增加菌体生物量。同时构建ppc过表达质粒,减少乙酸积累。发酵结果显示,构建的重组菌株产酸量大幅减少,pH值平均升高20%,菌体浓度增加了50%,生长特性得到了强化。     

线粒体热化学研究Ⅴ.鳙鱼肝脏线粒体代谢热动力学

用微量热法研究了不同温度下鳙鱼肝脏线粒体代谢的产热曲线,建立了热动力学方程,得到了线粒体在活性增长期的增长速率常数ｋ,平均比增长速率ｋ／ｍ等．实验结果表明,同一动物的同一器官线粒体,其不同温度下的产热曲线的形状相同,反映了线粒体的固有特征;不同温度下线粒体代谢各阶段的时间不同,其单位线粒体产热量亦不同     

Metabolic network analysis of the causes and evolution of enzyme dispensability in yeast

Under laboratory conditions 80% of yeast genes seem not to be essential for viability. This raises the question of what the mechanistic basis for dispensability is, and whether it is the result of selection for buffering or an incidental side product. Here we analyse these issues using an in silico flux model of the yeast metabolic network. The model correctly predicts the knockout fitness effects in 88% of the genes studied and in vivo fluxes. Dispensable genes might be important, but under conditions not yet examined in the laboratory. Our model indicates that this is the dominant explanation for apparent dispensability, accounting for 37-68% of dispensable genes, whereas 15-28% of them are compensated by a duplicate, and only 4-17% are buffered by metabolic network flux reorganization. For over one-half of those not important under nutrient-rich conditions, we can predict conditions when they will be important. As expected, such condition-specific genes have a more restricted phylogenetic distribution. Gene duplicates catalysing the same reaction are not more common for indispensable reactions, suggesting that the reason for their retention is not to provide compensation. Instead their presence is better explained by selection for high enzymatic flux.     

红芪组织培养中次生代谢产物的研究Ⅰ.红芪愈伤组织异柠檬酸裂解酶活性的变化

在红芪不同生长状态的愈伤组织中,异柠檬酸裂解酶（ＩＣＬ）活性差异很大,其中以分化愈伤组织中活性最高．与此相平行在红芪不同培养时期的愈伤组织中,不饱和脂肪酸含量降低,多糖含量升高．由此可以肯定异柠檬酸裂解酶为红芪次生代谢产生多糖过程中的一个重要酶．同时在无菌苗中异柠檬酸裂解酶活性也很高,而多糖没有检测到,说明异柠檬酸裂解酶也参加了植物细胞的初级代谢．我们认为初级和次级代谢过程中某些重要的甚至关键的酶是相同的,只是随着细胞的发育过程其活性发生了变化．     

Simulation and validation of modelled sphingolipid metabolism in Saccharomyces cerevisiae

Mathematical models have become a necessary tool for organizing the rapidly increasing amounts of large-scale data on biochemical pathways and for advanced evaluation of their structure and regulation. Most of these models have addressed specific pathways using either stoichiometric or flux-balance analysis, or fully kinetic Michaelis-Menten representations, metabolic control analysis, or biochemical systems theory. So far, the predictions of kinetic models have rarely been tested using direct experimentation. Here, we validate experimentally a biochemical systems theoretical model of sphingolipid metabolism in yeast. Simulations of metabolic fluxes, enzyme deletion and the effects of inositol (a key regulator of phospholipid metabolism) led to predictions that show significant concordance with experimental results generated post hoc. The model also allowed the simulation of the effects of acute perturbations in fatty-acid precursors of sphingolipids, a situation that is not amenable to direct experimentation. The results demonstrate that modelling now allows testable predictions as well as the design and evaluation of hypothetical 'thought experiments' that may generate new metabolomic approaches.     

手机辐射对大肠杆菌生长影响的研究初报

用手机对生长状态下的大肠杆菌进行不同辐射时间的处理.采用涂布平板法观察菌落数,辐射时间在60 min时,实验组菌落数多于对照组,辐射时间在80~140 min时间内,实验组菌落数都明显少于对照组;通过亚甲蓝法测定菌体活性,辐射时间为60 min的实验组,菌体活性与对照组差异不显著外,其他实验组与对照组差异都极显著.表明手机辐射对大肠杆菌的分裂、生长与代谢在一定的强度范围内可产生显著的影响,低剂量时可能对菌体分裂与生长有一定的促进作用,当剂量超过一定强度后,对菌体分裂与生长有一定抑制作用,甚至导致菌体死亡.     

酿酒酵母中心代谢网络动力学建模

以酿酒酵母中心代谢网络为研究对象进行代谢网络动力学模拟,将酶反应速率方程改写成普适的形式,模拟了以葡萄糖为碳源的一次生长代谢状态,计算得到了代谢网络达到稳态的代谢流分布以及全部代谢物浓度随时间变化的情况,并将模拟得到的代谢流分布与实验结果进行了比较,结果具有一致性.     

通气提高Klebsiella oxytoca HP1发酵产氢

研究了Klebsiella oxytoca HP1在不同通气条件下的生长和放氢特性．批式发酵实验结果表明：通氩气放氢效果最好，通二氧化碳放氢效果最差．提高氩气通气速率能显著提高产氢量。0．25L／Lmin条件下的产氢量达到1155mL是不通气时的2．41倍，但通气也影响延迟期的长短。氩气通量应该与微生物生长阶段相匹配．代谢产物分析表明：K．oxytoca HP1呈混合酸发酵特性，主要代谢产物有乙酸、乙醇、乳酸和2，3-丁二醇，氩气通气速率明显影响丙酮酸节点的代谢途径和通量，从高通气条件到不通气条件，代谢主要途径按乙酸途径→乙醇途径→乳酸途径的次序转变，通气速率是K．oxytoca HP1发酵产氢的重要调控因子．     

Substance graphs are optimal simple-graph representations of metabolism

One approach to study the system-wide organization of biochemistry is to use statistical graph theory. In such heavily simplified methods, which disregard most of the dynamic aspects of biochemistry, one is faced with fundamental questions. One such question is how the chemical reaction systems should be reduced to a graph retaining as much functional information as possible from the original reaction system. In these graph representations, should the edges go between substrates and products, or substrates and substrates, or both? Should vertices represent substances or reactions? Different representations encode different information about the reaction system and affect network measures in different ways. This paper investigates which representation reflects the functional organization of the metabolic system in the best way, according to the defined criteria. Four different graph representations of metabolism (three where the vertices are metabolites, one where the vertices are reactions) are evaluated using data from different organisms and databases. The graph representations are evaluated by comparing the overlap between clusters (network modules) and annotated functions, and also by comparing the set of identified currency metabolites with those that other authors have identified using qualitative biological arguments. It is found that a “substance network”, where all metabolites participating in a reaction are connected, is better than others, evaluated with respect to both the functional overlap between modules and functions and to the number and identity of the identified currency metabolites.     

几种浮游单细胞藻类磷代谢的初步研究

用３２ＰＯ４３－同位素稀释法研究了单种一次培养的几种浮游单细胞藻类的磷代谢．小球藻（Ｃｈｌｏｒｅｌｌａｓｐ．）和一种绿藻对ＰＯ４３－的最大吸收速率（Ｖｍａｘ）与细胞磷含量成负相关，半饱和速率常数（Ｋｓ）与藻类的种类相关．光照与黑暗对中华盒形藻（ＢｉｄｄｕｌｐｈｉａｓｉｎｅｎｓｉｓＧｒｅｖｉｌｅ）和金藻（Ｄｉｃｒａｔｅｒｉａｓｐ）ＰＯ４３－的吸收和再生代谢无明显影响，其昼夜变化是藻类细胞新陈代谢的反映；处于细胞周期Ｇ１期与Ｓ期的藻类有较高的磷吸收和再生代谢速率，而Ｇ２期与Ｍ期的磷代谢速率较低．藻类磷代谢的动力学及其昼夜变化是围绕着对营养盐磷最大程度地利用和维持藻类持续生长的机制展开的．     

重组Pichia酵母(Muts)发酵过渡阶段关键酶活分析

重组Pichia酵母表达系统的发酵存在从利用甘油为碳源生长到利用甲醇为碳源表达外源蛋白的发酵表达过渡阶段。Pichia酵母过渡阶段碳源代谢途径关键酶酶活分析表明：甲醇诱导3h AOX2酶活为0,4h时突然增加到0．05U,继续诱导,酶活缓慢增加;在过渡阶段甲醛脱氢酶和6-P-葡萄糖脱氢酶分别增加了6．1倍、2．5倍,而丙酮酸脱氢酶和异柠檬酸脱氢酶分别下降为原酶活的29．4％及16．4％,表明甲醇诱导后甲醇完全氧化代谢途径得到强化,而糖酵解途径和三羧酸循环途径代谢作用减弱。     

Antibiotics in early life alter the murine colonic microbiome and adiposity

Antibiotics administered in low doses have been widely used as growth promoters in the agricultural industry since the 1950s, yet the mechanisms for this effect are unclear. Because antimicrobial agents of different classes and varying activity are effective across several vertebrate species, we proposed that such subtherapeutic administration alters the population structure of the gut microbiome as well as its metabolic capabilities. We generated a model of adiposity by giving subtherapeutic antibiotic therapy to young mice and evaluated changes in the composition and capabilities of the gut microbiome. Administration of subtherapeutic antibiotic therapy increased adiposity in young mice and increased hormone levels related to metabolism. We observed substantial taxonomic changes in the microbiome, changes in copies of key genes involved in the metabolism of carbohydrates to short-chain fatty acids, increases in colonic short-chain fatty acid levels, and alterations in the regulation of hepatic metabolism of lipids and cholesterol. In this model, we demonstrate the alteration of early-life murine metabolic homeostasis through antibiotic manipulation.