基于GC-MS的阿维链霉菌代谢物组学研究方法的建立

基于GC-MS分析平台,建立了阿维链霉菌代谢物组样品制备过程中的菌体分离、细胞清洗、细胞淬灭及代谢物提取条件,并对阿维菌素高产菌株和野生菌株胞内代谢物组进行了初步分析。采用"冷冻离心-细胞清洗(3次)-液氮淬灭(5min)-珠磨破壁(3个循环,每个循环48 s)-提取(氯仿∶乙醇∶水=2∶2∶1)"的方法,可以定性和定量检测阿维链霉菌胞内包括氨基酸、有机酸、脂肪酸、糖类等59种差异化合物。建立的方法能够有效地用于阿维链霉菌胞内代谢物的分析。     

链霉菌次级代谢调控相关的双组分系统研究进展

链霉菌具有强大的次级代谢能力, 能够产生众多具有生物活性的次级代谢产物, 如目前广泛应用的抗生素、抗肿瘤药物以及免疫抑制剂等。在链霉菌中, 次级代谢产物的生物合成受到包括途径特异性、多效性以及全局性调控基因在内的多层次严格调控。关键调控基因的缺失或过表达可以显著影响次级代谢产物的生物合成, 提示对于链霉菌次级代谢重要调控基因的功能及其作用机制的研究具有巨大的潜在应用价值。其中, 作为细菌信号传导系统的双组分系统(Two-component system, TCS)一直是大家研究的关注点。越来越多的研究表明TCS在链霉菌次级代谢过程中发挥着全局性的调控功能。本文重点介绍链霉菌模式菌株——天蓝色链霉菌中TCS(包括典型TCS)、孤立的组氨酸蛋白激酶(HK)以及应答调控蛋白(RR)参与次级代谢调控的研究进展。这些TCS的功能鉴定及机制解析为工业链霉菌的定向遗传改造以提高重要次级代谢产物的含量提供了理论依据。     

聚苹果酸生产菌株出芽短梗霉菌代谢组学样品前处理方法的优化

为了得到更加准确和完整的胞内代谢物信息,从而更加真实的反映微生物胞内代谢情况,基于GC-MS平台,从淬灭剂、提取剂和溶解剂3个方面对代谢组学样品制备过程进行了系统性的优化。对比了不同淬灭剂对胞内代谢物的渗出及细胞损伤情况,结果表明60%甲醇/0.9%Na Cl效果最佳。研究了不同提取剂、溶解剂的提取和溶解效果,结果显示60%甲醇提取效果显著,吡啶溶解效果突出。最终确定了出芽短梗霉菌代谢组样品最适前处理方法 :预冷的60%甲醇/0.9%Na Cl淬灭细胞,液氮研磨和反复冻融破碎细胞,预冷的60%甲醇提取,最后采用吡啶溶解提取物并采用MSTFA 40℃衍生90 min。采用该方法可以检测出300多种代谢组分,包括大量氨基酸、有机酸和糖类物质。本方法可以有效的应用于出芽短梗霉菌代谢组学样品的研究。     

苜蓿中华根瘤菌代谢组学样品制备方法的研究

代谢组样品制备是代谢组学研究的基础。本文以维生素B12生产菌株苜蓿中华根瘤菌Sinorhizobium meliloti 320为研究对象,通过检测细胞损伤、ATP泄漏、代谢物回收效率以及细胞代谢淬灭效率综合评价细胞淬灭方法,同时对5种提取试剂的提取效率进行比较优化胞内代谢物的提取方法。最终获得苜蓿中华根瘤菌S.meliloti 320的胞内代谢组学样品制备较佳条件:即-20℃40%甲醇淬灭细胞,过滤收集淬灭细胞,甲醇/乙腈/水(体积比为2∶2∶2,外加0.1%的甲酸)与50%甲醇相结合提取胞内代谢物。实验结果显示-20℃的40%甲醇(通过过滤收集细胞)对细胞膜的损伤较小,且细胞代谢淬灭效率和回收效率较高;甲醇/乙腈/水(体积比为2∶2∶2,外加0.1%的甲酸)与50%的甲醇对胞内代谢物的提取效率较高且有互补作用。     

基于比较代谢组学的理性优化方法提高阿维菌素产量

【目的】利用比较代谢组学的分析方法,研究不同发酵培养基中阿维链霉菌的胞内代谢差异,揭示合成阿维菌素的关键代谢物和代谢途径,再通过理性优化添加主要关键代谢物,提高阿维菌素产量。【方法】对M1和M2培养基中生长的菌体进行基于GC-MS的胞内代谢组学分析,通过理性添加强化前体代谢物,确定阿维菌素高产培养基。【结果】GC-MS共检测到232种物质,能够精确匹配70种胞内代谢物,通过PCA和PLS分析,最终确定了21种已知的胞内代谢物与阿维菌素的生物合成密切相关。其中乳酸、丙酮酸、琥珀酸、苏氨酸、异亮氨酸、缬氨酸和油脂类物质对阿维菌素的产量影响较为显著。通过单独或组合优化添加这些前体,阿维菌素的产量从5.36 g/L提高到了5.92 g/L,增加了10.4%。【结论】基于比较代谢组学分析的理性优化培养基的方法可有效提高阿维菌素的产量,并为提高当下生物基产品的产量提供了新思路。     

放线菌中亮氨酸应答调控蛋白的生物学功能及其调控机理

放线菌是一类革兰氏阳性细菌,可产生氨基酸等初级代谢产物和抗生素等次级代谢产物,其广泛用于食品、医药、添加剂及化妆品行业。此外,还有少数放线菌,如分枝杆菌等,是可以引起人和动植物病害的病原菌。亮氨酸应答调控蛋白(Leucine-responsive regulatory protein,Lrp)是一类在氨基酸代谢及其相关代谢过程中的重要转录调控子,能够应答各种氨基酸,参与调控微生物细胞的多个生理过程,例如氨基酸代谢和转运、中心代谢、细菌的持久性和毒力等。本文总结了放线菌Lrp的生物学功能,并综述了放线菌中不同种属Lrp以及天蓝色链霉菌和红色糖多孢菌Lrp调控机理的研究进展。     

链霉菌的全局调控蛋白DasR

链霉菌能够产生多种次级代谢产物,在临床、农牧业、生物技术等领域具有重要应用价值;对链霉菌的调控网络进行深入研究有助于提高次级代谢产物产量并发现新的次级代谢产物.链霉菌中次级代谢产物生物合成按调控通路分为全局调控与途径特异性调控,其中全局调控蛋白可靶向多种通路特异调控基因和生物合成基因,在链霉菌的生命活动中发挥着更为普遍...     

链霉菌转运系统研究进展

链霉菌是具有复杂生命周期的革兰氏阳性细菌,其丰富多样的次级代谢产物使得链霉菌成为天然抗生素来源最为广泛的菌株。目前链霉菌研究主要集中在次级代谢和形态分化过程。近年来,因链霉菌转运系统在次级代谢和形态分化等过程中发挥重要作用而得到研究者们的极大关注。链霉菌转运系统不仅能够通过信号转导系统直接参与菌丝体形态分化过程,而且能够运送抗生素等次级代谢产物,使其在链霉菌工业生产方面具有极大的应用前景。除此之外,转运系统作为菌体“第一道门”,能够运送营养物质来改变初级代谢过程,进而影响形态分化和次级代谢等过程,使菌体能迅速适应复杂的环境。因此,链霉菌中转运系统的研究对其形态分化调控通路的阐释具有重要的指导意义。     

刺糖多孢菌中铵载体蛋白基因amtS的克隆及功能研究

目的:铵离子是细胞内合成各种核酸、氨基酸和辅助因子等含氮化合物的重要原料之一。微生物细胞膜上的铵载体蛋白介导了铵离子的转运。通过异源表达刺糖多孢菌中铵载体蛋白基因,研究其对链霉菌产孢能力和次级代谢产物产量的影响。方法:从刺糖多孢菌S04-41菌株中克隆铵载体蛋白基因amt S,通过接合转移导入天蓝色链霉菌M145和变铅青链霉菌TK24中,分析比较amt S基因的异源表达对其产孢能力和次级代谢产物产量的影响。结果:天蓝色链霉菌重组菌株M145/p MF-amt S和变铅青链霉菌重组菌株TK24/p MF-amt S中放线紫红素的产量分别提高了2.85倍和30.02倍。结论:刺糖多孢菌中的铵载体蛋白能够提高链霉菌中次生代谢产物的产量,为进一步研究该基因的功能与对刺糖多孢菌中多杀菌素合成的作用奠定了重要基础。     

无机磷酸盐对链霉菌合成次级代谢产物的影响

链霉菌一个突出的特征是具有合成丰富的次级代谢产物的能力,许多次级代谢产物,如抗生素、免疫抑制剂、抗癌物质等在临床医药、水产养殖业等领域具有重要的应用价值。链霉菌次级代谢产物的合成常与环境中的营养因子有着密切的关系,在代谢水平上综述了无机磷酸盐对链霉菌合成次级代谢产物的影响,并在转录水平上阐释了双组分信号转导系统PhoR-PhoP的分子调控机制。PhoR-PhoP能够感应环境中的无机磷酸盐信号,当无机磷酸盐的浓度低于一定"阈值"时,PhoP蛋白作为主导的调控因子将抑制参与中心代谢和次级代谢等一系列基因的转录表达,减慢磷酸盐的消耗,并激活磷酸盐的摄取和转运系统及时补充胞内的磷酸盐,最终影响链霉菌次级代谢产物的合成和形态发育分化。     

IncP-7 naphthalene-degradative plasmids from Pseudomonas putida

Abstract Seven naphthalene-degrading and two naphthalene and camphor-degrading Pseudomonas putida strains were isolated from marine sediments. Most of them carried two plasmids, of molecular size 60 and 200 kb. The naphthalene and salicylate metabolism determinants were transferred to a P. putida strain by conjugation, and the transconjugants acquired either both plasmids or only the 200-kb one. These plasmids appear to belong to the IncP-7 group and encode for catabolism of naphthalene and salicylate, but not camphor.     

城市代谢研究评述:内涵与方法

随着城市发展面临的生态问题日益显著,部分研究者试图通过对自然生态系统进行类比来寻求解决途径,城市代谢理论应运而生。当以系统科学来研究城市生态系统时,哲学思想的引入为探索和城市及城市代谢的内涵提供了最原始的桥梁。因此,在介绍城市代谢内涵与研究进展的基础上,融合产业、家庭、社会等多尺度代谢理论,对城市代谢的边界进行扩展,将其分为狭义与广义两类,结合亚里士多德的"四因说"对其质料因(组分)、形式因(结构)、动力因(驱动力)和目的因(功能)进行识别分析,据此将城市代谢研究方法归纳为质料、形式和混合研究方法三类,并提出未来研究的主要动向和解决手段。城市代谢"四因图"可为相关研究者提供参考。     

脂质代谢在乳腺癌发病中的作用

乳腺癌已经成为全球第一大癌症,其发病机制及治疗方法的探索越来越受到人们重视。脂质代谢异常是癌细胞中最突出的代谢改变之一,探索乳腺癌细胞中脂质代谢的改变,以寻找新的诊断指标和治疗靶点是至关重要的。本文从脂肪酸代谢、甘油三酯代谢、胆固醇代谢和脂质代谢信号通路4个方面介绍脂质代谢异常在乳腺癌中的研究进展,为靶向脂质代谢治疗乳腺癌提供新思路和新方法。     

A pilot study of the effects of pioglitazone and rosiglitazone on de novo lipogenesis in type 2 diabetes

Treatment of type 2 diabetes mellitus (T2DM) patients with pioglitazone results in a more favorable lipid profile, and perhaps more favorable cardiac outcomes, than treatment with rosiglitazone. Pioglitazone treatment increases VLDL-triacylglycerol clearance, but the role of de novo lipogenesis (DNL) has not been explored, and no direct comparison has been made between the thiazolidinediones (TZDs). Twelve subjects with T2DM and hypertriacylglyceridemia were randomized to either rosiglitazone or pioglitazone treatment. Stable isotope infusion studies were performed at baseline and after 20 weeks of treatment. Both treatments reduced glucose and HbA(1c) concentrations equally. Pioglitazone treatment resulted in a 40% reduction in hepatic DNL (P < 0.01) and in a 25% reduction in hepatic glucose production (P < 0.05), while rosiglitazone did not significantly change either parameter, although comparisons of changes between treatments were not significantly different. These pilot results indicate that pioglitazone reduces hepatic DNL while rosiglitazone does not. Larger follow-up studies are required to confirm differential effects of these agents definitively. The reduction in DNL may underlie altered assembly or atherogenicity of lipoprotein particles and may reflect PPARalpha or other non-PPARgamma actions on the liver by pioglitazone. These differences might help explain previously reported differences in lipid profiles and cardiovascular disease outcomes for rosiglitazone and pioglitazone.     

Effects of Cadmium on the Metabolic Activity of Avena sativa Plants Grown in Soil or Hydroponic Culture

Oat (Avena sativa L.) plants cultured in soil and hydroponic culture were treated with cadmium [0.154 mg g^–1 (dry soil) and 100 M CdSO₄, respectively] for 21 d and growth rate and various biochemical processes were studied. Applied cadmium reduced plant growth and chlorophyll content. Changes in activity of enzymes involved in C, N and S metabolism and in guaiacol peroxidase activity were observed. In particular, O-acetylserine sulphydrylase (OASS; EC 4.2.99.8) activity was increased by Cd exposure in both growth conditions, probably as a resistance mechanism to cadmium based on the production of phytochelatins. Results show that both field and hydroponic conditions represent suitable systems for investigating Cd effects on plant growth and metabolism.     

Macrophage polarization state affects lipid composition and the channeling of exogenous fatty acids into endogenous lipid pools

Adipose-tissue-resident macrophages (ATMs) maintain metabolic homeostasis but also contribute to obesity-induced adipose tissue inflammation and metabolic dysfunction. Central to these contrasting effects of ATMs on metabolic homeostasis is the interaction of macrophages with fatty acids. Fatty acid levels are increased within adipose tissue in various pathological and physiological conditions, but appear to initiate inflammatory responses only upon interaction with particular macrophage subsets within obese adipose tissue. The molecular basis underlying these divergent outcomes is likely due to phenotypic differences between ATM subsets, although how macrophage polarization state influences the metabolism of exogenous fatty acids is relatively unknown. Herein, using stable isotope-labeled and nonlabeled fatty acids in combination with mass spectrometry lipidomics, we show marked differences in the utilization of exogenous fatty acids within inflammatory macrophages (M1 macrophages) and macrophages involved in tissue homeostasis (M2 macrophages). Specifically, the accumulation of exogenous fatty acids within triacylglycerols and cholesterol esters is significantly higher in M1 macrophages, while there is an increased enrichment of exogenous fatty acids within glycerophospholipids, ether lipids, and sphingolipids in M2 macrophages. Finally, we show that functionally distinct ATM populations in vivo have distinct lipid compositions. Collectively, this study identifies new aspects of the metabolic reprogramming that occur in distinct macrophage polarization states. The channeling of exogenous fatty acids into particular lipid synthetic pathways may contribute to the sensitivity/resistance of macrophage subsets to the inflammatory effects of increased environmental fatty acid levels.     

Effects of triacylglycerol and diacylglycerol oils on blood clearance, tissue uptake, and hepatic apolipoprotein B secretion in mice

Prior studies have suggested that FAs liberated in the small intestine from ingested 1,3-diacylglycerol (DAG) are inefficiently incorporated into triglyceride (TG) in enterocytes, with less chylomicron TG entering the circulation postprandially. We found less TG, but more monacylglyerol and DAG, with similar total acylglycerol in newly secreted chylomicrons after oral DAG or triacylglycerol (TAG). However, clearance of DAG-chylomicrons was more rapid than that of TAG-chylomicrons; this was associated with more efficient in vitro LPL-mediated lipolysis of DAG-derived chylomicrons. Intravenously infused DAG was also cleared faster than TAG in normal mice, via both LPL-mediated lipolysis and apolipoprotein E (apoE)-dependent hepatic uptake. Infusions of TAG, but not DAG, increased plasma TG levels. Greater delivery of DAG-derived FA to the liver during infusion of DAG led to greater TG secretion versus TAG; this allowed the maintenance of similar hepatic TG levels after DAG and TAG infusions. Of note, apoB secretion was similar after DAG versus TAG, indicating the assembly of larger very low density lipoproteins after DAG. In conclusion, reduced plasma TG levels, after oral or intravenous DAG, result from more efficient clearance of DAG by both LPL lipolysis and apoE-mediated hepatic endocytosis. DAG emulsions may by useful for intravenous nutrition in people with preexisting hypertriglyceridemia.     

Mechanisms for the prevention of vitamin E excess

The liver is at the nexus of the regulation of lipoprotein uptake, synthesis, and secretion, and it is the site of xenobiotic detoxification by cytochrome P450 oxidation systems (phase I), conjugation systems (phase II), and transporters (phase III). These two major liver systems control vitamin E status. The mechanisms for the preference for α-tocopherol relative to the eight naturally occurring vitamin E forms largely depend upon the liver and include both a preferential secretion of α-tocopherol from the liver into the plasma for its transport in circulating lipoproteins for subsequent uptake by tissues, as well as the preferential hepatic metabolism of non-α-tocopherol forms. These mechanisms are the focus of this review.     

The effect of different growth conditions on dark and light carbon assimilation in Littorella uniflora

The effect of long-term exposure to different inorganic carbon, nutrient and light regimes on CAM activity and photosynthetic performance in the submerged aquatic plant, Littorella uniflora (L.) Aschers was investigated. The potential CAM activity of Littorella was highly plastic and was reduced upon exposure to low light intensities (43 μmol m⁻² s⁻¹), high CO₂ concentrations (5.5 mM, pH 6.0) or low levels of inorganic nutrients, which caused a 25–80% decline in the potential maximum CAM activity relative to the activity in the control experiments (light: 450 μmol m⁻² s⁻¹; free CO₂: 1.5 mM). The CAM activity was regulated more by light than by CO₂, while nutrient levels only affected the activity to a minor extent. The minor effect of low nutrient regimes may be due to a general adaptation of isoetid species to low nutrient levels.
The photosynthetic capacity and CO₂ affinity was unaffected or increased by exposure to low CO₂, irrespective of nutrient levels. High CO₂, low nutrient and low light, however, reduced the capacity by 22–40% and the CO₂ affinity by 35-45%, relative to control.
The parallel effect of growth conditions on CAM activity and photosynthetic performance of Littorella suggest that light and dark carbon assimilation are interrelated and constitute an integrated part of the carbon assimilation physiology of the plant. The results are consistent with the hypothesis that CAM is a carbon-conserving mechanism in certain aquatic plants. The investment in the CAM enzyme system is beneficial to the plants during growth at high light and low CO₂ conditions.