proC及putP基因的敲除对钝齿棒杆菌产L-精氨酸生理代谢的影响

为了选育精氨酸高产菌株,基于谷氨酸棒杆菌的基因组尺度代谢网络模型的指导,以钝齿棒杆菌(Corynebacterium crenatum)MT-M4为出发菌株,通过基因敲除技术构建了pro C和put P敲除菌株。摇瓶发酵结果表明,pro C敲除菌株精氨酸产量达到9.94g/L,较出发菌株提高了15.90%,葡萄糖转化率提高了26.02%。由于其生长受到明显抑制,因此在发酵液中外源添加24mmol/L的脯氨酸,结果发现其精氨酸产量达到12.22g/L,且菌株恢复生长。put P敲除菌株精氨酸产量达到12.23g/L,较出发菌株提高了42.70%,葡萄糖转化率提高了49.31%。以上结果显示,put P的敲除比pro C的敲除更有利于精氨酸的合成,put P的敲除对菌株的生理代谢基本无影响且无需外添加脯氨酸。     

The role of inorganic phosphate in the regulation of C4 photosynthesis

Inorganic phosphate participates in many fundamental processes within the plant cell. Its broad influence on plant metabolism is related to such key operations as metabolite transport, enzyme regulation and carbohydrate metabolism in general. This review discusses these topics with special emphasis on the role assigned to this ubiquitous anion within the C₄ pathway of photosynthesis.Abbreviations DHAP dihydroxyacetone phosphate - Ga3P glyceraldehyde-3-phosphate - NAD(P)-ME-NAD(P) dependent malic enzyme - PEP phosphoenolpyruvate - 3-PGA 3-phosphoglycerate - PFK and PFP-ATP- and PPi dependent fructose-6-phosphate 1-phosphotransferase - PPDK pyruvate:orthophosphate dikinase - RPPC reductive pentose-phosphate cycle - RuBisCO ribulose bisphosphate carboxylase-oxygenase - SPS sucrose-6-phosphate synthase     

Plasmid RFLP profiling and DNA homology inThermus isolated from hot springs of different geographical areas

Thirty-four strains belonging to various species of the genus Thermus (T. aquaticus, "T. thermophilus," "T. brockianus," T. scotoductus, and genomic species 2) isolated from hot springs of different geographical areas were examined for plasmid content and restriction fragment length polymorphism (RFLP) of plasmid DNAs. The four strains of the numerical taxonomy cluster E of genomic species 2 did not harbor plasmid DNA. Overall examination of the HindIII-RFLP profiling of plasmid DNA showed considerable variability between and within genomic species, with the exception of presumed clonal isolates. In spite of this heterogeneity, HindIII plasmid digests within a numerical taxonomic cluster gave a subset of restriction fragments of similar or identical length. Strains belonging to genomic species 2 or unclassified isolates from S. Pedro do Sul that harbored plasmid DNA (7 of the 14 strains studied) exhibited strong DNA homology between plasmid regions. No homologous sequences to these plasmid regions were found in chromosomal DNA from strains isolated from S. Pedro do Sul in which no plasmids were detected. The strains belonging to T. scotoductus formed two plasmid DNA homology groups, as estimated by probing with a plasmid fragment that coincided with the two numerical taxonomy clusters proposed previously. Among the other species, homology of plasmid regions was also found between some strains. Strong homology was also found between plasmid regions from some strains of different taxonomic groups, isolated from the same and from different sources, suggesting that these sequences are highly conserved in plasmids present in Thermus. For plasmid-containing strains, results of plasmid RFLP profiling/DNA homology appear promising for the typing of Thermus at the level of biotypes or of individual strains, namely, for monitoring the diversity and frequency of isolates from a particular hot spring. Received: 24 October 1994 / Accepted: 6 March 1995     

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.     

黑线仓鼠繁殖输出与基础代谢率的关系

为了解黑线仓鼠繁殖输出与基础代谢率(BMR)的关系,阐明最大持续能量收支(SusMR)的限制水平, 揭示哺乳期能量收支对策,本文测定了哺乳期黑线仓鼠的体重、摄食量、BMR 和身体组成,以及哺乳期的胎仔数、胎仔重和泌乳能量支出(MEO)。结果显示,黑线仓鼠哺乳期体重降低了15.0 ± 0.8% , 摄食量显著增加, 哺乳高峰期平均摄食量为13.9 ± 0.3 g /d, 摄入能为222.1 ± 5.3 kJ/ d, 比哺乳初期增加121% , 比对照组高288% ;哺乳高峰期MEO 为62.4 ± 2.3 kJ/ d, 哺乳末期BMR 为49.7 ± 1.1 kJ/ d; 断乳时平均胎仔数4.7 ± 0.2、窝胎仔重50.5 ±1.6 g; 哺乳末期BMR 比对照组增加48% ,BMR 与消化系统各器官的相关性高于对照组; BMR 与胎仔数、胎仔重、乳腺重量和MEO 显著正相关。结果表明:初次繁殖的黑线仓鼠哺乳期SusMR 限制为4.47 ×BMR, 在自身维持和繁殖输出之间采取了“权衡分配”的原则,通过体重降低以减少BMR 的增加幅度, 从而有利于繁殖输出。     

Activation of peroxisome proliferator-activated receptor-alpha stimulates both differentiation and fatty acid oxidation in adipocytes

Peroxisome proliferator-activated receptor-α (PPARα) is a dietary lipid sensor, whose activation results in hypolipidemic effects. In this study, we investigated whether PPARα activation affects energy metabolism in white adipose tissue (WAT). Activation of PPARα by its agonist (bezafibrate) markedly reduced adiposity in KK mice fed a high-fat diet. In 3T3-L1 adipocytes, addition of GW7647, a highly specific PPARα agonist, during adipocyte differentiation enhanced glycerol-3-phosphate dehydrogenase activity, insulin-stimulated glucose uptake, and adipogenic gene expression. However, triglyceride accumulation was not increased by PPARα activation. PPARα activation induced expression of target genes involved in FA oxidation and stimulated FA oxidation. In WAT of KK mice treated with bezafibrate, both adipogenic and FA oxidation-related genes were significantly upregulated. These changes in mRNA expression were not observed in PPARα-deficient mice. Bezafibrate treatment enhanced FA oxidation in isolated adipocytes, suppressing adipocyte hypertrophy. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα was recruited to promoter regions of both adipogenic and FA oxidation-related genes in the presence of GW7647 in 3T3-L1 adipocytes. These findings indicate that the activation of PPARα affects energy metabolism in adipocytes, and PPARα activation in WAT may contribute to the clinical effects of fibrate drugs.     

Microbial production of propionic acid from propionibacteria: Current state,challenges and perspectives

Propionic acid (PA) is an important building block chemical and finds a variety of applications in organic synthesis, food, feeding stuffs, perfume, paint and pharmaceutical industries. Presently, PA is mainly produced by petrochemical route. With the continuous increase in oil prices, public concern about environmental pollution, and the consumers’ desire for bio-based natural and green ingredients in foods and pharmaceuticals, PA production from propionibacteria has attracted considerable attention, and substantial progresses have been made on microbial PA production. However, production of PA by propionibacteria is facing challenges such as severe inhibition of end-products during cell growth and the formation of by-products (acetic acid and succinic acid). The integration of reverse metabolic engineering and systematic metabolic engineering provides an opportunity to significantly improve the acid tolerance of propionibacteria and reduce the formation of by-products, and makes it feasible to strengthen the commercial competition of biotechnological PA production from propionibacteria to be comparable to the petrochemical route.     

Modeling of yeast metabolism and process dynamics in batch fermentation

Much is known about yeast metabolism and the kinetics of industrial batch fermentation processes. In this study, however, we provide the first tool to evaluate the dynamic interaction that exists between them. A stoichiometric model, using wine fermentation as a case study, was constructed to simulate batch cultures of Saccharomyces cerevisiae. Five differential equations describe the evolution of the main metabolites and biomass in the fermentation tank, while a set of underdetermined linear algebraic equations models the pseudo-steady-state microbial metabolism. Specific links between process variables and the reaction rates of metabolic pathways represent microorganism adaptation to environmental changes in the culture. Adaptation requirements to changes in the environment, optimal growth, and homeostasis were set as the physiological objectives. A linear programming routine was used to define optimal metabolic mass flux distribution at each instant throughout the process. The kinetics of the process arise from the dynamic interaction between the environment and metabolic flux distribution. The model assessed the effect of nitrogen starvation and ethanol toxicity in wine fermentation and it was able to simulate fermentation profiles qualitatively, while experimental fermentation yields were reproduced successfully as well.     

白头鹎的代谢率与器官重量在季节驯化中的可塑性变化

动物能量代谢的生理生态特征与物种的分布和丰富度密切相关,基础代谢率(BMR)是恒温动物维持正常生理机能的最小产热速率,是动物在清醒时维持身体各项基本功能所需的最小能量值,是内温动物能量预算的重要组成部分.本研究测定了白头鹎(Pycnonotus sinensis)的BMR、内部器官(肝、心、肌胃、小肠、肾和整体消化道)和肌肉的重量,分析了白头鹎内部器官和肌肉重量的季节性变化及与BMR的关系.方差分析表明,白头鹎的BMR存在明显的季节性变化,冬季较高,夏季最低.其内部器官及肌肉重量的变化同样有明显的季节性.相关分析表明,白头鹎的BMR与肝、心、消化道等内部器官和肌肉重量存在明显的相关性.