Effects of Aeration on the Synthesis of Poly(3-Hydroxybutyrate) from Glycerol and Glucose in Recombinant Escherichia coli

Bioreactor cultures of Escherichia coli recombinants carrying phaBAC and phaP of Azotobacter sp. FA8 grown on glycerol under low-agitation conditions accumulated more poly(3-hydroxybutyrate) (PHB) and ethanol than at high agitation, while in glucose cultures, low agitation led to a decrease in PHB formation. Cells produced smaller amounts of acids from glycerol than from glucose. Glycerol batch cultures stirred at 125 rpm accumulated, in 24 h, 30.1% (wt/wt) PHB with a relative molecular mass of 1.9 MDa, close to that of PHB obtained using glucose.Polyhydroxyalkanoates (PHAs), accumulated as intracellular granules by many bacteria under unfavorable conditions (5, 8), are carbon and energy reserves and also act as electron sinks, enhancing the fitness of bacteria and contributing to redox balance (9, 11, 19). PHAs have thermoplastic properties, are totally biodegradable by microorganisms present in most environments, and can be produced from different renewable carbon sources (8).Poly(3-hydroxybutyrate) (PHB) is the best known PHA, and its accumulation in recombinant Escherichia coli from several carbon sources has been studied (1, 13). In the last few years, increasing production of biodiesel has caused a sharp fall in the cost of its main by-product, glycerol (22). Its use for microbial PHA synthesis has been analyzed for natural PHA producers, such as Methylobacterium rhodesianum, Cupriavidus necator (formerly called Ralstonia eutropha) (3), several Pseudomonas strains (22), the recently described bacterium Zobellella denitrificans (7), and a Bacillus sp. (18), among others. Glycerol has also been used for PHB synthesis in recombinant E. coli (12, 15). PHAs obtained from glycerol were reported to have a significantly lower molecular weight than polymer synthesized from other substrates, such as glucose or lactose (10, 23).Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes that are involved in granule formation and/or have regulatory functions, such as phasins, granule-associated proteins that have been shown to enhance polymer synthesis and the number and size of PHA granules (17, 24). The phasin PhaP has been shown to exert a beneficial effect on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of strain K24KP, a recombinant E. coli that carries phaBAC and phaP of Azotobacter sp. FA8 (6).Because the redox state of the cells is known to affect the synthesis of PHB (1, 4, 14), the present study investigates the behavior of this recombinant strain under different aeration conditions, by using two substrates, glucose and glycerol, with different oxidation states.     

Redox driven metabolic tuning: carbon source and aeration affect synthesis of poly(3-hydroxybutyrate) in Escherichia coli

Growth and polymer synthesis were studied in a recombinant E. coli strain carrying phaBAC and phaP of Azotobacter sp. strain FA8 using different carbon sources and oxygen availability conditions. The results obtained with glucose or glycerol were completely different, demonstrating that the metabolic routes leading to the synthesis of the polymer when using glycerol do not respond to environmental conditions such as oxygen availability in the same way as they do when other substrates, such as glucose, are used. When cells were grown in a bioreactor using glucose the amount of polymer accumulated at low aeration was reduced by half when compared to high aeration, while glycerol cultures produced at low aeration almost twice the amount of polymer synthesized at the higher aeration condition. The synthesis of other metabolic products, such as ethanol, lactate, formate and acetate, were also affected by both the carbon source used and aeration conditions. In glucose cultures, lactate and formate production increased in low agitation compared to high agitation, while poly(3-hydroxybutyrate) synthesis decreased. In glycerol cultures, the amount of acids produced also increased when agitation was lowered, but carbon flow was mostly redirected towards ethanol and poly(3-hydroxybutyrate). These results indicated that carbon partitioning differed depending on both carbon source and oxygen availability, and that aeration conditions had different effects on the synthesis of the polymer and other metabolic products when glucose or glycerol were used.     

活体生物发光与荧光成像技术在干细胞研究中的应用

活体生物发光与荧光成像技术是近年发展起来的新兴技术,以其操作简便、灵敏度高、创伤性小在生命科学研究中有着较大的优势,目前已被广泛应用于基因标记、细胞凋亡、免疫细胞研究、肿瘤转移等诸多领域,尤其在新兴的干细胞研究方面更是发挥着不可替代的作用。综述了活体生物发光与荧光成像技术的原理、优势、应用范围及发展前景,特别对近年来该技术在胚胎干细胞的肝向分化、人造血干细胞重建小鼠造血系统、神经祖细胞治疗中枢神经系统肿瘤等方面的应用做了详细介绍。     

Modelling the freezing response of baker's yeast prestressed cells: a statistical approach

Aims: To study the effect of prestress conditions on the freezing and thawing (FT) response of two baker’s yeast strains and the use of statistical analysis to optimize resistance to freezing. Methods and Results: Tolerance to FT of industrial strains of Saccharomyces cerevisiae was associated to their osmosensitivity and growth phase. Pretreatments with sublethal stresses [40°C, 0·5 mol l^?1 NaCl, 1·0 mol l^?1 sorbitol or 5% (v/v) ethanol] increased freeze tolerance. Temperature or hyperosmotic prestresses increased trehalose contents, nevertheless no clear correlation was found with improved FT tolerance. Plackett–Burman design and response surface methodology were applied to improve freeze tolerance of the more osmotolerant strain. Optimal prestress conditions found were: 0·779 mol l^?1 NaCl, 0·693% (v/v) ethanol and 32·15°C. Conclusions: Ethanol, saline, osmotic or heat prestresses increased freezing tolerance of two phenotypically distinct baker’s yeast strains. A relationship among prestresses, survival and trehalose content was not clear. It was possible to statistically find optimal combined prestress conditions to increase FT tolerance of the osmotolerant strain. Significance and Impact of the Study: Statistically designed combination of prestress conditions that can be applied during the production of baker’s yeast could represent a useful tool to increase baker’s yeast FT resistance.     

Escherichia coli arcA mutants: metabolic profile characterization of microaerobic cultures using glycerol as a carbon source

ArcA is a global regulator that switches on the expression of fermentation genes and represses the aerobic pathways when Escherichia coli enters low oxygen growth conditions. The metabolic profile of E. coli CT1062 (DeltaarcA)and CT1061 (arcA2) grown in microaerobiosis with glycerol as carbon source were determined and compared with E. coli K1060, the arcA+ parent strain. Both arcA mutants achieved higher biomass yields than the wild-type strain. The production of acetate, formate, lactate, pyruvate, succinate and ethanol were determined in the supernatants of cultures grown on glycerol under microaerobic conditions for 48 h. The yield of extracellular metabolites on glycerol showed lower acid and higher ethanol values for the mutants. The ethanol/acetate ratio was 0.87 for the parent strain, 2.01 for CT1062, and 12.51 for CT1061. Accordingly, the NADH/NAD+ ratios were 0.18, 0.63, and 0.97, respectively. The extracellular succinate yield followed a different pattern, with yield values of 0.164 for K1060, 0.442 for CT1062 and 0.214 for CT1061. The dissimilarities observed can be attributed to the different effects exerted by the deletion and point mutations in a global regulator.     

人类胚胎心血管发生发育的形态学研究

心血管系统是胚胎第一个发生作用的系统,其形成是在一系列相关基因的调控作用下各种细胞迁移、分化和精确组合的结果.就目前人类胚胎心血管发生发育及其有关的影响因素等方面的研究进展做一简要的综述.     

青海栽培何首乌中蒽醌类成分的含量动态变化研究

采用RP-HPLC方法研究不同生长年份的栽培何首乌药用部位中蒽醌类物质的动态变化规律,来确定何首乌的最佳采收年份,为青海栽培何首乌作为药用提供科学依据.试验结果表明:不同采收年份的栽培何首乌药材中蒽醌类物质以8年生的含量最高.     

3320例儿童微量元素检测结果分析

目的:了解本院就诊儿童血微量元素水平,并分析其影响因素.方法:取儿童末梢血,采用原子吸收分光光度法检测,用统计软件进行分析.结果:男女儿童之间铅含量有差异,但铜、锌、钙、镁、铁、镉含量无差别.不同年龄组儿童之间微量元素含量有显著性差异,其中儿童锌、铁含量随着年龄的增长呈上升趋势,钙呈下降趋势.除镁外,不同年龄组儿童之间微量元素异常率有显著性差异,其中儿童钙、铁缺乏率随着年龄的增长分别呈上升、下降趋势,缺锌率呈波动状态,而儿童铜、镁缺乏率较低;儿童铅超标率在18.88%～29.37%之间,在4～5岁儿童时达到峰值;镉超标率在0.60～4.37%之间.结论:本院就诊儿童缺锌、钙、铁较普遍,但儿童铅中毒平均发生率低于全国平均水平.通过微量元素的检测,并有针对性地进行补充和排毒,将大大有利于儿童的健康成长.     

海南岛东寨港国家级自然保护区鸟类组成和区系的研究

报道了东寨港红树林自然保护区鸟类资源的调查,记录了该保护区鸟类１１８种,隶属１３目３５科,其中有５９种属冬候鸟,５７种属留鸟和２种属夏候鸟。根据中国动物地理区,东寨港保护区有６０种鸟类属古北界物种、４３种是东洋界物种和１５种是广布种。还分析了该保护区鸟类组成区系和鸟类生态群的特征。     

猪胎儿肾脏成纤维细胞体外培养体系的建立

本研究旨在建立猪胎儿肾脏成纤维细胞体外培养体系,并探讨其作为猪体细胞克隆供体的可能性。使用组织块培养法从体长为10cm以上的猪胎儿分离得到猪胎儿肾脏成纤维细胞,绘制了生长曲线,鉴定了细胞类型并且进行了细胞周期同期化效果的研究。结果表明:该培养体系可以支持猪胎儿肾脏成纤维细胞的体外生长,单个细胞均为梭形细胞,抗波形蛋白免疫荧光染色显示为阳性,而抗角形蛋白免疫荧光染色为阴性,分离到的细胞为胎儿肾脏成纤维细胞。使用血清饥饿法和接触抑制法诱导细胞进入G0/G1期,并且分别比较两者同期化效率,结果显示:血清饥饿2d和4d的同期化效率差异不显著,但都比8d组的高(88.97%和87.69%比82.45%,P<0.05);接触抑制4d、6d组间同期化效率差异不显著,但都比0d组的高(85.56%和85.89%比81.82%,P<0.05)。本研究在国内首次分离得到猪胎儿肾脏成纤维细胞,已经在体外传代培养到32代,其同期化效果好,可以作为体细胞克隆供体。