Fermentative production of isobutene

Isobutene (2-methylpropene) is one of those chemicals for which bio-based production might replace the petrochemical production in the future. Currently, more than 10 million metric tons of isobutene are produced on a yearly basis. Even though bio-based production might also be achieved through chemocatalytic or thermochemical methods, this review focuses on fermentative routes from sugars. Although biological isobutene formation is known since the 1970s, extensive metabolic engineering is required to achieve economically viable yields and productivities. Two recent metabolic engineering developments may enable anaerobic production close to the theoretical stoichiometry of 1isobutene + 2CO₂ + 2H₂O per mol of glucose. One relies on the conversion of 3-hydroxyisovalerate to isobutene as a side activity of mevalonate diphosphate decarboxylase and the other on isobutanol dehydration as a side activity of engineered oleate hydratase. The latter resembles the fermentative production of isobutanol followed by isobutanol recovery and chemocatalytic dehydration. The advantage of a completely biological route is that not isobutanol, but instead gaseous isobutene is recovered from the fermenter together with CO₂. The low aqueous solubility of isobutene might also minimize product toxicity to the microorganisms. Although developments are at their infancy, the potential of a large scale fermentative isobutene production process is assessed. The production costs estimate is 0.9 € kg⁻¹, which is reasonably competitive. About 70% of the production costs will be due to the costs of lignocellulose hydrolysate, which seems to be a preferred feedstock.     

羊栖菜多糖诱导lovo细胞凋亡及其机理探讨

本课题研究羊栖菜多糖(Sargassum Fusiforme Polysaccharides,SFPS)诱导人大肠癌lovo细胞凋亡及凋亡过程中Caspase-3的变化及其意义。MTT法检测SFPS对大肠癌细胞增殖的抑制率;通过电镜、琼脂糖凝胶电泳、流式细胞术鉴定细胞凋亡;应用Western blot法测定Caspase-3酶原的变化; RT-PCR检测Caspase-3 mRNA表达。结果显示:SFPS作用lovo细胞24,36,48和72h的IC_(50)分别为375,355,178和60mg/L,表明对lovo细胞具有显著生长抑制作用。在电镜下,可见明显的细胞凋亡特征:细胞膜表面微绒毛减少、染色质固缩、边集,凋亡小体形成。在琼脂糖凝胶电泳中,药物浓度为5-300mg/L作用24h后,显示有凋亡细胞特有的DNA梯状条带;而500mg/L处理后梯状条带模糊,开始出现“涂片状”,表明在高药物浓度的作用下,细胞有坏死。流式细胞仪测得细胞凋亡率有剂量的依赖性;DNA直方图出现亚G1峰,但细胞周期时相的分布无明显改变。SFPS处理lovo细胞后,发现Caspase-3酶原蛋白表达降低,Caspase-3的mRNA高表达,并具有剂量和时间的依赖性。实验结果提示,SFPS在体外能够诱导lovo细胞凋亡,这可能是SFPS抑制肿瘤增殖的机制之一,而Caspase-3的活化参与了SFPS诱导lovo细胞凋亡的调控。     

Effects of methotrexate on the viscoelastic properties of single cells probed by atomic force microscopy

Methotrexate is a commonly used anti-cancer chemotherapy drug. Cellular mechanical properties are fundamental parameters that reflect the physiological state of a cell. However, so far the role of cellular mechanical properties in the actions of methotrexate is still unclear. In recent years, probing the behaviors of single cells with the use of atomic force microscopy (AFM) has contributed much to the field of cell biomechanics. In this work, with the use of AFM, the effects of methotrexate on the viscoelastic properties of four types of cells were quantitatively investigated. The inhibitory and cytotoxic effects of methotrexate on the proliferation of cells were observed by optical and fluorescence microscopy. AFM indenting was used to measure the changes of cellular viscoelastic properties (Young’s modulus and relaxation time) by using both conical tip and spherical tip, quantitatively showing that the stimulation of methotrexate resulted in a significant decrease of both cellular Young’s modulus and relaxation times. The morphological changes of cells induced by methotrexate were visualized by AFM imaging. The study improves our understanding of methotrexate action and offers a novel way to quantify drug actions at the single-cell level by measuring cellular viscoelastic properties, which may have potential impacts on developing label-free methods for drug evaluation.     

一个水稻显性高秆突变体的遗传分析和基因定位

从水稻(Oryza sativa L.)的两个半矮秆籼稻品种6442S-7和蜀恢881杂交F2代群体中发现一个高秆突变体D111,其株高和秆长分别比亲本蜀恢881增加63.0%和87.0%。用205个微卫星标记分析D111及其原始亲本6442S-7和蜀恢881之间的基因组DNA多态性,结果未发现D111具有2个原始亲本都没有的新带型,证明D111的确是6442S-7和蜀恢881的杂交后代发生基因突变产生的。将D111分别与蜀恢881、蜀恢527、明恢63、9311、IR68、G46B等6个半矮秆品种和高秆对照品种南京6号杂交,分析F1和F2代株高的遗传行为,结果表明D111的高秆性状由一对显性基因控制,且该基因与南京6号的高秆基因紧密连锁或等位。以蜀恢527/D111 F2群体为定位群体,运用微卫星标记将D111显性高秆突变基因定位于水稻第一染色体长臂,与RM212、RM302和RM472的遗传距离分别是27.7 cM、25.5 cM和6.0 cM,该基因暂命名为LC(t)。认为D111是首例从半矮秆品种自然突变产生的水稻显性高秆突变体,LC(t)为首次定位的水稻显性高秆突变基因。此外,将上述基因定位结果与Causse等(1994)和Temnykh等(2000; 2001)发表的水稻分子连锁图谱进行比较,发现LC(t)基因恰巧位于与水稻“绿色革命基因”sd1相同或十分相近的染色体区域,因此,还就LC(t)基因与sd1基因之间的可能关系进行了讨论。     

HFRSV在平皿表面室温暴露后存活力的定量研究

将肾综合症出血热病毒布于玻璃平皿表面,室温条件下分别暴露0、30、60、90和120min,观察病毒的定量存活情况.结果在经过120min的暴露后,HFRSV的效价仍高达104.23 TCID50.这一结果为HFRS的流行病学及其防治研究提供了重要的基础数据.     

Biodegradation of pyridine by an isolated bacterial consortium/strain and bio-augmentation of strain into activated sludge to enhance pyridine biodegradation

Pyridine and pyridine based products are of major concern as environmental pollutants due to their recalcitrant, persistent, toxic and teratogenic nature. In this study, we describe biodegradation of pyridine by an isolated consortium/strain under aerobic condition. Batch experiment results reveal that at lower initial pyridine concentrations (1-20 mg l(-1)), almost complete degradation was observed whereas at higher concentration (30-50 mg l(-1)), the degradation efficiency was dropped significantly. This may be due to inhibitory effect of pyridine at higher concentrations. The value of decay and yield coefficient was also determined. Furthermore, the bio-augmentation of isolated consortium/strain into the activated sludge consortium in different quantity has been also done and the effect of bio-augmentation on degradation has been studied. The results reveal that as the quantity of bio-augmentation increases, the degradation of pyridine increases. At 25% bio-augmentation, complete degradation of 20 mg l(-1) of pyridine can be achieved within 96 h of incubation. Thus, the study concluded that the bio-augmentation of the isolated consortium/strain into the sludge enhances the pyridine degradation efficiency of the biomass.     

Use of diluted medium in repeated-batch fermentation for production of lignin peroxidase by <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Lignin peroxidase has been extensively studied due to the potential use of this enzyme in environmental pollution control. Important aspects of the production of the enzyme by the white rot fungus, Phanerochaete chrysosporium, include the improvement of yield results and cell maintenance. In the present work, Phanerochaete chrysosporium was immobilized in polyurethane foam and used for repeated-batch fermentations with various dilution of the initial medium (D), and lignin peroxidase production was investigated. The peak of 283 ± 17.5 U lignin peroxidase/l production rate was obtained at a D of 1/5, with significantly lower production rates seen at higher and lower dilution ratios. When six cycles of repeated-batch fermentation were conducted using a D of 1/5, the results revealed that at least four cycles of repeated-batch fermentation were possible with a high lignin peroxidase production rate under a cut-off value of 178 ± 3.87 U/l. Furthermore, the cell-free culture broth could be successfully concentrated to 2,800 U/l by ultrafiltration. Thus, the present study shows that optimizing the dilution of the utilized nutritional medium can improve repeated batch production of lignin peroxidase from immobilized P. chrysosporium, in terms of both cycle number and output.     

Glucose ester enabled acylation in plant specialized metabolism

Acylation of core compound skeletons, together with other modifications, plays a significant role in producing the incredible diversity of plant specialized metabolites. Two major classes of acyltransferases, the BAHD and serine carboxypeptidase-like (SCPL) acyltransferases, can bring together through acylation compounds from the same or divergent metabolic pathways. BAHD acyltransferases (BAHD-ATs) employ CoA thioesters as the activated substrate, SCPL acyltransferases (SCPL-ATs), on the other hand, utilize β-acetal esters, typically glucose esters formed by UDP glycosyltransferases (UGTs). While the general trend of high energy glucose ester enabled acyltransfers is seen throughout the spermatophytes (seed plants), the specific metabolites that are conjugated appear to be lineage specific. In this review, we examine the reaction mechanism, biochemical property and evolutionary relationship of SCPL-ATs that utilize various glucose ester donors and acceptors from the same or different plant specialized metabolic pathways. The occurrence and taxonomic distribution of galloylated flavan-3-ols, hydrolyzable tannins and galloylated flavonols are also evaluated. Furthermore, glucose ester (acyl donor)-forming UGT activities and the subcellular localization of the UGT and SCPL-AT catalyzed reactions are discussed.