Selective production of acetone during continuous synthesis gas fermentation by engineered biocatalyst Clostridium sp. MAceT113

Aims: To engineer acetogen biocatalyst capable of fermenting synthesis gas blend to acetone as the only liquid carbonaceous product. Methods and Results: The metabolic engineering comprised inactivation of phosphotransacetylase via integration of a cassette comprising synthetic genes erm(B), thiolase and HMG‐CoA synthase. Acetaldehyde dehydrogenase was inactivated via integration of a cassette consisting of synthetic genes cat, HMG‐CoA lyase and acetoacetate decarboxylase. The engineered biocatalyst Clostridum sp. MAceT113 lost production of 253 mmol l^?1 ethanol and 296 mmol l^?1 acetate and started producing 1·8 mol l^?1 acetone in single‐stage continuous syngas fermentation. Conclusions: The acetone concentration in culture broth is economical for bulk manufacture because it is about twenty times of that achieved with known acetone–butanol–ethanol fermentation of sugars. Significance and Impact of the Study: The process shows the opportunity to produce acetone from synthesis gas at concentrations comparable with production of acetone from products of petroleum cracking. This is the first report on elimination of acetate and acetaldehyde production and directing carbon flux from Acetyl‐CoA to acetone via a non‐naturally occurring in acetogen acetone biosynthesis pathway identified in eukaryotic organisms.     

小麦幼穗蛋白质双向电泳条件的优化

本研究以温光敏小麦为试材,用TCA/丙酮和酚提取法提取小麦幼穗蛋白样品,进行了双向电泳优化分析,并对双向电泳过程中出现的问题进行了讨论。结果表明,用TCA/丙酮法提取小麦幼穗蛋白质其产率(浓度)高于酚提取法。SDS-PAGE电泳显示,用TCA/丙酮提取法提取的蛋白质能获得较清晰条带,分辨率较高,而酚提取法提取的蛋白质其条带模糊,分辨率低。对蛋白质纯化除盐可以提高分辨率,减少横竖纹,获得背景清晰的圆形蛋白点。通过ImageMasterTM 2D Platinum5.0软件分析凝胶图谱,结果显示纯化后可降低噪点,纯化后蛋白点数可从未纯化蛋白点数的216增加到583。显然,采用TCA/丙酮法可获得高浓度高质量的蛋白质,而进一步纯化、除盐离子可进一步获得背景清晰可高重复性的电泳图谱。在双向电泳实验过程中,观察到一些异常缺陷胶的出现,如双向电泳图谱中蛋白点扩散,蛋白聚集形成斑点串,没有点或点很少,出现纵纹横纹及图谱扭曲等影响图谱质量的严重问题,本研究对这些问题做了分析并提出了解决方案。     

Ectoine disperses keratin and alters hydration kinetics in stratum corneum

Moisturizing compounds are commonly applied topically to human stratum corneum (SC). Many types of molecular species are employed, most commonly including humectants and occlusives. We find new evidence of keratin dispersion caused by the moisturizing compound ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid), and provide the first characterization of its impacts on the hydration kinetics and biomechanics of SC. A second compound, 2-(2-hydroxyethoxy)ethylguanidine succinate (HEG) was investigated for comparison. A suite of biomechanical and biochemical assays including FTIR, drying stress, and cellular cohesion were used. Studies were conducted on normal, lipid-extracted, and lipid plus natural moisturizing factor extracted SC. Ectoine was found to improve the dispersity and hydration of keratin bundles in corneocytes. It also decreased rates of stress development in lipid extracted SC when exposed to a dry environment by ～30% while improving stress reduction during rehydration by ～20%. Peak stresses were increased in harsh drying environments of <5% RH, but SC swelling measurements suggest that water retention was improved in ambient conditions. Further, changes up to ～4 J/m² were seen in cohesion after ectoine treatments, suggesting corneodesmosome interactions. HEG was tested and found to disperse keratin without impacting corneodesmosomes. These results indicate that keratin dispersants produce beneficial effects on SC hydration kinetics, ultimately resulting in higher SC hydration under ambient conditions.     

The effects of protein synthesis inhibitors on oxidative phosphorylation by plant mitochondria

Inhibitors can be successfully used if they are specific for only one process. Published data suggest that some inhibitors of protein synthesis may also inhibit respiration or oxidative phosphorylation. The effect of a range of protein synthesis inhibitors on respiration and phosphorylation has been studied, using tightly coupled mitochondria from several plant species including turnips (Brassica napus).Puromycin, actinomycin D, lincomycin, mitomycin C and d-serine did not uncouple or inhibit respiration. Cycloheximide caused a partial inhibition (maximum 22% at 3 mM) of malate but not succinate-driven respiration. Chloramphenicol was a potent inhibitor of electron transport, but not of phosphorylation. The activity of the isomers of chloramphenicol varied in the order l-threo >d-threo >l-erythro >d-erythro. From evidence presented it is concluded that chloramphenicol has three sites of action, the flavoprotein level being most sensitive, the second site of variable sensitivity lies between cytochromes b and c and the third site at the cytochrome a level is only slightly affected by the inhibitor.     

The metabolism of acetone in the pregnant rat

The administration of a single dose of acetone (100 mg/kg bw) to virgin and 21-day pregnant rats resulted in the appearance of relatively high concentrations of 1,2-propanediol, acetol and methylglyoxal in plasma and liver. In the fetuses no methylglyoxal was detectable. The acetone metabolism curves tend to indicate that the capacity for acetone disposal may be enhanced in the 48 hr-fasted pregnant rat, thus enabling the animal to re-use acetone metabolically, possibly for accelerated gluconeogenesis.     

Continuous biomanufacturing with microbes — upstream progresses and challenges

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Modulation of peroxisomal and microsomal fatty acid oxidation by acetone. A comparative study between liver and kidney

The effect of acetone consumption on some microsomal and peroxisomal activities was studied in rat kidney and these results were compared with data from former investigations in liver. Acetone increased the microsomal lauric acid hydroxylation, the aminopyrine N-demethylation catalyzed by cytochrome P450 and the microsomal UDP-glucuronyltransferase activity. Also, acetone increased the peroxisomal β-oxidation of palmitoyl CoA and catalase activities in kidney. These studies suggest that acetone is a common inducer of the microsomal and peroxisomal fatty acid oxidation, as previously shown in both starved and ethanol treated rats. Our results support the hypothesis that microsomal fatty acid ω-hydroxylation results in the generation of substrates being supplied for peroxisomal β-oxidation. We propose that the final purpose of these linked fatty acid oxidations could be the catabolism of fatty acids or the generation of a substrate for the synthesis of glucose from fatty acids. This pathway would be triggered by acetone treatment in a similar way in liver and kidney.     

Effects of tsetse targets on mammals and birds in Kasungu National Park, Malawi

Possible effects on wildlife of targets baited with an attractive odour (acetone), impregnated with deltamethrin and used to control tsetse flies (Glossina morsitans morsitans), were investigated in the Kasungu National Park, Malawi. Mammals and birds were censused simultaneously along transects in 'Miombo' woodland or 'Dambo' grassland/mixed woodland, with and without targets. Mammals were also monitored by surveys of their spoor on experimentally cleared plots (5 m in diameter) and a target relocation experiment was conducted when the targets were removed from the test transects and transferred to the controls. Significantly fewer small antelopes (e.g. common Duiker Sylvicapra grimmia) were detected in plots along transects with targets (tests) than along control transects. The presence of targets affected the frequency of occurrence of antelopes, suids and large herbivores in the experimental plots, but small carnivores, monkeys, rodents and hares were unaffected. A conclusion of the relocation experiment was that the deltamethrin-impregnated cloth was responsible for the observed effects and not the acetone. A total number of 23 species of birds meeting a criterion for their abundance in the areas surveyed were selected for detailed analyses. Lower indices in the test areas than in the controls were recorded for 15 of these 23 species in the Dambos and for 10 of 21 species in the Miombo woodland. Matched paired comparisons revealed significantly lower numbers in the test areas than in the controls in both habitats only for black-headed Oriole Oriolus larvatus, but for Grey Lourie Corythaixoides concolor, little bee-eater Merops pusillus, fork-tailed Drongo Dicrurus adsimilis and combined data on three species of sunbirds in the Dambos. The black-eyed bulbul Pycnonotus barbatus had significantly lower numbers in the tests than in the controls in Miombo woodland. The numbers of three species of dove were higher in the tests than in the controls in both habitats, significantly so for the Cape turtle dove Streptopelia capicola, and numbers of the Flappet Lark Mirafra rufocinnamomea were significantly higher in the tests than in the controls in Dambos. The results are discussed in the light of previous studies on environmental effects of tsetse control, including effects of tsetse targets on pollinators especially non-target horseflies.