Identification of a carboxylesterase expressed in protoplasts using fluorescence-activated cell sorting

A method was developed to identify plant carboxylesterases using a homologous expression system with the capacity for high-throughput screening based on fluorescence-activated cell sorting (FACS). Protoplasts of Arabidopsis thaliana were prepared and transfected with a mutated (Cys59Ser) Arabidopsis S-formylglutathione hydrolase ( atsfghm ), which encoded a carboxylesterase highly active in the hydrolysis of the vital marker methylumbelliferyl acetate (MUA) to the fluorophore methylumbelliferone (MU). Unlike all other Arabidopsis carboxylesterases studied to date, At SFGH and its more stable mutant variant At SFGHm are insensitive to inhibition by organophosphate insecticides, such as paraoxon. By making use of the combined traits of a high carboxylesterase activity towards MUA and a lack of sensitivity to paraoxon, FACS was employed to selectively collect catalytically active atsfghm -transformed protoplasts. A population of 400 000 protoplasts containing 8000 sfghm transformants was treated with paraoxon to inhibit endogenous esterase activity and then fed with MUA. Fluorescent cells expressing the At SFGHm enzyme were then collected by FACS, and the presence of the respective transgene was confirmed by polymerase chain reaction, with 9.6% of the transformants recovered. We suggest that the use of FACS to identify other carboxylesterases which can be catalytically determined using plant cell fluorescence-based assays could be a powerful method for the high-throughput screening of new enzymes, especially those which do not express well in microbial hosts.     

厌氧甲烷氧化微生物物质代谢与能量代谢研究进展

甲烷既是一种温室气体,也是一种潜在的能源物质,其源与汇的平衡对地球化学循环及工程应用均有重要意义。厌氧甲烷氧化(anaerobic oxidation of methane,AOM)过程是深海、湿地和农田等自然生境中重要的甲烷汇,在缓解温室气体排放方面发挥了巨大作用。AOM微生物的中枢代谢机制及其能量转化途径则是介导厌氧甲烷氧化耦合其他物质还原的关键所在。因此,本文从电子受体多样性的视角,主要分析了硫酸盐型,硝酸盐/亚硝酸盐型,金属还原型厌氧甲烷氧化微生物的生理生化过程及环境分布,并对近些年发现的新型厌氧甲烷氧化进行了梳理;重点总结了厌氧甲烷氧化微生物细胞内电子传递路径以及胞外电子传递方式;根据厌氧甲烷氧化微生物环境分布及反应特征,就其生态学意义及在污染治理与能源回收方面的潜在应用价值进行了展望。本综述以期深化对厌氧甲烷氧化过程的微生物学认知,并为其潜在的工程应用方向提供新的思路。     

The impact of HPV infection on human glycogen and lipid metabolism – a review

Reinterpretation of the Wartburg effect leads to understanding aerobic glycolysis as a process that provides considerable amount of molecular precursors for the production of lipids, nucleotides and amino acids that are necessary for continuous growth and rapid proliferation characteristic for cancer cells.Human papilloma virus (HPV) is a number one cause of cervical carcinoma with 99% of the cervical cancer patients being HPV positive. This tight link between HPV and cancer raises the question if and how HPV impact cells to reprogram their metabolism? Focusing on early phase proteins E1, E2, E5, E6 and E7 we demonstrate that HPV activates plethora of metabolic pathways and directly influences enzymes of the glycolysis pathway to promote the Warburg effect by increasing glucose uptake, activating glycolysis and pentose phosphate pathway, increasing the level of lactate dehydrogenase A synthesis and inhibiting β-oxidation. Our considerations lead to conclusion that HPV is substantially involved in metabolic cell reprogramming toward neoplastic phenotype and its metabolic activity is the fundamental reason of its oncogenicity.     

The lifestyle of prokaryotic organisms influences the repertoire of promiscuous enzymes

The metabolism of microbial organisms and its diversity are partly the result of an adaptation process to the characteristics of the environments that they inhabit. In this work, we analyze the influence of lifestyle on the content of promiscuous enzymes in 761 nonredundant bacterial and archaeal genomes. Promiscuous enzymes were defined as those proteins whose catalytic activities are defined by two or more different Enzyme Commission (E.C.) numbers. The genomes analyzed were categorized into four lifestyles for their exhaustive comparisons: free‐living, extremophiles, pathogens, and intracellular. From these analyses we found that free‐living organisms have larger genomes and an enrichment of promiscuous enzymes. In contrast, intracellular organisms showed smaller genomes and the lesser proportion of promiscuous enzymes. On the basis of our data, we show that the proportion of promiscuous enzymes in an organism is mainly influenced by the lifestyle, where fluctuating environments promote its emergence. Finally, we evidenced that duplication processes occur preferentially in metabolism of free‐living and extremophiles species. Proteins 2015; 83:1625–1631. © 2015 Wiley Periodicals, Inc.