Improving simvastatin bioconversion in Escherichia coli by deletion of bioH

Simvastatin is an important cholesterol lowering compound and is currently synthesized from the natural product lovastatin via multistep chemical synthesis. We have previously reported the use of an Escherichia coli strain BL21(DE3)/pAW31 as the host for whole-cell biocatalytic conversion of monacolin J acid to simvastatin acid. During fermentation and bioconversion, unknown E. coli enzyme(s) hydrolyzed the membrane permeable thioester substrate dimethylbutyryl-S-methyl mercaptopropionate (DMB-S-MMP) to the free acid, significantly decreased the efficiencies of the whole-cell bioconversion and the downstream purification steps. Using the Keio K-12 Singe-Gene Knockout collection, we identified BioH as the sole enzyme responsible for the observed substrate hydrolysis. Purification and reconstitution of E. coli BioH activity in vitro confirmed its function. BioH catalyzed the rapid hydrolysis of DMB-S-MMP with kcat and Km values of 260+/-45 s(-1) and 229+/-26 microM, respectively. This is in agreement with previous reports that BioH can function as a carboxylesterase towards fatty acid esters. YT2, which is a delta bioH mutant of BL21(DE3), did not hydrolyze DMB-S-MMP during prolonged fermentation and was used as an alternative host for whole-cell biocatalysis. The rate of simvastatin acid synthesis in YT2 was significantly faster than in BL21(DE3) and 99% conversion of 15 mM simvastatin acid in less than 12 h was achieved. Furthermore, the engineered host required significantly less DMB-S-MMP to be added to accomplish complete conversion. Finally, simvastatin acid synthesized using YT2 can be readily purified from fermentation broth and no additional steps to remove the hydrolyzed dimethylbutyryl-S-mercaptopropionic acid is required. Together, the proteomic and metabolic engineering approaches render the whole-cell biocatalytic process more robust and economically attractive.     

Effect of 1-methylcyclopropene on postharvest physiological changes of ‘Zaohong’ plum

Plum is a highly perishable fruit and postharvest fruit softening limits its shelf life. The aim of this work was to study the specific effects of 1-methylcyclopropene (1-MCP) treatment on physiological changes in ‘Zaohong’ plums. Plums were treated with 500 nL L⁻¹ 1-MCP at 20°C for 18 h followed by 20°C storage. The results showed that 1-MCP treatment significantly reduced endogenous ethylene production and the activities of ethylene biosynthetic enzymes’ (1-aminocyclopropane-1-carboxylic acid synthase, ACS and 1-aminocyclopropane-1-carboxylic acid oxidase, ACO) in plum fruit during storage when compared with untreated fruit. Although 1-MCP treatment inhibited ethylene production and 1-aminocyclopropane-1-carboxylic acid (ACC) accumulation, it did not inhibit the accumulation of N-malonyl-ACC (MACC). Higher firmness was also found in 1-MCP-treated plums than in controls. During storage, superoxide anion (O₂^−·) and hydrogen peroxide (H₂O₂) levels decreased in 1-MCP-treated fruit. 1-MCP treatment also regulated superoxide dismutase (SOD) and catalase (CAT) activities during storage. Xylanase activity was upregulated while activities of polygalacturonase (PG), pectin methyl esterase (PME) and cellulase enzymes in the fruit were downregulated by 1-MCP treatment. In conclusion, 1-MCP might be a potent compound for extending both storage period and shelf life of ‘Zaohong’ plums by suppressing ethylene biosynthesis, regulating cell wall degradation enzymes and reducing fruit softening.     

Effects of organic matter incorporation on nitrous oxide emissions from rice-wheat rotation ecosystems in China

Organic matter addition is thought to be an important regulator of nitrous oxide (N₂O) emissions from croplands. Contradictory effects, however, were reported in previous studies. To investigate the effects of crop residue management on N₂O emissions from rice-wheat rotation ecosystems, we conducted field experiments at three sites (Suzhou, Wuxi and Jiangdu) in the Yangtze River Delta, using static chamber and gas chromatography methods. Our data show that N₂O emissions throughout the rice season from plots treated with wheat straw application at a high rate (WS) prior to rice transplanting (1.1–2.0 kg N ha^?1) were significantly lower (P?<?0.05) than those from the control plots without organic matter addition or added with wheat straw at a moderate rate (1.6–2.9 kg N ha^?1). Furthermore, the WS treatments had a residual inhibitory effect on N₂O emissions in the following non-rice season, which consistently resulted in significantly lower emissions (P?<?0.05) compared to the control treatments (2.2–3.1 vs. 3.9–5.6 kg N ha^?1). In comparison to the control treatments, the WS treatments reduced both the seasonal and annual direct emission factors of the applied nitrogen (EF_d) by 50–68% (mean: 57%). The addition of compost (aerobically composted rice or wheat straw harvested in the last rotation) reduced the seasonal and annual EF_ds by 29–32%. Over the entire rice-wheat rotation cycle, annual N₂O emissions from the fertilized fields at the three sites ranged from 3.3?±?0.3 to 16.8?±?0.6 kg N ha^?1, with a coefficient of variation (CV) of 61%. Similarly, the EF_ds during the rice-wheat rotation cycle ranged from 0.4% to 2.5%, with a CV of 67%. These high spatial variations might have been related to: variations in soil properties, such as texture and soil organic carbon; management practices, such as straw treatments (i.e., compost versus fresh straw) and weather conditions, such as precipitation and rainfall distribution. Our results indicate that the incorporation of fresh wheat straw at a high rate during the rice season is an effective management practice for the mitigation of N₂O emissions in rice-wheat rotation systems. Whether this practice is also effective in reducing the overall global warming potential of net N₂O, CH₄ and CO₂ emissions needs to be seen through further studies.     

Identification and characterization of adipose triglyceride lipase (ATGL) gene in birds

Adipose triglyceride lipase (ATGL) is a triglyceride hydrolysis lipase and is generally related to lipid metabolism in animals. The ATGL gene was well studied in mammals, however very less was known in birds that differed significantly with mammals for lipid metabolism. In this study, cloning, mRNA real time and association analysis was performed to characterize the ATGL gene in birds. Results showed that the obtained ATGL gene cDNA of parrot, quail, duck were 1,651 bp (NCBI accession number: GQ221784), 1,557 bp (NCBI accession number: GQ221783) and 1,440 bp each, encoded 481-, 482- and 279-amino acid (AA) peptide, respectively. The parrot ATGL (pATGL) gene was found to predominantly express in breast muscle and leg muscle, and very higher ATGL mRNA level was also found in heart, abdominal fat and subcutaneous fat. The quail ATGL (qATGL) gene was also predominantly expressed in breast muscle and leg muscle, and then to a much lesser degree in heart. The duck ATGL (dATGL) gene was found to predominantly express in subcutaneous fat and abdominal fat, quite higher ATGL mRNA was also found in heart, spleen, breast muscle and leg muscle. Blast analyses indicated the high homology of ATGL and its patatin region, and moreover, and the active serine hydrolase motif (“GASAG” for “GXSXG”) and the glycine rich motif (“GCGFLG” for “GXGXXG”) were completely conservative among 14 species. Association analyses showed that c.950+24C>A, c.950+45C>G, c.950+73G>A, c.950+83C>T and c.950+128delA of chicken ATGL gene (cATGL) were all significantly or highly significantly with cingulated fat width (CFW) (P < 0.05 or P < 0.01), and c.777−26C>A, c.950+45C>G, c.950+73G>A and c.950+118C>T were all significantly or highly significantly with pH value of breast muscle (BMPH) (P < 0.05).     

创新性教育在长学制医学生人体解剖学教学中的探索与实践

人体解剖学是医学科学中一门重要的基础课程.长学制医学教育是一种新模式,创新性教育是一种新理念.为了探索长学制医学生人体解剖学的创新性教育,我系在过去五年中以国家精品课程建设为契机.以"培养创新人才"为指导思想,更新教学思想,实行PBL教学,引入"RA"的训练体系,推行青年教师培养的"六个一工程"和国内、国外互动发展的人才培养新模式,取得了初步成果.本文对此进行总结,以期抛砖引玉,引起更多同行的共鸣而促进<人体解剖学>课程的建设.     

The RBCC gene RFP2 (Leu5) encodes a novel transmembrane E3 ubiquitin ligase involved in ERAD

RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-delta, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.     

Angiostatin production in cultivation of recombinant Pichia pastoris fed with mixed carbon sources

A recombinant strain of Pichia pastoris with a phenotype of Mut^S was used to produce angiostatin. Due to the low methanol consumption rate of this strain, both methanol and glycerol feedings, that produced oscillation in dissolved O₂ concentration, were used during the expression phase to improve cell growth and angiostatin expression. However, enhanced cell growth led to nitrogen limitation that suppressed further production of angiostatin, but addition of ammonia allowed angiostatin concentration to reach 108 mg l^–1 after an expression period of 96 h. The ratio of consumed glycerol to methanol of 1.5:1 (w/w) in the expression phase suggested that methanol played an important role in the metabolism of carbon sources.