Uricase production by a recombinant <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> strain harboring <Emphasis Type="Italic">Candida utilis</Emphasis> uricase gene

Uricase is an important medical enzyme which can be used to determine urate in clinical analysis, to therapy gout, hyperuricemia, and tumor lysis syndrome. Uricase of Candida utilis was successfully expressed in Hansenula polymorpha under the control of methanol oxidase promoter using Saccharomyces cerevisiae alpha-factor signal peptide as the secretory sequence. Recombinant H. polymorpha MU200 with the highest extracellular uricase production was characterized with three copies of expression cassette and selected for process optimization for the production of recombinant enzyme. Among the parameters investigated in shaking flask cultures, the pH value of medium and inoculum size had great influence on the recombinant uricase production. The maximum extracellular uricase yield of 2.6 U/ml was obtained in shaking flask culture. The yield of recombinant uricase was significantly improved by the combined use of a high cell-density cultivation technique and a pH control strategy of switching culture pH from 5.5 to 6.5 in the induction phase. After induction for 58 h, the production of recombinant uricase reached 52.3 U/ml (about 2.1 g/l of protein) extracellularly and 60.3 U/ml (about 2.4 g/l) intracellularly in fed-batch fermentation, which are much higher than those expressed in other expression systems. To our knowledge, this is the first report about the heterologous expression of uricase in H. polymorpha.     

Rapid response to changing environments during biological invasions: DNA methylation perspectives

Dissecting complex interactions between species and their environments has long been a research hot spot in the fields of ecology and evolutionary biology. The well‐recognized Darwinian evolution has well‐explained long‐term adaptation scenarios; however, “rapid” processes of biological responses to environmental changes remain largely unexplored, particularly molecular mechanisms such as DNA methylation that have recently been proposed to play crucial roles in rapid environmental adaptation. Invasive species, which have capacities to successfully survive rapidly changing environments during biological invasions, provide great opportunities to study molecular mechanisms of rapid environmental adaptation. Here, we used the methylation‐sensitive amplified polymorphism (MSAP) technique in an invasive model ascidian, Ciona savignyi, to investigate how species interact with rapidly changing environments at the whole‐genome level. We detected quite rapid DNA methylation response: significant changes of DNA methylation frequency and epigenetic differentiation between treatment and control groups occurred only after 1 hr of high‐temperature exposure or after 3 hr of low‐salinity challenge. In addition, we detected time‐dependent hemimethylation changes and increased intragroup epigenetic divergence induced by environmental stresses. Interestingly, we found evidence of DNA methylation resilience, as most stress‐induced DNA methylation variation maintained shortly (~48 hr) and quickly returned back to the control levels. Our findings clearly showed that invasive species could rapidly respond to acute environmental changes through DNA methylation modifications, and rapid environmental changes left significant epigenetic signatures at the whole‐genome level. All these results provide fundamental background to deeply investigate the contribution of DNA methylation mechanisms to rapid contemporary environmental adaptation.     

嗜酸乳杆菌NCFM粘附宿主后对其粘附相关基因表达的影响

摘要：【目的】益生菌粘附于肠道上皮细胞上是它的一种益生作用。本研究通过体内外实验,分析嗜酸乳杆菌NCFM对粘附相关基因的影响。【方法】利用GO (Gene Ontolog) 分类筛选Human Genome U133 Plus 2.0 Array基因表达谱芯片中的粘附相关基因,通过体外Caco-2细胞培养模型和体内小鼠粘附模型,采用Real-time PCR方法对粘附相关基因进行验证分析。【结果】经NCFM作用后,12个粘附相关基因呈上调表达。利用Real-time PCR验证,12个基因在体内和体外经嗜酸乳杆菌NCFM作用后亦均同样为上调表达,其中CCL2基因上调表达最为明显。【结论】经体内外研究表明,嗜酸乳杆菌NCFM粘附肠上皮细胞后能够引起宿主粘附相关基因出现特定表达变化,为今后深入揭示其粘附作用提供必要基础。     

甾醇C-24甲基转移酶基因在酿酒酵母中的内源表达及工程菌麦角甾醇的合成

通过高保真PCR克隆到含酿酒酵母甾醇C-24甲基转移酶基因编码序列及终止子序列的DNA片段ERG6, 以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体, 磷酸甘油酸激酶基因PGK1启动子为上游调控元件构建了酵母菌表达质粒pPERG6。通过同源重组, 以铜离子螯合蛋白基因CUP1替换染色体上ERG6基因内部序列获得ERG6破坏菌株YS58-erg6, 其中麦角甾醇的合成被阻断, 同时细胞的生长也受到明显抑制。表达质粒pPERG6转化破坏菌株YS58-erg6后, 不但使细胞恢复了合成麦角甾醇的能力, 细胞生物量也得到明显提高, 这说明表达质粒上的ERG6基因得到了功能性的表达。分别用载体质粒YEp352和表达质粒pPERG6转化酿酒酵母单倍体菌株YS58, 获得对照菌株YS58(YEp352)和重组菌株YS58(pPERG6)。重组菌株YS58(pPERG6) 生物量和麦角甾醇含量分别是对照菌YS58(YEp352)的1.23和1.32倍。可见甾醇C-24甲基转移酶基因的高表达可以增强酵母细胞麦角甾醇的合成能力。     

Molecular cloning and functional characterization of a RabGTPase in large yellow croaker (Pseudosciaena crocea)

The molecular mechanisms of the immune system against pathogens in large yellow croaker (Pseudosciaena crocea) are not well known, despite its economic importance as an aquaculture species. In this investigation, a Rab gene (named as LycRab gene) was obtained from this fish, which exhibited high homology with Rab8 of other species. It was expressed in Escherichia coli, and the specific antibody was raised using the purified fusion protein (GST-LycRab). The LycRab protein, containing characteristic signatures of Rab proteins with 5 GTP-binding domains, had GTP-binding activity. The LycRab gene was ubiquitously expressed in all analyzed tissues as revealed by Western blot, although expression levels varied from tissue to tissue. Real-time PCR revealed that the LycRab gene was up-regulated after immunization with poly I:C, formalin-inactive Gram-negative bacterium Vibrio parahaemolyticus or bacterial lipopolysaccharides (LPS), suggesting that LycRab protein might play an important role in large yellow croaker defense against pathogens infection. This discovery might contribute better understanding to the molecular events involved in fish immune responses.     

Ergosterol production from molasses by genetically modified <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ergosterol is an economically important metabolite produced by fungi. Recombinant Saccharomyces cerevisiae YEH56(pHXA42) with increased capacity of ergosterol formation was constructed by combined overexpression of sterol C-24(28) reductase and sterol acyltransferase in the yeast strain YEH56. The production of ergosterol by this recombinant strain using cane molasses (CM) as an inexpensive carbon source was investigated. An ergosterol content of 52.6 mg/g was obtained with 6.1 g/l of biomass from CM medium containing 60 g/l of total sugar in 30 h in shake flask. The ergosterol yield was enhanced through the increasing cell biomass by supplementation of urea to a concentration of 6 g/l in molasses medium. Fermentation was performed in 5-l bioreactor using the optimized molasses medium. In batch fermentation, the effect of agitation velocity on ergosterol production was examined. The highest ergosterol yield was obtained at 400 rpm that increased 60.4 mg/l in comparison with the shake flask culture. In fed-batch fermentation, yeast cells were cultivated, firstly, in the starting medium containing molasses with 20 g/l of total sugar, 1.68 g/l of phosphate acid, and 6 g/l of urea (pH 5.4) for 5 h, then molasses containing 350 g/l of total sugar was fed exponentially into the bioreactor to keep the ethanol level in the broth below 0.5%. After 40 h of cultivation, the ergosterol yield reached 1,707 mg/l, which was 3.1-fold of that in the batch fermentation.     

Speciation of chromium in chromium yeast

High-performance liquid chromatography was used to separate Cr(III) and Cr(VI) in samples with detection by inductively coupled plasma mass spectrometry(ICP-MS). The separation was achieved on a weak anion exchange column. The mobile phase was pH 7.0 ammonium nitrate solution. The redox reaction between Cr(III) and Cr(VI) was avoided during separation and determination. This separation method could be used to separate the samples with large concentration differences between Cr(III) and Cr(VI). The alkaline digestion was used to extract chromium in solid sample, which had no effect on the retention time and the peak area of the Cr(VI). However, the conversion of Cr(VI) from Cr(III) was observed during alkaline digestion, which displayed positive relation with the ratio of Cr(III) and Cr(VI) in samples. Both Cr(III) and Cr(VI) contents of chromium yeasts cultured in media with different chromium additions were determined. The spike recoveries of Cr(VI) for chromium yeasts were in the range of 95–108 %.     

衔接重复序列对酵母菌絮凝蛋白功能的调控作用

【目的】了解絮凝基因FLO1中重复DNA序列B和D对絮凝蛋白Flo1p功能的影响,为构建遗传稳定的最小絮凝功能基因奠定理论基础。【方法】通过PCR和融合PCR方法分别克隆到完整的絮凝基因FLO1、重复DNA序列B和D分别缺失的衍生基因FLO1b和FLO1d,分析这些基因在非絮凝酵母中表达对细胞絮凝特性的影响。【结果】与完整絮凝基因相比,重复DNA序列B和D分别缺失后对酵母细胞絮凝强度没有明显影响,但不同基因在酵母菌中表达产生的絮凝特性受环境因素,如甘露糖浓度和pH等的影响有明显差异。FLO1中重复DNA序列B和D缺失后,细胞絮凝特性受甘露糖抑制的敏感性降低;同时对环境pH的改变具有更广泛的适应性。【结论】重复DNA序列B和D对絮凝蛋白Flo1p结构和功能具有调控作用,二者缺失后,特别是D缺失后会使絮凝蛋白在极端酸碱环境下更稳定。