Pachlinger Robert, Mitterbauer Rudolf, Adam Gerhard, Strauss Joseph
Institut für Angewandte Genetik und Zellbiologie, BOKU-University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
Appl Environ Microbiol. 2005 Feb;71(2):672-8. doi: 10.1128/AEM.71.2.672-678.2005.
Filamentous fungi are well-established expression hosts often used to produce extracellular proteins of use in the food and pharmaceutical industries. The expression systems presently used in Aspergillus species rely on either strong constitutive promoters, e.g., that for glyceraldehyde-3-phosphate dehydrogenase, or inducible systems derived from metabolic pathways, e.g., glaA (glucoamylase) or alc (alcohol dehydrogenase). We describe for Aspergillus nidulans and Aspergillus niger a novel expression system that utilizes the transcriptional activation of the human estrogen receptor by estrogenic substances. The system functions independently from metabolic signals and therefore can be used with low-cost, complex media. A combination of positive and negative regulatory elements in the promoter drives the expression of a reporter gene, yielding a linear dose response to the inducer. The off status is completely tight, yet the system responds within minutes to induction and reaches a level of expression of up to 15% of total cell protein after 8 h. Both Aspergillus species are very sensitive to estrogenic substances, and low-cost inducers function in the picomolar concentration range, at which estrogenic substances also can be found in the environment. Given this high sensitivity to estrogens, Aspergillus cells carrying estrogen-responsive units could be used to detect xenoestrogens in food or in the environment.
丝状真菌是成熟的表达宿主,常用于生产食品和制药行业中使用的细胞外蛋白质。目前在曲霉菌种中使用的表达系统依赖于强组成型启动子,例如甘油醛-3-磷酸脱氢酶的启动子,或源自代谢途径的诱导型系统,例如glaA(葡糖淀粉酶)或alc(乙醇脱氢酶)。我们为构巢曲霉和黑曲霉描述了一种新型表达系统,该系统利用雌激素物质对人雌激素受体的转录激活。该系统独立于代谢信号起作用,因此可用于低成本的复杂培养基。启动子中正负调控元件的组合驱动报告基因的表达,对诱导剂产生线性剂量反应。关闭状态完全紧密,但该系统在几分钟内对诱导作出反应,并在8小时后达到总细胞蛋白表达水平的15%。这两种曲霉菌种对雌激素物质都非常敏感,低成本诱导剂在皮摩尔浓度范围内起作用,在该浓度范围内环境中也能发现雌激素物质。鉴于对雌激素的这种高敏感性,携带雌激素反应单元的曲霉细胞可用于检测食品或环境中的外源性雌激素。
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