L-Methionine repressible promoters for tuneable gene expression in Trichoderma reesei.
L-Methionine repressible promoters for tuneable gene expression in Trichoderma reesei.
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DOI：
10.1186/s12934-015-0308-3
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发表时间：
2015-08-14
影响因子：
6.4
通讯作者：
Seiboth B
中科院分区：
文献类型：
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作者：
Bischof RH;Horejs J;Metz B;Gamauf C;Kubicek CP;Seiboth B
里氏木霉是生物炼油业中植物生物质水解所需的木质纤维素酶的主要生产者。尽管里氏锥虫的分子工具箱已经开发得很好，但仍然缺乏用于菌株工程和功能基因组研究的阻遏启动子。一种广泛用于酵母的这样的启动子是编码三磷酸腺苷硫化酶的L-蛋氨酸可抑制的met3基因的启动子。结果表明，MET3系统仅适用于以纤维素酶诱导的碳源乳糖为碳源的里氏木霉，而不适用于以麦草为底物的木质纤维素酶产生菌。因此，我们对小麦秸秆培养中L-蛋氨酸可抑制的基因进行了转录筛选。这项分析检索了50个差异表达基因，其中33个基因表达下调。其中，编码转运蛋白的基因以及含铁的DszA样单加氧酶和TauD样双加氧酶的表达明显过高。我们发现其中一种双加氧酶的启动子区域可以用于黑曲霉SuA编码的胞外转化酶在里氏小麦秸秆培养物中的强抑制表达。该系统也可移植到其他碳源，包括d-葡萄糖和甘油，这一点从大肠杆菌LacZ编码的半乳糖苷酶在T.reesei中的可阻遏表达中得到了证明。我们描述了一组新颖的、通用的里氏木霉启动子，可以用来在不同表达强度的小麦秸秆培养物中以L-蛋氨酸可抑制的方式驱动重组基因的表达。我们详细研究的双加氧酶启动子还与不同的碳源相容，因此适用于控制蛋白质生产以及与里氏木霉的功能基因组学。本文的在线版本(doi：10.1186/s12934-015-0308-3)包含补充材料，授权用户可以使用。
Trichoderma reesei is the main producer of lignocellulolytic enzymes that are required for plant biomass hydrolysis in the biorefinery industry. Although the molecular toolbox for T. reesei is already well developed, repressible promoters for strain engineering and functional genomics studies are still lacking. One such promoter that is widely employed for yeasts is that of the l-methionine repressible MET3 gene, encoding ATP sulphurylase. We show that the MET3 system can only be applied for T. reesei when the cellulase inducing carbon source lactose is used but not when wheat straw, a relevant lignocellulosic substrate for enzyme production, is employed. We therefore performed a transcriptomic screen for genes that are l-methionine repressible in a wheat straw culture. This analysis retrieved 50 differentially regulated genes of which 33 were downregulated. Among these, genes encoding transport proteins as well as iron containing DszA like monooxygenases and TauD like dioxygenases were strongly overrepresented. We show that the promoter region of one of these dioxygenases can be used for the strongly repressible expression of the Aspergillus niger sucA encoded extracellular invertase in T. reesei wheat straw cultures. This system is also portable to other carbon sources including d-glucose and glycerol as demonstrated by the repressible expression of the Escherichia coli lacZ encoded ß-galactosidase in T. reesei. We describe a novel, versatile set of promoters for T. reesei that can be used to drive recombinant gene expression in wheat straw cultures at different expression strengths and in an l-methionine repressible manner. The dioxygenase promoter that we studied in detail is furthermore compatible with different carbon sources and therefore applicable for manipulating protein production as well as functional genomics with T. reesei. The online version of this article (doi:10.1186/s12934-015-0308-3) contains supplementary material, which is available to authorized users.