Characterization of a copper responsive promoter and its mediated overexpression of the xylanase regulator 1 results in an induction-independent production of cellulases in Trichoderma reesei
Characterization of a copper responsive promoter and its mediated overexpression of the xylanase regulator 1 results in an induction-independent production of cellulases in Trichoderma reesei
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铜响应启动子的表征及其介导的木聚糖酶调节剂 1 的过表达导致里氏木霉中不依赖于诱导的纤维素酶产生
DOI：
10.1186/s13068-015-0249-4
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发表时间：
2015-04-14
影响因子：
6.3
通讯作者：
Liu, Weifeng
中科院分区：
文献类型：
--
作者：
Lv, Xinxing;Zheng, Fanglin;Liu, Weifeng
背景：里氏木霉是工业生产纤维素酶和其他蛋白质的重要原料。大规模生产通常以底物诱导的方式进行，通过一系列转录因子的微调合作来实现。然而，它们的生产和随后的分析通常要么难以操作，要么因同时生产其他可诱导蛋白而变得复杂。因此，在某些情况下，为了促进蛋白质的生化研究和生产者的基因工程，控制基因表达独立于营养状态的替代方案是首选的。结果：我们鉴定出一个铜转运蛋白基因tcu1 (jgi:Trire2:52315)，该基因的转录对铜的可利用性有高度响应。过量的铜抑制了T. reesei中tcu1的表达，而在培养基中消除铜的添加导致了tcu1的高水平转录。当以d -葡萄糖或甘油作为唯一碳源培养时，T. reesei中由tcu1启动子驱动的特定纤维素酶的高水平表达进一步证明了该系统的实用性。重组T. reesei菌株在tcu1启动子的控制下过表达水解酶的主要转录激活因子（木聚糖酶调节因子1），从而解除了碳分解代谢产物的抑制，从而表现出组成型纤维素酶的表达。此外，该菌株在甘油或葡萄糖上产生的纤维素酶的数量和活性完全再现了亲本菌株在Avicel上产生的纤维素酶的数量和活性。结论：T. reesei tcu1基因的表达受铜可用性的严格控制，并基于该启动子构建了同源蛋白表达体系。由tcu1启动子介导的XYR1（木聚糖酶调节因子1）的解除调控不仅克服了纤维素酶的碳分解代谢抑制，而且使其在非诱导碳源上充分表达。
Background: Trichoderma reesei represents an important workhorse for industrial production of cellulases as well as other proteins. The large-scale production is usually performed in a substrate-inducing manner achieved by a fine-tuned cooperation of a suite of transcription factors. Their production and subsequent analysis are, however, often either difficult to manipulate or complicated by the concomitant production of other inducible proteins. Alternatives to control gene expression independent of the nutritional state are thus preferred in some cases to facilitate not only biochemical studies of proteins but also genetic engineering of the producer.Results: We identified a copper transporter encoding gene tcu1 (jgi:Trire2:52315) in T. reesei, the transcription of which was highly responsive to copper availability. Whereas excess copper repressed the expression of tcu1 from T. reesei, eliminating copper addition in the medium resulted in a high-level transcription of tcu1. The usefulness of the system was further illustrated by the high-level expression of specific cellulases driven by the tcu1 promoter in T. reesei when cultivated on D-glucose or glycerol as the sole carbon source. A recombinant T. reesei strain, which overexpressed the main transcription activator of hydrolases (xylanase regulator 1) under the control of tcu1 promoter, was found to be relieved from the carbon catabolite repression and thus displayed a constitutive cellulase expression. Moreover, the amount and activities of cellulases produced by this strain on glycerol or glucose fully recapitulated those of the parental strain produced on Avicel.Conclusion: Expression of T. reesei tcu1 gene was tightly controlled by copper availability, and a homologous protein expression system was developed based on this promoter. Deregulation of XYR1 (xylanase regulator 1) mediated by the tcu1 promoter not only overcame the carbon catabolite repression of cellulases but also resulted in their full expression even on the non-inducing carbon sources.