A versatile system for fast screening and isolation of Trichoderma reesei cellulase hyperproducers based on DsRed and fluorescence-assisted cell sorting.
A versatile system for fast screening and isolation of Trichoderma reesei cellulase hyperproducers based on DsRed and fluorescence-assisted cell sorting.
复制标题
基于 DsRed 和荧光辅助细胞分选的多功能系统，用于快速筛选和分离里氏木霉纤维素酶高产者
DOI：
10.1186/s13068-018-1264-z
复制
发表时间：
2018
影响因子：
6.3
通讯作者：
Su X
中科院分区：
文献类型：
--
作者：
Gao F;Hao Z;Sun X;Qin L;Zhao T;Liu W;Luo H;Yao B;Su X
背景：在生物燃料工业中，纤维素酶在将纤维素水解成可发酵葡萄糖的过程中发挥着不可或缺的作用。里氏木霉是一种流行的丝状真菌，具有突出的产纤维素酶能力。虽然经典诱变和现代多重基因组工程都是提高纤维素酶产量的有效方法，但成功获得纤维素酶生产能力提高的菌株需要筛选大量菌株，这既费时又费力。 结果：在此，我们开发了一种在T中偶联表达红色荧光蛋白（DsRed）的通用方法。萌发孢子的研究和荧光辅助细胞分选（FACS）。该方法首先是通过在 T 中主要 cellulasecbh1 启动子的控制下在细胞内表达 DsRed 来建立的。里氏木霉，这使我们能够快速从木霉中分离出纤维素酶高产者。用专用转录激活因子 3 和常压和室温等离子体产生的突变体 T 转化的 reesei 后代。里斯图书馆。由于细胞内表达的 DsRed 预计会分离出主要影响纤维素酶转录的突变，因此通过在 T 上展示 DsRed 进一步改进了该方法。 reeseicell 表面，能够分离具有有益遗传改变（过度表达 hac1 和 bip1）的菌株，影响转录以外的调控阶段。使用这种方法，T.还从农杆菌介导的随机插入突变体文库中成功分离出瑞丝纤维素酶高产菌。结论：耦合的DsRed-FACS高通量筛选方法被证明是快速分离瑞丝纤维素酶的有效策略。丝状纤维素酶高产者，也可应用于其他工业上重要的丝状真菌。
Background:In the biofuel industry, cellulase plays an indispensable role in hydrolyzing cellulose into fermentable glucose.Trichoderma reeseiis a popular filamentous fungus with prominent ability to produce cellulase. While classical mutagenesis and modern multiplex genome engineering are both effective ways to improve cellulase production, successful obtaining of strains with improved cellulase-producing ability requires screening a large number of strains, which is time-consuming and labor intensive.Results:Herein, we developed a versatile method coupling expression of the red fluorescence protein (DsRed) inT. reeseiand fluorescence-assisted cell sorting (FACS) of germinated spores. This method was first established by expressing DsRed intracellularly under the control of the major cellulasecbh1promoter inT. reesei, which allowed us to rapidly isolate cellulase hyperproducers fromT. reeseiprogenies transformed with a dedicated transcriptional activatorace3and from an atmospheric and room temperature plasma-created mutantT. reeseilibrary. Since intracellularly expressed DsRed was expected to isolate mutations mainly affecting cellulase transcription, this method was further improved by displaying DsRed on theT. reeseicell surface, enabling isolation of strains with beneficial genetic alterations (overexpressinghac1andbip1) affecting regulatory stages beyond transcription. Using this method,T. reeseicellulase hyperproducers were also successfully isolated from anAgrobacterium-mediated random insertional mutant library.Conclusions:The coupled DsRed-FACS high-throughput screening method proved to be an effective strategy for fast isolation ofT. reeseicellulase hyperproducers and could also be applied in other industrially important filamentous fungi.
登录
查看更多内容
影响因子：
6.3
作者：
Kubicek CP;Mikus M;Schuster A;Schmoll M;Seiboth B
通讯作者：
Seiboth B
影响因子：
6.3
作者：
Häkkinen M;Valkonen MJ;Westerholm-Parvinen A;Aro N;Arvas M;Vitikainen M;Penttilä M;Saloheimo M;Pakula TM
通讯作者：
Pakula TM
DOI：
10.1073/pnas.97.22.11984
发表时间：
2000-10-24
影响因子：
11.1
作者：
Baird, GS;Zacharias, DA;Tsien, RY
通讯作者：
Tsien, RY
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
4.4
作者：
Nakari-Setala, Tiina;Paloheimo, Marja;Saloheimo, Markku
通讯作者：
Saloheimo, Markku