A versatile system for fast screening and isolation of Trichoderma reesei cellulase hyperproducers based on DsRed and fluorescence-assisted cell sorting

Abstract

BackgroundIn the biofuel industry, cellulase plays an indispensable role in hydrolyzing cellulose into fermentable glucose. Trichoderma reesei is 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.ResultsHerein, we developed a versatile method coupling expression of the red fluorescence protein (DsRed) in T. reesei and fluorescence-assisted cell sorting (FACS) of germinated spores. This method was first established by expressing DsRed intracellularly under the control of the major cellulase cbh1 promoter in T. reesei, which allowed us to rapidly isolate cellulase hyperproducers from T. reesei progenies transformed with a dedicated transcriptional activator ace3 and from an atmospheric and room temperature plasma-created mutant T. reesei library. Since intracellularly expressed DsRed was expected to isolate mutations mainly affecting cellulase transcription, this method was further improved by displaying DsRed on the T. reesei cell surface, enabling isolation of strains with beneficial genetic alterations (overexpressing hac1 and bip1) affecting regulatory stages beyond transcription. Using this method, T. reesei cellulase hyperproducers were also successfully isolated from an Agrobacterium-mediated random insertional mutant library.ConclusionsThe coupled DsRed-FACS high-throughput screening method proved to be an effective strategy for fast isolation of T. reesei cellulase hyperproducers and could also be applied in other industrially important filamentous fungi.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1264-z) contains supplementary material, which is available to authorized users.

Genetic Engineering of Filamentous Fungi for Efficient Protein Expression and Secretion

“…In fact, the fluorescence-activated cell sorting (FACS) assisted the intracellular protein production has been extensively adopted in filamentous fungi, but such strategy is difficult to screen out the beneficial mutants with enhanced protein secretion capacity (Throndset et al, 2010). To solve this problem, displaying the fluorescence protein on the cell surface, coupled by FACS, allows screening of the cellulose hypersecretors from T. reesei (Gao et al, 2018). As a promising alternative, the droplet-based microfluidic high-throughput screening platform has been established in T. reesei and A. niger (Beneyton et al, 2016;He et al, 2019).…”

Global Reprogramming of Gene Transcription in Trichoderma reesei by Overexpressing an Artificial Transcription Factor for Improved Cellulase Production and Identification of Ypr1 as an Associated Regulator

“…Results above indicate the enhanced effect of endoplasmic reticulum-associated degradation (ERAD) on protein processing for glycoside hydrolases in T. reesei U5 at 24 h. However, at 24 h, the transcription levels of the genes encoding disulphide isomerase Pdi and ER-resident transmembrane kinase Ire1 proteins were not up-regulated, the transcript levels of hac1 gene encoding regulator for UPR, and bip1 gene encoding the luminal binding protein, an ER-localized member of the HSP70 family, also kept in similar level between T. reesei U5 and Rut-C30 at 24 h, which suggests that the unfolded protein response (UPR) was not changed in T. reesei U5. The positive function of genes above for protein processing in endoplasmic reticulum in T. reesei have been confirmed before (Saloheimo et al, 2003;Gao et al, 2018).…”

Simultaneous manipulation of multiple genes within a same regulatory stage for iterative evolution of Trichoderma reesei

“…Experimentally verified or predicted activators were overexpressed, while the repressors were silenced for their expression. The plasmids bearing the genes in one same stage were pooled and co-transformed into a T. reesei strain (SUS4) with surface-displayed DsRed [ 27 ]. Transformant spores or protoplasts were screened for cellulase hyperproducers via fluorescence-assisted cell sorting (FACS, Fig.…”