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.

Fei Zhang,Feng-Wu Bai,Jia-Xiang Li,Verawat Champreda,Chen-Guang Liu,Xin-Qing Zhao

doi:10.3389/fbioe.2020.00649

Abstract

Synthetic biology studies on filamentous fungi are providing unprecedented opportunities for optimizing this important category of microbial cell factory. Artificial transcription factor can be designed and used to offer novel modes of regulation on gene transcription network. Trichoderma reesei is commonly used for cellulase production. In our previous studies, a plasmid library harboring genes encoding artificial zinc finger proteins (AZFPs) was constructed for engineering T. reesei, and the mutant strains with improved cellulase production were selected. However, the underlying mechanism by which AZFP function remain unclear. In this study, a T. reesei Rut-C30 mutant strain T. reesei U5 bearing an AZFP named as AZFP-U5 was focused, which secretes high level protein and shows significantly improved cellulase and xylanase production comparing with its parental strain. In addition, enhanced sugar release was achieved from lignocellulosic biomass using the crude cellulase from T. reesei U5. Comparative transcriptome analysis was further performed, which showed reprogramming of global gene transcription and elevated transcription of genes encoding glycoside hydrolases by overexpressing AZFP-U5. Furthermore, 15 candidate regulatory genes which showed remarkable higher transcription levels by AZFP-U5 insertion were overexpressed in T. reesei Rut-C30 to examine their effects on cellulase biosynthesis. Among these genes, TrC30_93861 (ypr1) and TrC30_74374 showed stimulating effects on filter paper activity (FPase), but deletion of these two genes did not affect cellulase activity. In addition, increased yellow pigment production in T. reesei Rut-C30 by overexpression of gene ypr1 was observed, and changes of cellulase gene transcription were revealed in the ypr1 deletion mutant, suggesting possible interaction between pigment production and cellulase gene transcription. The results in this study revealed novel aspects in regulation of cellulase gene expression by the artificial regulators. In addition, the candidate genes and processes identified in the transcriptome data can be further explored for synthetic biology design and metabolic engineering of T. reesei to enhance cellulase production.

Highlights

Cellulase is widely used in detergents, textiles, pulp processing, food, and feed industries (Sharma et al, 2016)
The artificial zinc finger protein (AZFP) sequence from the transformant T. reesei U5 was named as AZFP-U5, and the corresponding gene AZFPU5 was found to integrate into the genome as a single copy
Transcriptome analysis was performed using T. reesei grown in different carbon source and lignocellulose substrates, and data analysis has been focused on the functions of known major regulators, such as Xyr1 and Cre1

Summary

Introduction

Cellulase is widely used in detergents, textiles, pulp processing, food, and feed industries (Sharma et al, 2016). Transcription factor Xyr is the major positive regulator governing a complex regulatory network for expression of (hemi-) cellulase genes, and recently, the function of Ace is characterized for cellulase production, and its cross talk with Xyr and Crt was revealed (Zhang et al, 2019). Other regulators, such as Ace, BglR, and Vib have been identified to regulate cellulase production (Aro et al, 2001; Nitta et al, 2012; Zhang et al, 2018a). The detailed regulatory mechanisms of cellulase production from T. reesei are known to be complicated and are still not fully clear

Methods

Results

Conclusion