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

Abstract

BackgroundTrichoderma 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.ResultsWe 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.ConclusionExpression 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.

Trichoderma reesei XYR1 recruits SWI/SNF to facilitate cellulase gene expression

“…). XYR1 overexpression has been reported to lead to high‐level cellulase gene expression even under repressing conditions (Lv et al , ). In accordance with this, TrSWI1 binding was found to occur even when cultured with glucose as the sole carbon source wherein XYR1 was expressed simultaneously (Fig.…”

“…To determine the subcellular localization of GFP‐TrSNF12, GFP‐TrSWI1 and GFP‐TrSNF5, the Trsnf12 , Trswi1 and Trsnf5 coding sequences were amplified from QM9414 cDNA, digested with Not I and Spe I, and then ligated into the pMDP tcu1 ‐ gfp ‐T trpC plasmid (Lv et al , ) to generate the pMDP tcu1 ‐ gfp ‐ Trsnf12 , ‐ gfp ‐ Trswi1 and ‐gfp ‐ Trsnf5 ‐T trpC respectively. These plasmids were then individually co‐transformed with P tcu1 ‐mCherry‐ xyr1 ‐ pyr4 (Zheng et al , ) into QM9414Δ pyr4 .…”

The Post-genomic Era of Trichoderma reesei : What's Next?

“…The use of inducible systems has mainly focused on naturally strong promoters such as Pcbh1 and Pcbh2 [61][62][63][64][65]. Additionally, genomic and transcriptomic approaches have been used to identify novel inducible systems, such as copper and L-methionine inducible promoters with strong activity in T. reesei [66,67]; both systems display high performance for constructing stable phenotypes. On the other hand, several studies have tried to identify and characterize strong constitutive expression systems for T. reesei, using, for example, promoters from ribosomal proteins, such as Prp2 [68], the pyruvate decarboxylase promoter Ppdc [69], and the enolase promoter Peno [70].…”

Rational Engineering A Xylanase Hyper-Producing System in Trichoderma Reesei for Efficient Biomass Degradation

“…The global transcription factor XYR1 participated in the regulation of cellulase and xylanase production. Overexpression of XYR1 showed a signi cant increase in cellulase/xylanase production [31] combined with derepressed cellulase/xylanase production on repressing medium. In contrast, the deletion of xyr1 would lead to despaired cellulase/xylanase production [32] and slower growth in xylose containing medium.…”