In Vitro Characterization of a Nuclear Receptor-like Domain of the Xylanase Regulator 1 from Trichoderma reesei

Abstract

Engineering transcription factors is an interesting research target gaining increasing attention, such as in the case of industrially used organisms. With respect to sustainability, biomass-degrading saprophytic fungi, such as Trichoderma reesei, are promising industrial work horses because they exhibit a high secretory capacity of native and heterologously expressed enzymes and compounds. A single-point mutation in the main transactivator of xylanase and cellulase expressions in T. reesei Xyr1 led to a strongly deregulated and enhanced xylanase expression. Circular dichroism spectroscopy revealed a change in secondary structure caused by this mutation. According to electrophoretic mobility shift assays and determination of the equilibrium-binding constants, the DNA-binding affinity of the mutated Xyr1 was considerably reduced compared to the wild-type Xyr1. Both techniques were also used to investigate the allosteric response to carbohydrates (D-glucose-6-phosphate, D-xylose, and sophorose) signalling the repression or induction of Xyr1 target genes. The mutated Xyr1 no longer exhibited a conformational change in response to these carbohydrates, indicating that the observed deregulation is not a simple matter of a change in DNA-binding of the transactivator. Altogether, we postulate that the part of Xyr1 where the mutation is located functions as a nuclear receptor-like domain that mediates carbohydrate signals and modulates the Xyr1 transactivating activity.

Single Mutation in Transcriptional Activator Xyr1 Enhances Cellulase and Xylanase Production in Trichoderma reesei on Glucose

“…So far, several studies have focused on the N-terminal DNA-binding domain and the C-terminal putative transcriptional activating region of Xyr1. ,, In contrast, little was known about the function of the middle homology region of Xyr1, although various studies have pointed to a role of this region not only in assisting DNA targeting discrimination but also in playing an important role in tuning the transcriptional activity. , A novel Xyr1 R434L mutant was first discovered with a single mutation in the middle homology region of Xyr1. A824V mutation in Xyr1 has been assumed to result in an activated conformation but with a reduced DNA-binding affinity regardless of inducing or repressing conditions . However, the activating mechanism of Xyr1 R434L is still not clear yet.…”

“…17 Since both mutations are found in the same predicted α-helix of Xyr1, the increased Xyr1 transcriptional activity has been assumed to result from an activated conformation regardless of inducing or repressing carbohydrates. 15 The results also implied that the C-terminus of Xyr1 may contain an autoregulatory domain. 19 Further analysis with a series of Xyr1 C-terminal truncations has revealed a coiled-coil domain that mediates its own homodimerization, which is essential for cellulase expression, but exerts no effect on the induced xylanase gene expression.…”

“…Similar to Xyr1 V821F , Xyr1 A824V is also capable of causing xylanase expression deregulation and improved basal cellulase biosynthesis even on repressing sugars . Since both mutations are found in the same predicted α-helix of Xyr1, the increased Xyr1 transcriptional activity has been assumed to result from an activated conformation regardless of inducing or repressing carbohydrates . The results also implied that the C-terminus of Xyr1 may contain an autoregulatory domain .…”

“…It has thus been shown that the deletion of xyr1 abolishes the induced biosynthesis of cellulases or xylanases . On the other hand, quite a few studies have focused on improved cellulase production under repressing conditions by the constitutive expression of Xyr1 or its mutant derivatives. − Although cellulase expression has been revealed to be positively correlated with Xyr1 levels, no such correlation has been found for xylanases, indicating that differential regulatory patterns acting on Xyr1 may exist to coordinate cellulase and xylanase biosynthesis …”

“…11−14 Although cellulase expression has been revealed to be positively correlated with Xyr1 levels, no such correlation has been found for xylanases, indicating that differential regulatory patterns acting on Xyr1 may exist to coordinate cellulase and xylanase biosynthesis. 15 Together with the middle homology region common to many fungi-specific Zn 2 Cys 6 -type TFs, the C-terminal transactivation domain of Xyr1 has not been fully characterized yet. 16,17 So far, two mutations in Xyr1, A824V, and V821F, which individually lead to improved transcriptional activity, have been reported.…”