Overexpression of the Transcription Factor Azf1 Reveals Novel Regulatory Functions and Impacts ß-Glucosidase Production in Trichoderma reesei.

The fungus Trichoderma reesei is an essential producer of enzymes that degrade lignocellulosic biomass to produce value-added bioproducts. The cellulolytic system of T. reesei is controlled by several transcription factors (TFs) that efficiently regulate the production of these enzymes. Recently, a new TF named Azf1 was identified as a positive regulator of cellulase expression. Here, we investigated novel regulatory functions of Azf1 by its overexpression. In the mutant strain OEazf1, overexpression of azf1 was achieved under both repression and induction conditions. Although azf1 was more abundant in transcript and protein, overexpression of this TF did not activate transcription of the cellulase gene in the presence of the repressor glucose, suggesting that Azf1 may be subject to posttranslational regulation. In cellulose, the expression of swo, encoding the accessory protein swollenin, and the ß-glucosidases cel1a, cel1b, cel3b, and cel3g increases in the early stages of cultivation. The increased production of these ß-glucosidases increases the hydrolysis rate of cellobiose and sophorose, which activates carbon catabolite repression (CCR) and causes repression of cellulase genes and the regulator Xyr1 in the later stages of cultivation. Moreover, overexpression of azf1 led to increased cellulase activity in T. reesei during long-term cultivation in cellulose and sugarcane bagasse. Our results provide new insights into the mechanisms regulating Azf1 and novel genes that are important targets of this TF. This work contributes to a better understanding of the complex mechanisms regulating cellulase expression in T. reesei. It will contribute to the development of strains with higher production of these essential enzymes.

The Cerato-Platanin EPL2 from Trichoderma reesei Is Not Directly Involved in Cellulase Formation but in Cell Wall Remodeling.

Trichoderma reesei is a saprophytic fungus that produces large amounts of cellulases and is widely used for biotechnological applications. Cerato-platanins (CPs) are a family of proteins universally distributed among Dikarya fungi and have been implicated in various functions related to fungal physiology and interaction with the environment. In T. reesei, three CPs are encoded in the genome: Trire2_111449, Trire2_123955, and Trire2_82662. However, their function is not fully elucidated. In this study, we deleted the Trire2_123955 gene (named here as epl2) in the wild-type QM6aΔtmus53Δpyr4 (WT) strain and examined the behavior of the Δepl2 strain compared with WT grown for 72 h in 1% cellulose using RNA sequencing. Of the 9143 genes in the T. reesei genome, 760 were differentially expressed, including 260 only in WT, 214 only in Δepl2, and 286 in both. Genes involved in oxidative stress, oxidoreductase activity, antioxidant activity, and transport were upregulated in the Δepl2 mutant. Genes encoding cell wall synthesis were upregulated in the mutant strain during the late growth stage. The Δepl2 mutant accumulated chitin and glucan at higher levels than the parental strain and was more resistant to cell wall stressors. These results suggest a compensatory effect in cell wall remodeling due to the absence of EPL2 in T. reesei. This study is expected to contribute to a better understanding of the role of the EPL2 protein in T. reesei and improve its application in biotechnological fields.

Use of carbohydrate-directed enzymes for the potential exploitation of sugarcane bagasse to obtain value-added biotechnological products.

Microorganisms, such as fungi and bacteria, are crucial players in the production of enzymatic cocktails for biomass hydrolysis or the bioconversion of plant biomass into products with industrial relevance. The biotechnology industry can exploit lignocellulosic biomass for the production of high-value chemicals. The generation of biotechnological products from lignocellulosic feedstock presents several bottlenecks, including low efficiency of enzymatic hydrolysis, high cost of enzymes, and limitations on microbe metabolic performance. Genetic engineering offers a route for developing improved microbial strains for biotechnological applications in high-value product biosynthesis. Sugarcane bagasse, for example, is an agro-industrial waste that is abundantly produced in sugar and first-generation processing plants. Here, we review the potential conversion of its feedstock into relevant industrial products via microbial production and discuss the advances that have been made in improving strains for biotechnological applications.

Corrigendum to analysis of the phosphorylome of Trichoderma reesei cultivated on sugarcane bagasse suggests post-translational regulation of the secreted glycosyl hydrolase Cel7A.

[This corrects the article DOI: 10.1016/j.btre.2021.e00652.].

Analysis of the phosphorylome of trichoderma reesei cultivated on sugarcane bagasse suggests post-translational regulation of the secreted glycosyl hydrolase Cel7A.

Trichoderma reesei is one of the major producers of holocellulases. It is known that in T. reesei, protein production patterns can change in a carbon source-dependent manner. Here, we performed a phosphorylome analysis of T. reesei grown in the presence of sugarcane bagasse and glucose as carbon source. In presence of sugarcane bagasse, a total of 114 phosphorylated proteins were identified. Phosphoserine and phosphothreonine corresponded to 89.6% of the phosphosites and 10.4% were related to phosphotyrosine. Among the identified proteins, 65% were singly phosphorylated, 19% were doubly phosphorylated, 12% were triply phosphorylated, and 4% displayed even higher phosphorylation. Seventy-five kinases were predicted to phosphorylate the sites identified in this work, and the most frequently predicted serine/threonine kinase was PKC1. Among phosphorylated proteins, four glycosyl hydrolases were predicted to be secreted. Interestingly, Cel7A activity, the most secreted protein, was reduced to approximately 60% after in vitro dephosphorylation, suggesting that phosphorylation might alter Cel7A structure, substrate affinity, and targeting of the substrate to its carbohydrate-binding domain. These results suggest a novel post-translational regulation of Cel7A.