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Electrophysiological characterization of a diverse group of sugar transporters from Trichoderma reesei.

ABSTRACT: Trichoderma reesei is an ascomycete fungus known for its capability to secrete high amounts of extracellular cellulose- and hemicellulose-degrading enzymes. These enzymes are utilized in the production of second-generation biofuels and T. reesei is a well-established host for their production. Although this species has gained considerable interest in the scientific literature, the sugar transportome of T. reesei remains poorly characterized. Better understanding of the proteins involved in the transport of different sugars could be utilized for engineering better enzyme production strains. In this study we aimed to shed light on this matter by characterizing multiple T. reesei transporters capable of transporting various types of sugars. We used phylogenetics to select transporters for expression in Xenopus laevis oocytes to screen for transport activities. Of the 18 tested transporters, 8 were found to be functional in oocytes. 10 transporters in total were investigated in oocytes and in yeast, and for 3 of them no transport function had been described in literature. This comprehensive analysis provides a large body of new knowledge about T. reesei sugar transporters, and further establishes X. laevis oocytes as a valuable tool for studying fungal sugar transporters.

SUBMITTER: Havukainen S

PROVIDER: S-EPMC8290022 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Electrophysiological characterization of a diverse group of sugar transporters from Trichoderma reesei.

Havukainen Sami S Pujol-Giménez Jonai J Valkonen Mari M Westerholm-Parvinen Ann A Hediger Matthias A MA Landowski Christopher P CP

Scientific reports 20210719 1

Trichoderma reesei is an ascomycete fungus known for its capability to secrete high amounts of extracellular cellulose- and hemicellulose-degrading enzymes. These enzymes are utilized in the production of second-generation biofuels and T. reesei is a well-established host for their production. Although this species has gained considerable interest in the scientific literature, the sugar transportome of T. reesei remains poorly characterized. Better understanding of the proteins involved in the t ...[more]

PMID: 34282161

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Project description:BackgroundGlobal climate change and fossil fuels limitations have boosted the demand for robust and efficient microbial factories for the manufacturing of bio-based products from renewable feedstocks. In this regard, efforts have been done to enhance the enzyme-secreting ability of lignocellulose-degrading fungi, aiming to improve protein yields while taking advantage of their ability to use lignocellulosic feedstocks. Access to sugars in complex polysaccharides depends not only on their release by specific hydrolytic enzymes, but also on the presence of transporters capable of effectively transporting the constituent sugars into the cell. This study aims to identify and characterize xylose transporters from Aspergillus niger and Trichoderma reesei, two fungi that have been industrially exploited for decades for the production of lignocellulose-degrading hydrolytic enzymes.ResultsA hidden Markov model for the identification of xylose transporters was developed and used to analyze the A. niger and T. reesei in silico proteomes, yielding a list of candidate xylose transporters. From this list, three A. niger (XltA, XltB and XltC) and three T. reesei (Str1, Str2 and Str3) transporters were selected, functionally validated and biochemically characterized through their expression in a Saccharomyces cerevisiae hexose transport null mutant, engineered to be able to metabolize xylose but unable to transport this sugar. All six transporters were able to support growth of the engineered yeast on xylose but varied in affinities and efficiencies in the uptake of the pentose. Amino acid sequence analysis of the selected transporters showed the presence of specific residues and motifs recently associated to xylose transporters. Transcriptional analysis of A. niger and T. reesei showed that XltA and Str1 were specifically induced by xylose and dependent on the XlnR/Xyr1 regulators, signifying a biological role for these transporters in xylose utilization.ConclusionsThis study revealed the existence of a variety of xylose transporters in the cell factories A. niger and T. reesei. The particular substrate specificity and biochemical properties displayed by A. niger XltA and XltB suggested a possible biological role for these transporters in xylose uptake. New insights were also gained into the molecular mechanisms regulating the pentose utilization, at inducer uptake level, in these fungi. Analysis of the A. niger and T. reesei predicted transportome with the newly developed hidden Markov model showed to be an efficient approach for the identification of new xylose transporting proteins.

Project description:BackgroundOn-site cellulase production using locally available lignocellulosic biomass (LCB) is essential for cost-effective production of 2nd-generation biofuels. Cellulolytic enzymes (cellulases and hemicellulases) must be produced in fed-batch mode in order to obtain high productivity and yield. To date, the impact of the sugar composition of LCB hydrolysates on cellulolytic enzyme secretion has not been thoroughly investigated in industrial conditions.ResultsThe effect of sugar mixtures (glucose, xylose, inducer) on the secretion of cellulolytic enzymes by a glucose-derepressed and cellulase-hyperproducing mutant strain of Trichoderma reesei (strain CL847) was studied using a small-scale protocol representative of the industrial conditions. Since production of cellulolytic enzymes is inducible by either lactose or cellobiose, two parallel mixture designs were performed separately. No significant difference between inducers was observed on cellulase secretion performance, probably because a common induction mechanism occurred under carbon flux limitation. The characteristics of the enzymatic cocktails did not correlate with productivity, but instead were rather dependent on the substrate composition. Increasing xylose content in the feed had the strongest impact. It decreased by 2-fold cellulase, endoglucanase, and cellobiohydrolase activities and by 4-fold β-glucosidase activity. In contrast, xylanase activity was increased 6-fold. Accordingly, simultaneous high β-glucosidase and xylanase activities in the enzymatic cocktails seemed to be incompatible. The variations in enzymatic activity were modelled and validated with four fed-batch cultures performed in bioreactors. The overall enzyme production was maintained at its highest level when substituting up to 75% of the inducer with non-inducing sugars.ConclusionsThe sugar substrate composition strongly influenced the composition of the cellulolytic cocktail secreted by T. reesei in fed-batch mode. Modelling can be used to predict cellulolytic activity based on the sugar composition of the culture-feeding solution, or to fine tune the substrate composition in order to produce a desired enzymatic cocktail.

Dataset Information

Electrophysiological characterization of a diverse group of sugar transporters from Trichoderma reesei.

Publications

Electrophysiological characterization of a diverse group of sugar transporters from Trichoderma reesei.

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