“…For Trire2_62380, _3405 and _67752, we used the genes from the RUT-C30 genome assembly 72 (TrireRUT-C30_95062, _109243, and _79984, respectively). RUT-C30 versions of Trire2_3405 and _67752 have been shown to be functional in previous studies 20 , 21 . Sequence for Trire2_67469 was manually annotated based on a homolog identified from other species of Trichoderma , as shown in Supplementary Figure S10a .…”
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.
“…For Trire2_62380, _3405 and _67752, we used the genes from the RUT-C30 genome assembly 72 (TrireRUT-C30_95062, _109243, and _79984, respectively). RUT-C30 versions of Trire2_3405 and _67752 have been shown to be functional in previous studies 20 , 21 . Sequence for Trire2_67469 was manually annotated based on a homolog identified from other species of Trichoderma , as shown in Supplementary Figure S10a .…”
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.
“…1 b, alignment was done with MUSCLE [ 24 ]). Homologs of the passive facilitator CDT-2 include putative lactose transporters trire2_77517 and trire2_79202 [ 15 ], while closest functionally characterized homolog for the proton symporter CDT-1 is trire2_67752 [ 25 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…7 c). The previously mentioned disaccharide transporters might include the two putative lactose transporters identified by Porciuncula et al [ 15 ] (trire2_79202 and 77517) and the two recently identified transporters which both have been shown to have cellobiose transport activity when expressed in yeast (trire2_69957 and 67752) [ 25 , 35 ]. The genes coding for these transporters are expressed in cellulase-inducing conditions.…”
Background
Trichoderma reesei is an ascomycete fungus that has a tremendous capability of secreting extracellular proteins, mostly lignocellulose-degrading enzymes. Although many aspects of the biology of this organism have been unfolded, the roles of the many sugar transporters coded in its genome are still a mystery with a few exceptions. One of the most interesting sugar transporters that has thus far been discovered is the cellulose response transporter 1 (CRT1), which has been suggested to be either a sugar transporter or a sensor due to its seemingly important role in cellulase induction.
Results
Here we show that CRT1 is a high-affinity cellobiose transporter, whose function can be complemented by the expression of other known cellobiose transporters. Expression of two sequence variants of the crt1 gene in Saccharomyces cerevisiae revealed that only the variant listed in the RUT-C30 genome annotation has the capability to transport cellobiose and lactose. When expressed in the $$\Delta$$
Δ
crt1 strain, the variant listed in the QM6a genome annotation offers partial complementation of the cellulase induction, while the expression of the RUT-C30 variant or cellobiose transporters from two other fungal species fully restore the cellulase induction.
Conclusions
These results add to our knowledge about the fungal metabolism of cellulose-derived oligosaccharides, which have the capability of inducing the cellulase production in many species. They also help us to deepen our understanding of the T. reesei lactose metabolism, which can have important consequences as this sugar is used as the inducer of protein secretion in many industrial processes which employ this species.
“…One of the approaches aimed at exploring novel cellodextrin transporters and intracellular BGLs in order to confer S. cerevisiae cells the ability to ferment cellobiose more efficiently. In this regard, diverse cellobiose permeases from yeast or from other fungi different from N. crassa have been successfully expressed in S. cerevisiae (Sadie et al 2011;Ha et al 2013b;Li et al 2013;Zhang et al 2013;Bae et al 2014;dos Reis et al 2016;Casa-Villegas, Polaina and Marín-Navarro 2018;Nogueira et al 2018), as well as alternative intracellular BGLs from yeast, bacteria and fungi (Bae et al 2014;Fan et al 2016; Casa-Villegas, Polaina and Marín-Navarro 2018) (Table 2).…”
First-generation (1G) fuel ethanol production in sugarcane-based biorefineries is an established economic enterprise in Brazil. Second-generation (2G) fuel ethanol from lignocellulosic materials, though extensively investigated, is currently facing severe difficulties to become economically viable. Some of the challenges inherent to these processes could be resolved by efficiently separating and partially hydrolysing the cellulosic fraction of the lignocellulosic materials into the disaccharide cellobiose. Here, we propose an alternative biorefinery, where the sucrose-rich stream from the 1G process is mixed with a cellobiose-rich stream in the fermentation step. The advantages of mixing are 3-fold: (i) decreased concentrations of metabolic inhibitors that are typically produced during pretreatment and hydrolysis of lignocellulosic materials; (ii) decreased cooling times after enzymatic hydrolysis prior to fermentation; and (iii) decreased availability of free glucose for contaminating microorganisms and undesired glucose repression effects. The iSUCCELL platform will be built upon the robust Saccharomyces cerevisiae strains currently present in 1G biorefineries, which offer competitive advantage in non-aseptic environments, and into which intracellular hydrolyses of sucrose and cellobiose will be engineered. It is expected that high yields of ethanol can be achieved in a process with cell recycling, lower contamination levels and decreased antibiotic use, when compared to current 2G technologies.
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