Carbon catabolite repression(CCR),which is mainly mediated by Cre1 and triggered by glucose,leads to a decrease in cellulase production in Trichoderma reesei.Many studies have focused on modifying Cre1 for alleviating...Carbon catabolite repression(CCR),which is mainly mediated by Cre1 and triggered by glucose,leads to a decrease in cellulase production in Trichoderma reesei.Many studies have focused on modifying Cre1 for alleviating CCR.Based on the homologous alignment of CreA from wild-type Penicillium oxalicum 114–2(Po-0)and cellulase hyperproducer JUA10-1(Po-1),we constructed a C-terminus substitution strain—Po-2—with decreased transcriptional levels of cellulase and enhanced CCR.Results revealed that the C-terminal domain of CreAPo−1 plays an important role in alleviating CCR.Furthermore,we replaced the C-terminus of Cre1 with that of CreAPo−1 in T.reesei(Tr-0)and generated Tr-1.As a control,the C-terminus of Cre1 was truncated and Tr-2 was generated.The transcriptional profiles of these transformants revealed that the C-terminal chimera greatly improves cellulase transcription in the presence of glucose and thus upregulates cellulase in the presence of glucose and weakens CCR,consistent with truncating the C-terminus of Cre1 in Tr-0.Therefore,we propose constructing a C-terminal chimera as a new strategy to improve cellulase production and alleviate CCR in the presence of glucose.展开更多
This research identified four amino acid residues(Leu174,Asn297,Tyr301,and Gln291)that contribute to sub-strate recognition by the high-affinity glucose transporter Xltr1p from Trichoderma reesei.Potential hotspots af...This research identified four amino acid residues(Leu174,Asn297,Tyr301,and Gln291)that contribute to sub-strate recognition by the high-affinity glucose transporter Xltr1p from Trichoderma reesei.Potential hotspots af-fecting substrate specificity were selected through homology modeling,evolutionary conservation analyses,and substrate-docking modeling of Xltr1p.Variants carrying mutations at these hotspots were subsequently obtained via in silico screening.Replacement of Leu174 or Asn297 in Xltr1p with alanine resulted in loss of hexose trans-port activity,indicating that Leu174 and Asn297 play essential roles in hexose transport.The Y301W variant exhibited accelerated mannose transport,but lost galactose transport capacity,and mutation of Gln291 to ala-nine greatly accelerated mannose transport.These results suggest that amino acids located in transmembrane𝛼-helix 7(Asn297,Tyr301,and Gln291)play critical roles in substrate recognition by the hexose transporter Xltr1p.Our results will help expand the potential applications of this transporter and provide insights into the mechanisms underlying its function and specificity.展开更多
基金This work was supported by National Key R&D Program of China(No.2018YFA0901700)National Natural Science Foundation of China(No.31870785 and 31570040)+1 种基金the 111 Project(No.B16030)the State Key Laboratory of Microbial Technology Open Projects Fund.
文摘Carbon catabolite repression(CCR),which is mainly mediated by Cre1 and triggered by glucose,leads to a decrease in cellulase production in Trichoderma reesei.Many studies have focused on modifying Cre1 for alleviating CCR.Based on the homologous alignment of CreA from wild-type Penicillium oxalicum 114–2(Po-0)and cellulase hyperproducer JUA10-1(Po-1),we constructed a C-terminus substitution strain—Po-2—with decreased transcriptional levels of cellulase and enhanced CCR.Results revealed that the C-terminal domain of CreAPo−1 plays an important role in alleviating CCR.Furthermore,we replaced the C-terminus of Cre1 with that of CreAPo−1 in T.reesei(Tr-0)and generated Tr-1.As a control,the C-terminus of Cre1 was truncated and Tr-2 was generated.The transcriptional profiles of these transformants revealed that the C-terminal chimera greatly improves cellulase transcription in the presence of glucose and thus upregulates cellulase in the presence of glucose and weakens CCR,consistent with truncating the C-terminus of Cre1 in Tr-0.Therefore,we propose constructing a C-terminal chimera as a new strategy to improve cellulase production and alleviate CCR in the presence of glucose.
基金This work was supported by National Key R&D Program of China(No.2018YFA0901700)National Natural Science Foundation of China(No.32271526).
文摘This research identified four amino acid residues(Leu174,Asn297,Tyr301,and Gln291)that contribute to sub-strate recognition by the high-affinity glucose transporter Xltr1p from Trichoderma reesei.Potential hotspots af-fecting substrate specificity were selected through homology modeling,evolutionary conservation analyses,and substrate-docking modeling of Xltr1p.Variants carrying mutations at these hotspots were subsequently obtained via in silico screening.Replacement of Leu174 or Asn297 in Xltr1p with alanine resulted in loss of hexose trans-port activity,indicating that Leu174 and Asn297 play essential roles in hexose transport.The Y301W variant exhibited accelerated mannose transport,but lost galactose transport capacity,and mutation of Gln291 to ala-nine greatly accelerated mannose transport.These results suggest that amino acids located in transmembrane𝛼-helix 7(Asn297,Tyr301,and Gln291)play critical roles in substrate recognition by the hexose transporter Xltr1p.Our results will help expand the potential applications of this transporter and provide insights into the mechanisms underlying its function and specificity.
