Protein production and unfolded protein response in fermentations of Trichoderma reesei and its transformant expressing endoglucanase I with a hydrophobic tag

Research output: Contribution to conferenceConference articleScientific

Abstract

The effect of induction of protein production was studied in bioreactor cultures of the T. reesei strain Rut-C30 and its transformant expressing endoglucanase I (EGI, Cel7B) fused with a hydrophobic tag. The peptide tag was previously designed for efficient purification of the fusion protein in aqueous two-phase separation. The first phase of the bioreactor cultivations was carried out on glucose containing minimal medium. At the stage when glucose was nearly depleted, the medium was supplemented with rich medium containing lactose as a carbon source to induce production of cellulases. The transformant produced somewhat less secreted protein and cellobiohydrolase I (CBHI, Ce17A) activity than the parental strain. Western analysis of intracellular proteins showed that the fusion protein EGIcore-p5(WP)4 accumulated inside the cell, indicating impaired secretion of the protein. Two-dimensional gel analysis suggested that the fusion protein was possibly trapped early in the secretory pathway. The mRNA levels of the UPR (unfolded protein response) target genes, bipl and pdil, and the level of the activated hac1 transcript encoding the UPR transcription factor, increased at the same time with an increase in the transcript levels of cellulase genes, suggesting UPR activation in response to cellulase induction. However, only a minor increase in pdi1 and bip1 transcript level was observed in the transformant expressing the fusion protein compared to its parental strain. In addition, slightly lower CBHI production and cbhl mRNA levels were measured in the transformant as compared to the parental strain, indicating activation of the novel repression mechanism of genes encoding secreted proteins in response to secretion stress, RESS (repression under secretion stress).
Original languageEnglish
Publication statusPublished - 2004
EventPhysiology of Yeasts and Filamentous Fungi (PYFF2): 121th Event of the European Federation of Biotechnology - Anglet, France
Duration: 24 Mar 200428 Mar 2004

Conference

ConferencePhysiology of Yeasts and Filamentous Fungi (PYFF2)
CountryFrance
CityAnglet
Period24/03/0428/03/04

Fingerprint

unfolded protein response
Trichoderma reesei
endo-1,4-beta-glucanase
fermentation
proteins
bioreactors
secretion
cellulose 1,4-beta-cellobiosidase
glucose
protein secretion
cellulases
genes
lactose
transcription factors
gels
carbon

Keywords

  • protein production
  • secretion stress
  • unfolded protein response
  • two-phase separation

Cite this

@conference{2464d4c640cc43daa301c0da941e3bb4,
title = "Protein production and unfolded protein response in fermentations of Trichoderma reesei and its transformant expressing endoglucanase I with a hydrophobic tag",
abstract = "The effect of induction of protein production was studied in bioreactor cultures of the T. reesei strain Rut-C30 and its transformant expressing endoglucanase I (EGI, Cel7B) fused with a hydrophobic tag. The peptide tag was previously designed for efficient purification of the fusion protein in aqueous two-phase separation. The first phase of the bioreactor cultivations was carried out on glucose containing minimal medium. At the stage when glucose was nearly depleted, the medium was supplemented with rich medium containing lactose as a carbon source to induce production of cellulases. The transformant produced somewhat less secreted protein and cellobiohydrolase I (CBHI, Ce17A) activity than the parental strain. Western analysis of intracellular proteins showed that the fusion protein EGIcore-p5(WP)4 accumulated inside the cell, indicating impaired secretion of the protein. Two-dimensional gel analysis suggested that the fusion protein was possibly trapped early in the secretory pathway. The mRNA levels of the UPR (unfolded protein response) target genes, bipl and pdil, and the level of the activated hac1 transcript encoding the UPR transcription factor, increased at the same time with an increase in the transcript levels of cellulase genes, suggesting UPR activation in response to cellulase induction. However, only a minor increase in pdi1 and bip1 transcript level was observed in the transformant expressing the fusion protein compared to its parental strain. In addition, slightly lower CBHI production and cbhl mRNA levels were measured in the transformant as compared to the parental strain, indicating activation of the novel repression mechanism of genes encoding secreted proteins in response to secretion stress, RESS (repression under secretion stress).",
keywords = "protein production, secretion stress, unfolded protein response, two-phase separation",
author = "Anna Collen and Michael Bailey and Markku Saloheimo and Jaana Uusitalo and Merja Penttil{\"a} and Tiina Pakula",
note = "CA2: BEL2 CA: BEL; Physiology of Yeasts and Filamentous Fungi (PYFF2) : 121th Event of the European Federation of Biotechnology ; Conference date: 24-03-2004 Through 28-03-2004",
year = "2004",
language = "English",

}

Protein production and unfolded protein response in fermentations of Trichoderma reesei and its transformant expressing endoglucanase I with a hydrophobic tag. / Collen, Anna; Bailey, Michael; Saloheimo, Markku; Uusitalo, Jaana; Penttilä, Merja; Pakula, Tiina.

2004. Paper presented at Physiology of Yeasts and Filamentous Fungi (PYFF2), Anglet, France.

Research output: Contribution to conferenceConference articleScientific

TY - CONF

T1 - Protein production and unfolded protein response in fermentations of Trichoderma reesei and its transformant expressing endoglucanase I with a hydrophobic tag

AU - Collen, Anna

AU - Bailey, Michael

AU - Saloheimo, Markku

AU - Uusitalo, Jaana

AU - Penttilä, Merja

AU - Pakula, Tiina

N1 - CA2: BEL2 CA: BEL

PY - 2004

Y1 - 2004

N2 - The effect of induction of protein production was studied in bioreactor cultures of the T. reesei strain Rut-C30 and its transformant expressing endoglucanase I (EGI, Cel7B) fused with a hydrophobic tag. The peptide tag was previously designed for efficient purification of the fusion protein in aqueous two-phase separation. The first phase of the bioreactor cultivations was carried out on glucose containing minimal medium. At the stage when glucose was nearly depleted, the medium was supplemented with rich medium containing lactose as a carbon source to induce production of cellulases. The transformant produced somewhat less secreted protein and cellobiohydrolase I (CBHI, Ce17A) activity than the parental strain. Western analysis of intracellular proteins showed that the fusion protein EGIcore-p5(WP)4 accumulated inside the cell, indicating impaired secretion of the protein. Two-dimensional gel analysis suggested that the fusion protein was possibly trapped early in the secretory pathway. The mRNA levels of the UPR (unfolded protein response) target genes, bipl and pdil, and the level of the activated hac1 transcript encoding the UPR transcription factor, increased at the same time with an increase in the transcript levels of cellulase genes, suggesting UPR activation in response to cellulase induction. However, only a minor increase in pdi1 and bip1 transcript level was observed in the transformant expressing the fusion protein compared to its parental strain. In addition, slightly lower CBHI production and cbhl mRNA levels were measured in the transformant as compared to the parental strain, indicating activation of the novel repression mechanism of genes encoding secreted proteins in response to secretion stress, RESS (repression under secretion stress).

AB - The effect of induction of protein production was studied in bioreactor cultures of the T. reesei strain Rut-C30 and its transformant expressing endoglucanase I (EGI, Cel7B) fused with a hydrophobic tag. The peptide tag was previously designed for efficient purification of the fusion protein in aqueous two-phase separation. The first phase of the bioreactor cultivations was carried out on glucose containing minimal medium. At the stage when glucose was nearly depleted, the medium was supplemented with rich medium containing lactose as a carbon source to induce production of cellulases. The transformant produced somewhat less secreted protein and cellobiohydrolase I (CBHI, Ce17A) activity than the parental strain. Western analysis of intracellular proteins showed that the fusion protein EGIcore-p5(WP)4 accumulated inside the cell, indicating impaired secretion of the protein. Two-dimensional gel analysis suggested that the fusion protein was possibly trapped early in the secretory pathway. The mRNA levels of the UPR (unfolded protein response) target genes, bipl and pdil, and the level of the activated hac1 transcript encoding the UPR transcription factor, increased at the same time with an increase in the transcript levels of cellulase genes, suggesting UPR activation in response to cellulase induction. However, only a minor increase in pdi1 and bip1 transcript level was observed in the transformant expressing the fusion protein compared to its parental strain. In addition, slightly lower CBHI production and cbhl mRNA levels were measured in the transformant as compared to the parental strain, indicating activation of the novel repression mechanism of genes encoding secreted proteins in response to secretion stress, RESS (repression under secretion stress).

KW - protein production

KW - secretion stress

KW - unfolded protein response

KW - two-phase separation

M3 - Conference article

ER -