Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae

Penttilä, Merja; André, Lars; Lehtovaara, Päivi; Bailey, Michael; Teeri, Tuula T.; Knowles, Jonathan

doi:10.1016/0378-1119(88)90549-5

Cited by 170 publications

(91 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…S. cerevisiae does not itself produce practically any hydrolases attacking polymeric (hemi)cellulosic substrates and the possible background activities against oligosaccharides can be easily determined. Analysis of fungal extracellular hydrolases produced in S. cerevisiar has shown that their substrate specificities do not significantly differ from those produced in the native host (Penttila et al, 1988;Reinikainen et al, 1992;Margolles-Clark et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

Three α‐Galactosidase Genes of Trichoderma reesei Cloned by Expression in Yeast

Margolles-Clark

Tenkanen

Luonteri

et al. 1996

European Journal of Biochemistry

View full text Add to dashboard Cite

Three cx-galactosidase genes, agll, ag12 and ag13, were isolated from a cDNA expression library of Trichoderma reesei RutC-30 constructed in the yeast Saccharomyces cerevisiae by screening the library on plates containing the substrate 5-bromo-4-chloro-3-indolyl-a-D-gnlactopyranoside. The genes ugll, ag12 and agl3 encode 444, 746 and 624 amino acids, respectively, including the signal sequences. The deduced amino acid sequences of AGLI and AGLIII showed similarity with the a-galactosidases of plant, animal, yeast and filamentous fungal origin classified into family 27 of glycosyl hydrolases whereas the deduced amino acid sequence of AGLII showed similarity with the bacterial a-galactosidases of family 36. The enzymes produced by yeast were analysed for enzymatic activity against different substrates. AGLI, AGLII and AGLIII were able to hydrolyse the synthetic substrate p-nitrophenyl-a-D-galactopyranoside and the small galactose-containing oligosaccharides, melibiose and raffinose. They liberated galactose from polymeric galacto(g1uco)mannan with different efficiencies. The action of AGLI towards polymeric substrates was enhanced by the presence of the endo-l,4-/?-mannanase of T reesei. AGLII and AGLIII showed synergy in galacto(g1uco)mannan hydrolysis with the endo-l,4-/?-mannanase of 7: reesei and a B-mannosidase of Aspergillus niger. The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the a-galactosidase previously purified from 7: reesei.

show abstract

Section: Discussionmentioning

confidence: 99%

Three α‐Galactosidase Genes of Trichoderma reesei Cloned by Expression in Yeast

Margolles-Clark

Tenkanen

Luonteri

et al. 1996

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…The glutamic acids found in this study (Glu-126 in CBH I and Glu-127 in EG I) are obvious targets for site-directed mutagenesis, especially since several successful cloning experiments of these enzymes have been described [30].…”

Section: Isolation Of An Active-site Peptidementioning

confidence: 90%

Identification of a functionally important carboxyl group in cellobiohydrolase I from Trichoderma reesei

Tomme

Claeyssens

1989

FEBS Letters

View full text Add to dashboard Cite

Several aspects of the specific modification of cellobiohydrolase I (CBH I) from Trichoderma reesei with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-Y-sulfonate) are presented. The pH dependence of the resulting inactivation points to the implication of an ionising group with a pKa of approx. 5.5. The rapid inactivation kinetics, the specific protection and the stoichiometry of modification (3 versus 2 residues), together with the isolation and amino acid sequencing of the putative active site peptide, provide a large body of evidence for the presence of a catalytically important carboxyl residue in the 125-135 region of the CBH I amino acid sequence. From the striking homology between this peptide sequence and those of the active site regions of different lysozymes, glutamic acid 126 is retained as the most plausible catalytic residue (proton donor) in CBH I, equivalent to glutamic acid 35 in hen egg white lysozyme. Glutamic acid 127 is proposed as a potential active site residue to the homologous endoglucanase I (EG I) isolated from the same Trichoderma species.

show abstract

“…The secreted Tr-Cel7A from S. cerevisiae was proved to be Nhyperglycosylated [9][10][11]. Our previous result showed that the Nglycosidase F (PNGase F) removed almost all types of the N-linked glycosylation of recombinant Tr-Cel7A protein secreted by S. cerevisiae and lessened the molecular masses from over 100 kDa to two small types, which were ~90 and ~45 kDa [11].…”

Section: Resultsmentioning

confidence: 99%

“…One of the advantages of using budding yeast expression system for the production of heterologous glycoproteins is their capability to perform glycosylation, a process that does not occur in Escherichia coli [2]. In Saccharomyces cerevisiae, glycoprotein are normally modified by the core structure (Man 9 GlcNAc 2 ) which contains three α-1,2 mannosyl linkages in the endoplasmic reticulum (ER). One of α-1,2 mannosyl linkages is hydrolyzed by the site-specific α-1,2-mannosidase Mns1p just before the glycoprotein leaves for the Golgi apparatus.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Promotion of Extracellular Activity of Cellobiohydrolase I from Trichoderma reesei by Protein Glycosylation Engineering in Saccharomyces cerevisiae

Shen²,

Hou³

et al. 2013

Curr Synthetic Sys Biol

View full text Add to dashboard Cite

The N-glycosylation in Saccharomyces cerevisiae is of the high-mannose type, which affects the activity of the secreted heterologous glycoproteins. Cellobiohydrolase I (Tr-Cel7A) from Trichoderma reesei, is thus hyperglycosylated when expressed in S. cerevisiae. In the present work, three genes encoding the endogenous mannosyltransferases, Och1p, Mnn9p and Mnn1p, involved in glycoprotein processing in the S. cerevisiae Golgi apparatus, were individually or combinatorially disrupted to investigate the effect of the glycosylation extent on the activity of the secreted Tr-Cel7A. The glycosylation of the recombinant Tr-Cel7A was decreased and its extracellular activity was increased in all the deletion mutants. The simultaneous deletion of och1 and mnn1 has the most improvement on extracellular Tr-Cel7A activity. After expressed the α-1,2-mannosidase (Tr-Mds1p) from T. reesei in mnn1Δ/och1Δ strain, the Tr-Cel7A activity was further increased up to 320 ± 8% higher than that of the wild type strain. Such activity improvement was due not only to the higher secretion yield but also to the increased specific activity resulted from the changes in glycosylation. The results thus indicated that protein glycosylation engineering in S. cerevisiae was an effective approach to improve the extracellular activity of Tr-Cel7A.

show abstract

Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae

Cited by 170 publications

References 31 publications

Three α‐Galactosidase Genes of Trichoderma reesei Cloned by Expression in Yeast

Three α‐Galactosidase Genes of Trichoderma reesei Cloned by Expression in Yeast

Identification of a functionally important carboxyl group in cellobiohydrolase I from Trichoderma reesei

Promotion of Extracellular Activity of Cellobiohydrolase I from Trichoderma reesei by Protein Glycosylation Engineering in Saccharomyces cerevisiae

Contact Info

Product

Resources

About