Expression of novel β-glucanase Cel12A from Stachybotrys atra in bacterial and fungal hosts

Picart, Pere; Goedegebuur, Frits; Dı́az, Pilar; Pastor, F. I. Javier

doi:10.1016/j.funbio.2012.01.004

Cited by 12 publications

(15 citation statements)

References 42 publications

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“…To evaluate the ability of S. cerevisiae to secrete bglucanase Cel12A, cells from cultures of control W303-1A/pG-1 and the recombinant strain W303-1A/pG-1-Cel12A were collected and enzymatic activity on lichenan of both, culture supernatants and cell extracts, was evaluated as previously described (Picart et al 2012). Most of the activity was found in the extracellular fraction indicating that the signal peptide of Cel12A was well processed by the yeast translational system.…”

Section: Resultsmentioning

confidence: 99%

“…These results confirm the de novo secretion of an active form of Cel12A in yeasts. Supernatants from S. cerevisiae W303-1A and S. cerevisiae W303-1A/pG-1-Cel12A were analyzed by SDS-PAGE and zymography as described (Picart et al 2012) (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…To express cel12A in S. cerevisiae, a cDNA fragment corresponding to the entire coding region, including its own signal peptide (MKVGAFLVVALSPLAFAA), was amplified via PCR from the plasmid pET28a-PSCel12A (Picart et al 2012), cloned under the control of the S. cerevisiae glyceraldehyde 3-phosphate dehydrogenase strong constitutive promoter (TDH3 p ) and the 3-phosphoglycerate kinase I terminator (PGK1 t ) into the expression vector pG-1 (Schena et al 1991), and the resulting plasmid (pG-1-Cel12A) introduced into the yeast strain S. cerevisiae W303-1A by the method of Gietz and Woods (2002). The tryptophan prototrophic transformant S. cerevisiae W303-1A/pG-1-Cel12A was thus isolated and used to evaluate recombinant Cel12A production.…”

Section: Recombinant Strains Constructionmentioning

confidence: 99%

“…Lichenase activity was assayed by measuring the amount of reducing sugar released using the Nelson and Somogyi method as described (Picart et al 2012). The assay mixtures contained 1.5 % w/v lichenan in a final volume of 0.1 ml of 50 mM sodium phosphate buffer at pH 6.0.…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…awamori protease deficient industrial strain (AP4) under the control of the glucoamylase gene promoter, and in frame with the glucoamylase signal peptide. The recombinant enzyme showed a non-typical behavior, with low endoglucanase activity and substrate preference for barley b-glucan and lichenan (Picart et al 2012). We report here the production of Cel12A in two additional fungal hosts, Saccharomyces cerevisiae and Aspergillus nidulans and the characterization of the enzyme produced in both recombinant strains.…”

Section: Introductionmentioning

confidence: 98%

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Recombinant expression of a GH12 β-glucanase carrying its own signal peptide from Stachybotrys atra in yeast and filamentous fungi

Picart

Orejas

Pastor

2016

World J Microbiol Biotechnol

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The β-glucanase Cel12A gene from Stachybotrys atra has been cloned and heterologously expressed in Aspergillus nidulans and Saccharomyces cerevisiae. The recombinant strains constructed, contained the exonic sequence of cel12A including its own signal peptide coding sequence. SDS-PAGE and zymography revealed that recombinant Cel12A has a molecular mass of 24 kDa which agrees with that deduced from its amino acid sequence, indicating that it is expressed in the non-glycosylated active form. Recombinant A. nidulans showed about eightfold greater activity yield than S. cerevisiae recombinant strain, namely 0.71 and 0.09 β-glucanase Units/ml of culture, respectively. In both host strains most of the activity was secreted to the extracellular media, evidencing the functionality of Cel12A signal peptide in yeast and fungi. This novel signal peptide might facilitate the expression and efficient secretion of other recombinant proteins difficult to secrete.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Recombinant Strains Constructionmentioning

confidence: 99%

Section: Enzyme Assaysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

See 3 more Smart Citations

Recombinant expression of a GH12 β-glucanase carrying its own signal peptide from Stachybotrys atra in yeast and filamentous fungi

Picart

Orejas

Pastor

2016

World J Microbiol Biotechnol

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Molecular basis of substrate recognition and specificity revealed in family 12 glycoside hydrolases

Calzado

Prates

Gonçalves

et al. 2016

Biotech & Bioengineering

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Fungal GH12 enzymes are classified as xyloglucanases when they specifically target xyloglucans, or promiscuous endoglucanases when they exhibit catalytic activity against xyloglucan and β-glucan chains. Several structural and functional studies involving GH12 enzymes tried to explain the main patterns of xyloglucan activity, but what really determines xyloglucanase specificity remains elusive. Here, three fungal GH12 enzymes from Aspergillus clavatus (AclaXegA), A. zonatus (AspzoGH12), and A. terreus (AtEglD) were studied to unveil the molecular basis for substrate specificity. Using functional assays, site-directed mutagenesis, and molecular dynamics simulations, we demonstrated that three main regions are responsible for substrate selectivity: (i) the YSG group in loop 1; (ii) the SST group in loop 2; and (iii) loop A3-B3 and neighboring residues. Functional assays and sequence alignment showed that while AclaXegA is specific to xyloglucan, AtEglD cleaves β-glucan, and xyloglucan. However, AspzoGH12 was also shown to be promiscuous contrarily to a sequence alignment-based prediction. We find that residues Y111 and R93 in AtEglD harbor the substrate in an adequate orientation for hydrolysis in the catalytic cleft entrance and that residues Y19 in AclaXegA and Y30 in AspzoGH12 partially compensate the absence of the YSG segment, typically found in promiscuous enzymes. The results point out the multiple structural factors underlying the substrate specificity of GH12 enzymes. Biotechnol. Bioeng. 2016;113: 2577-2586. © 2016 Wiley Periodicals, Inc.

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Transcriptional analysis of the lichenase-like gene cel12A of the filamentous fungus Stachybotrys atra BP-A and its relevance for lignocellulose depolymerization

2021

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To rationally optimize the production of industrial enzymes by molecular means requires previous knowledge of the regulatory circuits controlling the expression of the corresponding genes. The genus Stachybotrys is an outstanding producer of cellulosedegrading enzymes. Previous studies isolated and characterized the lichenase-like/non-typical cellulase Cel12A of S. atra (AKA S. chartarum) belonging to glycosyl hydrolase family 12 (GH12). In this study, we used RT-qPCR to determine the pattern of expression of cel12A under different carbon sources and initial ambient pH. Among the carbon sources examined, rice straw triggered a greater increase in the expression of cel12A than 1% lactose or 0.1% glucose, indicating specific induction by rice straw. In contrast, cel12A was repressed in the presence of glucose even when combined with this inducer. The proximity of 2 adjacent 5′-CTGGGGTCTGGGG-3′ CreA consensus target sites to the translational start site of cel12A strongly suggests that the carbon catabolite repression observed is directly mediated by CreA. Ambient pH did not have a significant effect on cel12A expression. These findings present new knowledge on transcriptional regulatory networks in Stachybotrys associated with cellulose/hemicellulose depolymerization. Rational engineering of CreA to remove CCR could constitute a novel strategy for improving the production of Cel12A.

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Expression of novel β-glucanase Cel12A from Stachybotrys atra in bacterial and fungal hosts

Cited by 12 publications

References 42 publications

Recombinant expression of a GH12 β-glucanase carrying its own signal peptide from Stachybotrys atra in yeast and filamentous fungi

Recombinant expression of a GH12 β-glucanase carrying its own signal peptide from Stachybotrys atra in yeast and filamentous fungi

Molecular basis of substrate recognition and specificity revealed in family 12 glycoside hydrolases

Transcriptional analysis of the lichenase-like gene cel12A of the filamentous fungus Stachybotrys atra BP-A and its relevance for lignocellulose depolymerization

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