Abstract
To improve the general secretion ability of the biotechnologically relevant fungus Ashbya gossypii, random mutagenesis with ethyl methane sulfonate (EMS) was performed. The selection and screening strategy followed revealed mutants with improved secretion of heterologous Trichoderma reesei endoglucanase I (EGI), native α-amylase and/or native β-glucosidase. One mutant, S436, presented 1.4- to 2-fold increases in all extracellular enzymatic activities measured, when compared with the parent strain, pointing to a global improvement in protein secretion. Three other mutants exhibited 2- to 3-fold improvements in only one (S397, B390) or two (S466) of the measured activities. A targeted genetic approach was also followed. Two homologs of the Saccharomyces cerevisiae GAS1, AgGAS1A (AGL351W) and AgGAS1B (AGL352W), were deleted from the A. gossypii genome. For both copies deletion, a new antibiotic marker cassette conferring resistance to phleomycin, BLE3, was constructed. GAS1 encodes an β-1,3-glucanosyltransglycosylase involved in cell wall assembly. Higher permeability of the cell wall was expected to increase the protein secretion capacity. However, total protein secreted to culture supernatants and secreted EGI activity did not increase in the Aggas1AΔ mutants. Deletion of the AgGAS1B copy affected cellular morphology and resulted in severe retardation of growth, similarly to what has been reported for GAS1-defficient yeast. Thus, secretion could not be tested in these mutants.
| Original language | English |
|---|---|
| Pages (from-to) | 322-331 |
| Journal | Bioengineered |
| Volume | 4 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - 1 Jan 2013 |
| MoE publication type | A1 Journal article-refereed |
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Keywords
- Ashbya gossypii
- GAS1gene deletion
- increased protein production
- random mutagenesis
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Random and direct mutagenesis to enhance protein secretion in Ashbya gossypii. / Ribeiro, Orquídea; Magalhães, Frederico; Aguiar, Tatiana Q.; Wiebe, Marilyn G.; Penttilä, Merja; Domingues, Lucília (Corresponding Author).
In: Bioengineered, Vol. 4, No. 5, 01.01.2013, p. 322-331.Research output: Contribution to journal › Article › Scientific › peer-review
TY - JOUR
T1 - Random and direct mutagenesis to enhance protein secretion in Ashbya gossypii
AU - Ribeiro, Orquídea
AU - Magalhães, Frederico
AU - Aguiar, Tatiana Q.
AU - Wiebe, Marilyn G.
AU - Penttilä, Merja
AU - Domingues, Lucília
N1 - CA2: TK402 CA2: TK400 CA: Cluster4 SDA: BIC
PY - 2013/1/1
Y1 - 2013/1/1
N2 - To improve the general secretion ability of the biotechnologically relevant fungus Ashbya gossypii, random mutagenesis with ethyl methane sulfonate (EMS) was performed. The selection and screening strategy followed revealed mutants with improved secretion of heterologous Trichoderma reesei endoglucanase I (EGI), native α-amylase and/or native β-glucosidase. One mutant, S436, presented 1.4- to 2-fold increases in all extracellular enzymatic activities measured, when compared with the parent strain, pointing to a global improvement in protein secretion. Three other mutants exhibited 2- to 3-fold improvements in only one (S397, B390) or two (S466) of the measured activities. A targeted genetic approach was also followed. Two homologs of the Saccharomyces cerevisiae GAS1, AgGAS1A (AGL351W) and AgGAS1B (AGL352W), were deleted from the A. gossypii genome. For both copies deletion, a new antibiotic marker cassette conferring resistance to phleomycin, BLE3, was constructed. GAS1 encodes an β-1,3-glucanosyltransglycosylase involved in cell wall assembly. Higher permeability of the cell wall was expected to increase the protein secretion capacity. However, total protein secreted to culture supernatants and secreted EGI activity did not increase in the Aggas1AΔ mutants. Deletion of the AgGAS1B copy affected cellular morphology and resulted in severe retardation of growth, similarly to what has been reported for GAS1-defficient yeast. Thus, secretion could not be tested in these mutants.
AB - To improve the general secretion ability of the biotechnologically relevant fungus Ashbya gossypii, random mutagenesis with ethyl methane sulfonate (EMS) was performed. The selection and screening strategy followed revealed mutants with improved secretion of heterologous Trichoderma reesei endoglucanase I (EGI), native α-amylase and/or native β-glucosidase. One mutant, S436, presented 1.4- to 2-fold increases in all extracellular enzymatic activities measured, when compared with the parent strain, pointing to a global improvement in protein secretion. Three other mutants exhibited 2- to 3-fold improvements in only one (S397, B390) or two (S466) of the measured activities. A targeted genetic approach was also followed. Two homologs of the Saccharomyces cerevisiae GAS1, AgGAS1A (AGL351W) and AgGAS1B (AGL352W), were deleted from the A. gossypii genome. For both copies deletion, a new antibiotic marker cassette conferring resistance to phleomycin, BLE3, was constructed. GAS1 encodes an β-1,3-glucanosyltransglycosylase involved in cell wall assembly. Higher permeability of the cell wall was expected to increase the protein secretion capacity. However, total protein secreted to culture supernatants and secreted EGI activity did not increase in the Aggas1AΔ mutants. Deletion of the AgGAS1B copy affected cellular morphology and resulted in severe retardation of growth, similarly to what has been reported for GAS1-defficient yeast. Thus, secretion could not be tested in these mutants.
KW - Ashbya gossypii
KW - GAS1gene deletion
KW - increased protein production
KW - random mutagenesis
UR - http://www.scopus.com/inward/record.url?scp=84898389418&partnerID=8YFLogxK
U2 - 10.4161/bioe.24653
DO - 10.4161/bioe.24653
M3 - Article
VL - 4
SP - 322
EP - 331
JO - Bioengineered
JF - Bioengineered
SN - 2165-5979
IS - 5
ER -