title: Random and direct mutagenesis to enhance protein secretion in Ashbya gossypii.
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.
authors: Institute for Biotechnology and Bioengineering (IBB); Centre of Biological Engineering; Universidade do Minho; Braga, Portugal; VTT Technical Research Centre of Finland; Otaniemi, Finland.
authors: Orquídea
authors: O
authors: Ribeiro
authors: Frederico
authors: F
authors: Magalhães
authors: Tatiana Q
authors: TQ
authors: Aguiar
authors: Marilyn G
authors: MG
authors: Wiebe
authors: Merja
authors: M
authors: Penttilä
authors: Lucília
authors: L
authors: Domingues
mesh_terms: D002473:Cell Wall / Q000201:enzymology / Q000235:genetics*; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D025141:Drug Resistance, Fungal; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005819:Genetic Markers; D060847:Metabolic Engineering / Q000379:methods; D016296:Mutagenesis; D009154:Mutation; D010692:Phleomycins / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D004718:Saccharomycetales / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D014242:Trichoderma / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D005819:Genetic Markers; D010692:Phleomycins; D005020:Ethyl Methanesulfonate; C056374:endoglucanase 1; D000516:alpha-Amylases; D001617:beta-Glucosidase; D002480:Cellulase
keywords: Ashbya gossypii; GAS1gene deletion; increased protein production; random mutagenesis
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