期刊
BIOENGINEERED
卷 4, 期 5, 页码 322-331出版社
TAYLOR & FRANCIS INC
DOI: 10.4161/bioe.24653
关键词
Ashbya gossypii; random mutagenesis; increased protein production; GAS1 gene deletion
资金
- Fundacao para a Ciencia e a Tecnologia (FCT), Portugal
- project AshByofactory [PTDC/EBB-EBI/101985/2008-FCOMP-01-0124-FEDER-00971, SFRH/BD/30229/2006]
- MIT-Portugal Program Grant [SFRH/BD/39112/2007]
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 a-amylase and/or native beta-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 beta-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 Aggas1A Delta 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.
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