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    Improved production of a heterologous amylase in Saccharomyces cerevisiae by inverse metabolic engineering

    Access Status
    Open access via publisher
    Authors
    Liu, Z.
    Liu, Lifang
    Österlund, T.
    Hou, J.
    Huang, M.
    Fagerberg, L.
    Petranovic, D.
    Uhlén, M.
    Nielsen, J.
    Date
    2014
    Type
    Journal Article
    
    Metadata
    Show full item record
    Citation
    Liu, Z. and Liu, L. and Österlund, T. and Hou, J. and Huang, M. and Fagerberg, L. and Petranovic, D. et al. 2014. Improved production of a heterologous amylase in Saccharomyces cerevisiae by inverse metabolic engineering. Applied and Environmental Microbiology. 80 (17): pp. 5542-5550.
    Source Title
    Applied and Environmental Microbiology
    DOI
    10.1128/AEM.00712-14
    ISSN
    0099-2240
    School
    Centre for Crop Disease Management
    URI
    http://hdl.handle.net/20.500.11937/9968
    Collection
    • Curtin Research Publications
    Abstract

    The increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeast Saccharomyces cerevisiae is widely used as a cell factory for recombinant protein production. However, many recombinant proteins are produced at only 1% (or less) of the theoretical capacity due to the complexity of the secretory pathway, which has not been fully exploited. In this study, we applied the concept of inverse metabolic engineering to identify novel targets for improving protein secretion. Screening that combined UV-random mutagenesis and selection for growth on starch was performed to find mutant strains producing heterologous amylase 5-fold above the level produced by the reference strain. Genomic mutations that could be associated with higher amylase secretion were identified through whole-genome sequencing. Several single-point mutations, including an S196I point mutation in the VTA1 gene coding for a protein involved in vacuolar sorting, were evaluated by introducing these to the starting strain. By applying this modification alone, the amylase secretion could be improved by 35%. As a complement to the identification of genomic variants, transcriptome analysis was also performed in order to understand on a global level the transcriptional changes associated with the improved amylase production caused by UV mutagenesis. © 2014, American Society for Microbiology.

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