title: Evaluation of heterologous α-amylase production in two expression platforms dedicated for Yarrowia lipolytica: commercial Po1g-pYLSC (php4d) and custom-made A18-pYLTEF (pTEF).
abstract: In view of the constantly increasing demand for cost-effective, low-energy and environmentally friendly industrial processes and household care products, enzyme production occupies an essential place in the field of biotechnology. Along with increasing demand for industrial and household care enzymes, the demand for heterologous expression platforms has also increased. Apart from the conventional hosts, e.g. Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris, routinely used in heterologous protein expression, the non-conventional ones have become more and more exploited in this field. Among the available yeast host systems, Yarrowia lipolytica appears to be an attractive alternative. The aim of this study was to compare efficiency of two Yarrowia-based expression platforms, commercial Po1g-pYLSC and custom-made A18-pYLTEF, in expression of an insect-derived, raw-starch-digesting α-amylase, to select the 'champion' system for further studies on this valuable enzyme. Both expression platforms were compared with respect to copy number of the integrated expression cassette/transformed genome, and the recombinant strains performance (Po1g-pYLSC-derived 4.29 strain, and A18-pYLTEF-derived B9 strain) during batch bioreactor cultures. Our results demonstrate that the average number of integration events into the recipient's genome was comparable for both expression systems under investigation, but with varying distribution of the multicopy integrants; and the number of the recombinant gene copies was highly correlated with the acquired amylolytic activity of the strains. Due to severe susceptibility of the recombinant AMY1 polypeptide to native proteases of the custom-made expression system, the final yield of the enzyme was substantially lower when compared to the commercial Po1g-pYLSC (reaching a maximum level of 142.84 AU/l). Copyright © 2015 John Wiley & Sons, Ltd.
authors.affiliation: Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poland.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Celińska
authors.affiliation: Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Borkowska
authors.affiliation: Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poland.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Białas
mesh_terms: D019149:Bioreactors; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D000516:alpha-Amylases
keywords: Yarrowia lipolytica; amylase; plasmid loss; raw starch digesting enzyme; recombinant protein expression; yeast expression system