Monday, May 4, 2009
5-18
Pichia pastoris as host for the expression of lignocellulolytic enzymes: Expression of Trichoderma reesei cellobiohydrolase II (Cel6A) as a model case
Karlheinz Flicker1, Andrea Mellitzer2, Roland Weis3, and Anton Glieder1. (1) Institute of Molecular Biotechnology, Graz University of Technology and Research Centre Applied Biocatalysis, Petersgasse 14, Graz, 8010, Austria, (2) Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, Graz, 8010, Austria, (3) VTU Technology GmbH, Parkring 18, Grambach, 8074, Austria
Lignocellulose provides a globally available, renewable resource for energy and biofuel production. A sustainable exploitation of his natural resource relies on innovative technologies such as the application of robust enzymes for the hydrolysis of lignocellulose. Additionally, competitive industrial processes for bioethanol production require cheap and highly active enzymes to be available in large quantities.
Pichia pastoris is known as a suitable expression host for many different proteins. However, so far efficient production of lignocellulolytic enzymes in P. pastoris has only been shown in some cases and expression of such enzymes in T. reesei usually gives complex enzyme mixtures. Here we present Pichia pastoris as a suitable host for the heterologous expression of eukaryotic cellulases. Our effort to express a whole set of individual (hemi-)cellulolytic enzymes was initiated by the expression of active Cel6A from T. reesei. Using proprietary expression technology and a codon-optimized gene of TrCel6A, Pichia transformants were screened in 96-well format applying an adapted reducing sugar assay. Additionally, coexpression of protein disulfide isomerase resulted in a synergistic effect, leading to secretion of TrCel6A in high yield.
These results show that P. pastoris, in combination with our expression technology, can be used as high-level expression host for the production of functional TrCel6A. The enzyme is now available as a pure enzyme and in unlimited amounts, enabling detailed analysis of the specific properties and capabilities of this isolated enzyme.
Pichia pastoris is known as a suitable expression host for many different proteins. However, so far efficient production of lignocellulolytic enzymes in P. pastoris has only been shown in some cases and expression of such enzymes in T. reesei usually gives complex enzyme mixtures. Here we present Pichia pastoris as a suitable host for the heterologous expression of eukaryotic cellulases. Our effort to express a whole set of individual (hemi-)cellulolytic enzymes was initiated by the expression of active Cel6A from T. reesei. Using proprietary expression technology and a codon-optimized gene of TrCel6A, Pichia transformants were screened in 96-well format applying an adapted reducing sugar assay. Additionally, coexpression of protein disulfide isomerase resulted in a synergistic effect, leading to secretion of TrCel6A in high yield.
These results show that P. pastoris, in combination with our expression technology, can be used as high-level expression host for the production of functional TrCel6A. The enzyme is now available as a pure enzyme and in unlimited amounts, enabling detailed analysis of the specific properties and capabilities of this isolated enzyme.