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Expression of lignocellulolytic enzymes in Pichia pastoris

Andrea Mellitzer1, Roland Weis3, Anton Glieder2 and Karlheinz Flicker2*

Author Affiliations

1 Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria

2 ACIB GmbH, Austrian Centre of Industrial Biotechnology, Graz, Austria

3 VTU Technology GmbH, Grambach, Austria

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Microbial Cell Factories 2012, 11:61  doi:10.1186/1475-2859-11-61

Published: 14 May 2012



Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes.

Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic) proteins due to several advantages.


In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes we could develop strains capable of secreting gram quantities of enzyme per liter in fed-batch cultivations. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data.


In our experiments we could clearly show the importance of gene optimization and strain characterization for successfully improving secretion levels. We also present a basic guideline how to correctly interpret the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris.

xylanase; mannanase; cellobiohydrolase; synthetic gene; synthetic promoter; quantitative real time PCR; Pichia pastoris; fermentation; strain development