Expression of endoglucanases in Pichia pastoris under control of the GAP promoter

Microbial Cell Factories

Research

Campbell Tang¹  Andrew Atterton¹  Vincent GH Eijsink²  Geir Mathiesen²  Oskar Bengtsson²  Anikó Várnai² 

[1] Centre for Process Innovation Limited, Wilton Centre, Wilton, TS10 4RF, Redcar, UK;Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO Box 5003Chr. Magnus Falsens vei 1, N-1432, Ås, Norway;

关键词: Endoglucanase;    Heterologous protein expression;    Pichia pastoris;    PichiaPink;    AOX1 promoter;    GAP promoter;   

DOI  :  10.1186/1475-2859-13-57

 received in 2013-12-27, accepted in 2014-04-14,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundPlant-derived biomass is a potential alternative to fossil feedstocks for a greener economy. Enzymatic saccharification of biomass has been studied extensively and endoglucanases have been found to be a prerequisite for quick initial liquefaction of biomass under industrial conditions. Pichia pastoris, widely used for heterologous protein expression, can be utilized for fungal endoglucanase production. The recently marketed PichiaPink™ expression system allows for rapid clone selection, and employs the methanol inducible AOX1 promoter to ensure high protein expression levels. However, methanol is toxic and poses a fire hazard, issues which become more significant at an industrial scale. It is possible to eliminate these risks and still maintain high productivity by switching to the constitutive GAP promoter.ResultsIn the present study, a plasmid carrying the constitutive GAP promoter was created for PichiaPink™. We then studied expression of two endoglucanases, Af Cel12A from Aspergillus fumigatus and Ta Cel5A from Thermoascus aurantiacus, regulated by either the AOX1 promoter or the GAP promoter. Initial experiments in tubes and small bioreactors showed that the levels of Af Cel12A obtained with the constitutive promoter were similar or higher, compared to the AOX1 promoter, whereas the levels of Ta Cel5A were somewhat lower. After optimization of cultivation conditions using a 15-l bioreactor, the recombinant P. pastoris strains utilizing the GAP promoter produced ca. 3–5 g/l of total secreted protein, with CMCase activity equivalent to 1200 nkat/ml Af Cel12A and 170 nkat/ml Ta Cel5A.ConclusionsWe present a strategy for constitutive recombinant protein expression in the novel PichiaPink™ system. Both Af Cel12A and Ta Cel5A were successfully expressed constitutively in P. pastoris under the GAP promoter. Reasonable protein levels were reached after optimizing cultivation conditions.

【 授权许可】

CC BY   
© Várnai et al.; licensee BioMed Central Ltd. 2014