title: Expression of endoglucanases in Pichia pastoris under control of the GAP promoter.
abstract: BACKGROUND
  Plant-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.
  
  
  RESULTS
  In the present study, a plasmid carrying the constitutive GAP promoter was created for PichiaPink™. We then studied expression of two endoglucanases, AfCel12A from Aspergillus fumigatus and TaCel5A 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 AfCel12A obtained with the constitutive promoter were similar or higher, compared to the AOX1 promoter, whereas the levels of TaCel5A 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 AfCel12A and 170 nkat/ml TaCel5A.
  
  
  CONCLUSIONS
  We present a strategy for constitutive recombinant protein expression in the novel PichiaPink™ system. Both AfCel12A and TaCel5A were successfully expressed constitutively in P. pastoris under the GAP promoter. Reasonable protein levels were reached after optimizing cultivation conditions.
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO Box 5003Chr, Magnus Falsens vei 1, N-1432 Ås, Norway. aniko.varnai@nmbu.no.
authors.forename: Anikó
authors.initials: A
authors.lastname: Várnai
authors.forename: Campbell
authors.initials: C
authors.lastname: Tang
authors.forename: Oskar
authors.initials: O
authors.lastname: Bengtsson
authors.forename: Andrew
authors.initials: A
authors.lastname: Atterton
authors.forename: Geir
authors.initials: G
authors.lastname: Mathiesen
authors.forename: Vincent G H
authors.initials: VG
authors.lastname: Eijsink
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics; D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D010843:Pichia / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D002480:Cellulase
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