Abstract
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, 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.ConclusionsWe 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.
Highlights
Plant-derived biomass is a potential alternative to fossil feedstocks for a greener economy
Whilst the advantage of using the Alcohol oxidase I (AOX1) promoter is that it allows controlled expression of heterologous proteins that may be toxic to the host, the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter simplifies cultivation by avoiding the need for toxic methanol as a carbon source
The constructed plasmids were transformed into the double protease knock-out PichiaPinkTM Strain 4 and integrated into the yeast chromosome, giving four recombinant P. pastoris strains: AOX1/AfCel12A, AOX1/TaCel5A, GAP/AfCel12A and GAP/TaCel5A
Summary
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. Widely used for heterologous protein expression, can be utilized for fungal endoglucanase production. Over the last thirty years several promoters have been utilized for heterologous protein production in P. pastoris, but to date only the regulated alcohol oxidase I (AOX1) and the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoters are used regularly [10,11,12]. The AOX1 promoter controls expression of the alcohol oxidase (AOX) in the methanol-utilization pathway. Whilst the advantage of using the AOX1 promoter is that it allows controlled expression of heterologous proteins that may be toxic to the host, the GAP promoter simplifies cultivation by avoiding the need for toxic methanol as a carbon source
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