ArticleMicrobial cell factories2014
Expression of endoglucanases in Pichia pastoris under control of the GAP promoter.
Article in Microbial cell factories, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 36 papers.
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36 citing papers in PubMed, 78 citations in OpenAlex.
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- De novo biosynthesis of 2'-fucosyllactose in engineered Pichia pastoris.Biotechnology letters · 2023Article
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- Microfluidic screening and genomic mutation identification for enhancing cellulase production in Pichia pastoris.Biotechnology for biofuels and bioproducts · 2022Article
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- Quantifying Oxidation of Cellulose-Associated Glucuronoxylan by Two Lytic Polysaccharide Monooxygenases from Neurospora crassa.Applied and environmental microbiology · 2021Article
- Kinetic Characterization of a Putatively Chitin-Active LPMO Reveals a Preference for Soluble Substrates and Absence of Monooxygenase Activity.ACS catalysis · 2021Article
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- Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris.Biological procedures online · 2020Article
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- Establishing a EukaryoticFrontiers in bioengineering and biotechnology · 2020Article
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- Comparison of three seemingly similar lytic polysaccharide monooxygenases fromThe Journal of biological chemistry · 2019Article
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Authors and funding
6 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
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.
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