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Title
A novel bi-directional promotor system for tunable recombinant protein expression in Pichia pastoris / Karl Metzger
Additional Titles
Neuartige bi-direktionale Promotersystme für regulierbare rekombinante Proteinexpression in Pichia pastoris
AuthorMetzger, Karl
CensorSpadiut, Oliver
PublishedWien, 2016
Description86 Blätter : Illustrationen
Institutional NoteTechnische Universität Wien, Diplomarbeit, 2016
Annotation
Zusammenfassung in deutscher Sprache
Abweichender Titel nach Übersetzung der Verfasserin/des Verfassers
LanguageEnglish
Document typeThesis (Diplom)
Keywords (EN)Tunable protein expression / Bi-directional promotor systems / Pichia pastoris / Novel promotor systems
URNurn:nbn:at:at-ubtuw:1-3755 Persistent Identifier (URN)
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 The work is publicly available
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A novel bi-directional promotor system for tunable recombinant protein expression in Pichia pastoris [5.74 mb]
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Abstract (English)

Pichia pastoris has become an important recombinant production host in research and industry. Recombinant protein production in P. pastoris is commonly regulated by the methanol inducible PAOX1 and constitutive PGAP promoter systems. However, both are attributed to various disadvantages rendering limited degree of freedom for bioprocess engineers to tune productivity. A promising and innovative approach to tune and control productivity is the combination of two different promoter systems with varying characteristics forming a bi-directional promoter system. In this study, we successfully characterized a recombinant P. pastoris strain carrying both a de-repression promoter (PDC) and an inducible promoter (PAOX1). We cloned the model enzyme cellobiohydrolase 2 (CBH2) downstream each promoter variant and analyzed how different feeding regimes in the bioreactor activated these promoters and thus productivity. In fact, we were able to tune recombinant protein production using different feeding strategies. We determined optimal operating windows for both promoters to work individually but also concomitantly. Summarizing, novel bi-directional promoter systems allow tunable recombinant protein production in P. pastoris and thus allow a high degree of freedom for bioprocess development and optimization.

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