Presence of protein production enhancers results in significantly higher methanol-induced protein production in Pichia pastoris

Loknath Gidijala,Raymond M D Verhaert,Alexander Steinbüchel,Stefan Uthoff,Sebastiaan J Van Kampen

doi:10.1186/s12934-018-0961-4

Abstract

BackgroundThe yeast Komagataella phaffii, better known as Pichia pastoris, is a commonly used host for recombinant protein production. Here expression vectors are reported that address the different steps of the transcription–translation–secretion pathway of heterologous protein production.ResultsTranscription and translation enhancing elements were introduced in an expression cassette for the production of recombinant Aspergillus niger feruloyl esterase A. The yield was increased by threefold as compared to the yield without these elements. Multiple copy strains were selected using a zeocin resistance marker in the expression cassette and showed another sixfold higher yield. Modification of the C-terminal amino acid sequence of the secretion signal did not significantly improve the production yield. Similar data were obtained for the production of another protein, recombinant human interleukin 8. Upscaling to fed-batch fermentation conditions resulted in a twofold increase for reference strains, while for strains with enhancing elements a tenfold improvement was observed.ConclusionsPichia pastoris is used for recombinant protein production in industrial fermentations. By addressing the transcription and translation of mRNA coding for recombinant protein, significant yield improvement was obtained. The yield improvement obtained under microscale conditions was maintained under fed-batch fermentation conditions. These data demonstrate the potential of these expression vectors for large scale application as improved production of proteins has major implications on the economics and sustainability of biocatalyst dependent production processes e.g. for the production of pharmaceuticals and for the bioconversions of complex molecules.

Highlights

The yeast Komagataella phaffii, better known as Pichia pastoris, is a commonly used host for recombinant protein production
A series of expression vectors carrying these elements was generated to determine the effect on methanol-induced protein production of recombinant feruloyl esterase (FAE)
Independent transformants for each of the six vectors were isolated by selection for histidine biosynthesis complementation. In general this method results in the isolation of clones of which > 90% carry only one copy of the expression cassette integrated into the genome [17]

Summary

Introduction

The yeast Komagataella phaffii, better known as Pichia pastoris, is a commonly used host for recombinant protein production. Expression vectors are reported that address the different steps of the transcription– translation–secretion pathway of heterologous protein production. The methylotrophic yeast Pichia pastoris is known as an excellent host for recombinant protein production [1,2,3]. One is based on the methanol induced AOX1 promoter. The other exploits the constitutively expressed GAP promoter, which has a key role in glycolysis. The methanol inducible expression system is often preferred over the constitutive. Transcription factor involved in the activation of the methanol metabolism genes Cis [31]. AOX1 promoter library generated by systematic deletion of sequences within the PAOX1 [5]. Transcription factor binding site involved in the binding of the transcription factor AdrIp [6]

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