Promoter library designed for fine-tuned gene expression in Pichia pastoris

Abstract

Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (PAOX1) sequence. This first library initially spanned an activity range between ∼6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing a green fluorescent protein (GFP) variant, the new regulatory toolbox was successfully utilized in a ‘real case’, i.e. the expression of industrial enzymes. Characterization of the library under repressing, derepressing and inducing conditions displayed at least 12 cis-acting elements involved in PAOX1-driven high-level expression. Based on this deletion analysis, novel short artificial promoter variants were constructed by combining cis-acting elements with basal promoter. In addition to improving yields and quality of heterologous protein production, the new PAOX1 synthetic promoter library constitutes a basic toolbox to fine-tune gene expression in metabolic engineering and sequential induction of protein expression in synthetic biology.

Genome sequence of the recombinant protein production host Pichia pastoris

“…The commonly used methanol-inducible promoters in P. pastoris-the alcohol oxidase I promoter 10,24 and the formaldehyde dehydrogenase promoter 25 -drive the production of enzymes needed for methanol assimilation and therefore produce extremely high levels of these transcripts upon switching the carbon source to methanol. The genome sequence has allowed identification of all genes coding for enzymes involved in methanol assimilation and their promoters ( Fig.…”

“…Humanized glycosylation will further increase the importance of P. pastoris for biopharmaceutical production; indeed, proteins produced with this system are moving into clinical development 7 . Moreover, monoclonal antibodies can be made at gramper-liter scale in the humanized glycosylation-homogenous strains 8 .For further strain engineering, a better understanding of all aspects of the yeast's protein production machinery is needed, and a number of studies relating to P. pastoris's secretory system and engineered promoters have been forthcoming 9,10 . To facilitate the investigation of P. pastoris and other methylotrophic yeasts, we present the 9.43 Mbp genomic sequence of the GS115 strain of P. pastoris.…”

“…For further strain engineering, a better understanding of all aspects of the yeast's protein production machinery is needed, and a number of studies relating to P. pastoris's secretory system and engineered promoters have been forthcoming 9,10 . To facilitate the investigation of P. pastoris and other methylotrophic yeasts, we present the 9.43 Mbp genomic sequence of the GS115 strain of P. pastoris.…”

Analysis of the 5′ untranslated region (5′UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris

“…In order to increase the yield of the recombinant protein, most investigators have focused on altering the AOX1 promoter region or alternatively using a different promoter, such as glyceraldehyde-3-phosphate dehydrogenase ( GAP ) (Hartner et al, 2008; Qin et al, 2011). Little is known about the contribution of the AOX1 5′UTR to the expression of recombinant proteins.…”

Variable production windows for porcine trypsinogen employing synthetic inducible promoter variants in Pichia pastoris

“…Generally, the Pichia pastoris system is a faster, more simple and less expensive alternative to higher eukaryotic expression systems, such as mammalian cell cultures, still able to perform typical eukaryotic posttranslational modifications like glycosylation or disulfide bridge formation (Cregg et al 1985(Cregg et al , 1993. Although Pichia pastoris has often been used successfully in recombinant protein production still little is known about the regulation of the AOX1 promoter on a molecular level (Hartner et al 2008). Briefly, transcription can be regulated by positively and negatively acting sequences in the promoter (Verdier 1990).…”