Naturally occurring novel promoters around pyruvate branch-point for recombinant protein production in Pichia pastoris (Komagataella phaffii): Pyruvate decarboxylase- and pyruvate kinase- promoters

Massahi, Aslan
Çalık, Pınar
Novel strong promoter discovery is crucial for the design of novel strains of the industrial yeast Pichia pastoris for recombinant protein (r-protein) production. In order to remedy the deficiency, transcriptome and proteome data of P. pastoris were analysed. Genes having higher expression levels than glyceraldehyde-3-phosphate-dehydrogenase (GAP) gene were identified as promoter sources. Pyruvate kinase- (P-PYK) and pyruvate decarboxylase- (P-PDC) promoters around pyruvate-node were determined as promising candidates, and in silico analysis of the putative promoter regions was performed. The putative promoters were introduced into the plasmid pGAPZ alpha A::hGH harboring human growth hormone (hGH) gene, instead of P-GAP. Single-copy hGH gene carrying strains under P-PYK P-PDC, and for comparison under P-GAP were tested in high-cell-density rhGH fermentations, respectively, abbreviated as BRPYK, BRPDC. and BRGAP. Comparative promoter strength assessment was made by the mRNA-transcription-copy-number (mTCN) and r-protein concentration measurements, and by constrained flux analysis. P-PDC and P-PYK exhibited higher activity compared to P-GAP. Highest rhGH titer was obtained in BRPDC as 122 mg dm(-3) at t=15 h, in BRPYK as 101 mg dm(-3) at t=12 h, and in BRGAP as 58 mg dm(-3) at t=9 h; while, with P-PDC and P-GAP the mTCNs were high and close. The flux distributions demonstrated regulatory effects of the novel promoters in the engineered strains and validated cross-pathway regulatory interactions.


Engineering of alcohol dehydrogenase 2 hybrid-promoter architectures in Pichia pastoris to enhance recombinant protein expression on ethanol
Ergun, Burcu Gunduz; Gasser, Brigitte; Mattanovich, Diethard; Çalık, Pınar (2019-07-09)
The aim of this work is to increase recombinant protein expression in Pichia pastoris over the ethanol utilization pathway under novel-engineered promoter variants (NEPVs) of alcohol dehydrogenase 2 promoter (P-ADH2) through the generation of novel regulatory circuits. The NEPVs were designed by engineering of transcription factor binding sites (TFBSs) determined by in silico analyses and manual curation systematically, by (a) single-handedly replacement of specified TFBSs with synthetic motifs for Mxr1, Ca...
Ethanol fed-batch bioreactor operation to enhance therapeutic protein production in Pichia pastoris under hybrid-architectured ADH2 promoter
Wehbe, Omar; Yaman, Oğuz Ulaş; Çalık, Pınar (Elsevier BV, 2020-12-15)
We presented ethanol fed-batch bioreactor operations (FBBOs) designed for enhanced recombinant human growth hormone (rhGH) production in Pichia pastoris constructed with novel hybrid-architectured ADH2 promoter, PADH2-Cat8-L2. The parameters in the fed-batch fermentation of ethanol were investigated, and the boundaries of the production domain in terms of the design parameters were determined. Two FBBO design methods were used, and a platform aiming to adjust cellular metabolism for the generation of consta...
Naturally occurring novel promoters for recombinant protein production around pyruvate branch-point in Pichia pastoris
Çalık, Pınar (Elsevier BV, 2018-10-10)
Novel strong Pichia pastoris promoter discovery is crucial in metabolic engineering for recombinant protein (r-protein) production. Transcriptome and proteome data of P. pastoris were analysed and genes having higher expressions than glyceraldehyde-3-phosphate dehydrogenase (GAP) gene under limited-oxygen-transfer conditions were identified as promoter sources. Two promoters around pyruvate-node were determined as promising candidates, and in silico analysis of putative promoter regions was conducted. Promo...
Partial purification and characterization of neutral trehalase from commercial baker's yeast, Saccharomyces cerevisiae
Yarar, S; Hamamcı, Haluk; Bakir, U (2000-12-01)
The neutral trehalase of a commercial baker's yeast (S. cerevisiae) strain has been partially purified using ammonium sulfate fractionation and DEAE-cellulose column chromatography techniques. Trehalase was precipitated between 35-50% ammonium sulfate saturation and approximately 5-8 fold purification was achieved. The yeast cAMP-dependent protein kinase was also precipitated in the same fraction and these two proteins were separated by DEAE-cellulose column chromatography. Trehalase became totally inactive...
Engineered Deregulation of Expression in Yeast with Designed Hybrid-Promoter Architectures in Coordination with Discovered Master Regulator Transcription Factor
Ergun, Burcu Gunduz; Demir, Irem; Ozdamar, Tuncer H.; Gasser, Brigitte; Mattanovich, Diethard; Çalık, Pınar (2020-04-01)
Engineered promoters are key components in the cell-factory design, allowing precise and enhanced expression of genes. Promoters having exceptional strength are attractive candidates for designing metabolic engineering strategies for tailoring de novo production strategies that require directed evolution methods by engineering with de novo synthetic biology tools. Here, the custom-designed AOX1 hybrid-promoter architectures in coordination with targeted transcription factors are shown, transcriptionally rew...
Citation Formats
A. Massahi and P. Çalık, “Naturally occurring novel promoters around pyruvate branch-point for recombinant protein production in Pichia pastoris (Komagataella phaffii): Pyruvate decarboxylase- and pyruvate kinase- promoters,” BIOCHEMICAL ENGINEERING JOURNAL, pp. 111–120, 2018, Accessed: 00, 2020. [Online]. Available: