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
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, Cat8, and Aca1 binding, and synthetic TATA-box integration; and, (b) nucleosome optimization. PADH2-Cat8-L2 and PADH2-Cat8-L1 designed by the integration of synthetic Cat8 binding sites were superior, and then PADH2-NucOpt. Compared to that with P-ADH2 at t = 20 hr of the fermentations, PADH2-Cat8-L2 allowed the highest increase in enhanced green fluorescent protein expression as 4.8-fold on ethanol and 3.8-fold on methanol; and, PADH2-NucOpt upregulated the expression 1.5-fold on ethanol and enhanced 3.2-fold on methanol. Using the superior two tools, Cat8-L2 and NucOpt, we designed PADH2-NucOpt-Cat8-L2. With PADH2-NucOpt-Cat8-L2, the expression in the fermentation of ethanol was upregulated 3.7-fold that is distinctly higher than that with PADH2-NucOpt but lower than that with PADH2-Cat8-L2; while on methanol compared to that with P-ADH2, the expression was enhanced 8.8-fold. Extracellular recombinant human serum albumin production was also studied with PADH2-Cat8-L2 and PADH2-NucOpt, and average recombinant human serum albumin yield (Y-P/X) on ethanol was 1.13 and 0.38 mg/g(WCW,) respectively; whereas with P-ADH2, Y-P/X was 0.26 mg/g(WCW). We conclude that as upregulation of transcription and enhanced expression correlate with the sequence of synthetic motifs and their location in the hybrid-promoter architectures of NEPVs in coordination with trans-acting factors, which are the design parameters in the engineering of binding sites; the NEPVs generated promising recombinant protein production platforms with a high impact on industrial scale production processes, as well as would open up new avenues for research in P. pastoris.


Design and construction of double promoter systems and their use in pharmaceutical protein production in P. Pastoris
Demir, İrem; Çalık, Pınar; Department of Chemical Engineering (2019)
Intracellular phenomena such as promoter strength, mRNA secondary structure, translation efficiency and codon preference, 5′-untranslated region processing, and protein turnover, have impacts directly on the expression of heterologous genes. Design of multi-promoter expression systems with constituent strong promoters and engineered promoter variants is a novel metabolic engineering strategy for increasing the promoter strength further, and tuning the expression for recombinant protein (r-protein) productio...
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 (2018-10-15)
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...
Cloning, Expression and Characterization of Endo-beta-1,4-Mannanase from Aspergillus fumigatus in Aspergillus sojae and Pichia pastoris
Duruksu, Goekhan; Ozturk, Bengu; Biely, Peter; Bakir, Ufuk; Ögel, Zümrüt Begüm (2009-01-01)
To be utilized in biomass conversion, including ethanol production and galactosylated oligosaccharide synthesis, namely prebiotics, the gene of extracellular endo-beta-1,4-mannanase (EC of Aspergillus fumigatus IMI 385708 (formerly known as Thermomyces lanuginosus IMI 158749) was expressed first ill Aspergillus sojae and then in Pichia pastoris under the control of the glyceraldehyde triphosphate dehydrogenase ((gpdA) under bar) and the alcohol oxidase ((AOX1) under bar) promoters, respectively. T...
Hybrid-architectured double-promoter expression systems enhance and upregulate-deregulated gene expressions in Pichia pastoris in methanol-free media
Demir, Irem; Çalık, Pınar (Springer Science and Business Media LLC, 2020-10-01)
Double-promoter expression system (DPES) design as de novo metabolic engineering strategy enables fine-tuned and enhanced gene expression. We constructed a collection of monodirectional hybrid-architectured DPESs with engineered promoter variants P(ADH2-Cat8-L2)and P(mAOX1)and with the naturally occurring promoter P(GAP)to enhance and upregulate-deregulated gene expressions inPichia pastorisin methanol-free media. Reporter red fluorescent protein (mApple) and enhanced green fluorescent protein (eGFP) were e...
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...
Citation Formats
B. G. Ergun, B. Gasser, D. Mattanovich, and P. Çalık, “Engineering of alcohol dehydrogenase 2 hybrid-promoter architectures in Pichia pastoris to enhance recombinant protein expression on ethanol,” BIOTECHNOLOGY AND BIOENGINEERING, pp. 2674–2686, 2019, Accessed: 00, 2020. [Online]. Available: