Transcriptional engineering of the glyceraldehyde‐3‐phosphate dehydrogenase promoter for improved heterologous protein production in Pichia pastoris

Abstract

The constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (P ), which is one of the benchmark promoters of Pichia pastoris, was analyzed in terms of putative transcription factor binding sites. We constructed a synthetic library with distinct regulatory properties through deletion and duplication of these putative transcription factor binding sites and selected transcription factor (TF) genes were overexpressed or deleted to understand their roles on heterologous protein production. Using enhanced green fluorescent protein, an expression strength in a range between 0.35- and 3.10-fold of the wild-type P was obtained. Another model protein, recombinant human growth hormone was produced under control of selected promoter variants and 1.6- to 2.4-fold higher product titers were reached compared to wild-type P . In addition, a GAL4-like TF was found to be a crucial factor for the regulation of P , and its overexpression enhanced the heterologous protein production considerably (up to 2.2-fold compared to the parental strain). The synthetic P library generated enabled us to investigate the different putative transcription factors which are responsible for the regulation of P under different growth conditions, ergo recombinant protein production under P . Biotechnol. Bioeng. 2017;114: 2319-2327. © 2017 Wiley Periodicals, Inc.

Engineering of alcohol dehydrogenase 2 hybrid‐promoter architectures in Pichia pastoris to enhance recombinant protein expression on ethanol

“…The base plasmid pPICZα-A::eGFP (Ata et al, 2017) was used in the construction of recombinant eGFP expression cassettes. The list of primers designed and used is given in Method S1.…”

“…Efficient and tuneable promoter selection is crucial for recombinant protein production as it precedes and starts with the transcription of the target gene which is the first step of protein synthesis, wherein P. pastoris, these are often derived from carbon source utilization pathways. The results of the latter two work revealed that the strains constructed with P GAP variants and the NONPs produced considerable amounts of ethanol as a byproduct and, synthesized and secreted more recombinant protein than the strain constructed with P GAP (Ata et al, 2017;Massahi & Çalık, 2018). P GAPbased fermentation processes in glucose-based defined media are advantageous for the production of biopharmaceuticals compared to P AOX1 -based fermentations using methanol, since nontoxic carbon source containing media also may simplify downstream purification and regulatory documentation.…”

“…The highlighted yeast platform Pichia pastoris (syn. Recently we have analyzed P GAP in terms of putative transcription factor binding sites (TFBSs) and constructed a synthetic library with distinct regulatory properties through deletion and duplication of the determined putative TFBSs, empowering design of novel strains being more favorable for recombinant protein production (Ata, Prielhofer, Gasser, Mattanovich, & Çalık, 2017). Efficient and tuneable promoter selection is crucial for recombinant protein production as it precedes and starts with the transcription of the target gene which is the first step of protein synthesis, wherein P. pastoris, these are often derived from carbon source utilization pathways.…”

“…Promoter engineering includes approaches such as chimeric promoter design (Blazeck, Garg, Reed, & Alper, 2012;Blazeck, Liu, Redden, & Alper, 2011), random mutagenesis (Alper, Fischer, Nevoigt, & Stephanopoulos, 2005;Portela, Vogl, Ebner, Oliveira, & Glieder, 2018;Qin et al, 2011), modification of transcription factor binding sites (Ata et al, 2017;Hartner et al, 2008;Prielhofer et al, 2018), and synthetic promoter design (Curran et al, 2014;Portela et al, 2017;Redden & Alper, 2015;Vogl, Ruth, Pitzer, Kickenweiz, & Glieder, 2014). Synthetic biology extends on promoter engineering aiming to increase the transcription rate through designing regulatory circuit(s) by engineering binding of activating TF(s).…”

Hybrid fed‐batch bioreactor operation design: control of substrate uptake enhances recombinant protein production in high‐cell‐density fermentations

“…These HFBB design parameters change depending on the promoter architecture ( i.e . by promoter variants and transcriptional engineering) and with novel promoter systems due to changes occurring in the flow of C molecules through the pathways. A related article concerning the latter is under preparation by our research group.…”