Recombinant protein production in Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter: From carbon source metabolism to bioreactor operation parameters

Çalı́k, Pınar; Ata, Özge; Güneş, Hande; Massahi, Aslan; Boy, Erdem; Keskin, Abdullah; Öztürk, Sibel; Zerze, Gül H.; Özdamar, Tunçer H.

doi:10.1016/j.bej.2014.12.003

Cited by 92 publications

(86 citation statements)

References 147 publications

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“…The glyceraldehyde‐3‐phosphate dehydrogenase GAP promoter (P GAP ), which is involved in a key step of the glycolysis pathway, was the first to emerge as a benchmark for efficient protein expression on various carbon sources. Thus, by avoiding all methanol‐related drawbacks, P GAP‐ based bioprocesses present relevant advantages for large‐scale production (Zhang et al , ; Ahmad et al , ; Çalık et al , ).…”

Section: Introductionmentioning

confidence: 99%

“…It reflects the equilibrium between the various steps until the product is secreted, as a balance of the different processes involved during the protein synthesis, folding and secretion. This relationship is crucial to bioprocess development and optimization (Potvin et al , ; Looser et al , ; Çalik et al, ). Thus, Garcia‐Ortega et al () and Maurer et al () characterized P GAP ‐based strains producing an antibody fragment and obtained robust results with them in chemostat systems; so, they found q P to increase eight times with increasing μ. Rebnegger et al () examined the response of this expression system producing human serum albumin (HSA) at different specific growth rates at transcriptomic level and observed marked upregulation of genes involved in translation.…”

Section: Introductionmentioning

confidence: 99%

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Rationale‐based selection of optimal operating strategies and gene dosage impact on recombinant protein production in Komagataella phaffii (Pichia pastoris)

Nieto-Taype

Garrigós-Martínez

Sánchez-Farrando

et al. 2019

Microbial Biotechnology

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Summary Its features as a microbial and eukaryotic organism have turned Komagataella phaffii (Pichia pastoris) into an emerging cell factory for recombinant protein production (RPP). As a key step of the bioprocess development, this work aimed to demonstrate the importance of tailor designing the cultivation strategy according to the production kinetics of the cell factory. For this purpose, K. phaffii clones constitutively expressing (PGAP) Candida rugosa lipase 1 (Crl1) with different gene dosage were used as models in continuous and fed‐batch cultures. Production parameters were much greater with a multicopy clone (MCC) than with the single‐copy clone (SCC). Regarding production kinetics, the specific product generation rate (qP) increased linearly with increasing specific growth rate (µ) in SCC; by contrast, qP exhibited saturation in MCC. A transcriptional analysis in chemostat cultures suggested the presence of eventual post‐transcriptional bottlenecks in MCC. After the strain characterization, in order to fulfil overall development of the bioprocess, the performance of both clones was also evaluated in fed‐batch mode. Strikingly, different optimal strategies were determined for both models due to the different production kinetic patterns observed as a trade‐off for product titre, yields and productivity. The combined effect of gene dosage and adequate µ enables rational process development with a view to optimize K. phaffii RPP bioprocesses.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Rationale‐based selection of optimal operating strategies and gene dosage impact on recombinant protein production in Komagataella phaffii (Pichia pastoris)

Nieto-Taype

Garrigós-Martínez

Sánchez-Farrando

et al. 2019

Microbial Biotechnology

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“…Factors that influence the expression level of the target peptide include inhibition of host growth by AMPs natural activities, AMP instability against bacterial and yeast proteases, and the inability to perform appropriate post-translational modifications (such as disulphide bonds) and produce recombinant proteins in the form of inactive inclusion bodies [14,15]. Furthermore, due to the requirement for fermentation, the large-scale production of AMPs in bacteria and yeast systems is limited, and the expensive downstream process significantly increases the production costs [16,17].…”

Section: Introductionmentioning

confidence: 99%

Recombinant expression of LFchimera antimicrobial peptide in a plant-based expression system and its antimicrobial activity against clinical and phytopathogenic bacteria

Chahardoli

Fazeli

Niazi‎

et al. 2018

Biotechnology & Biotechnological Equipment

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Antimicrobial peptides (AMPs) with their broad and selective antimicrobial activity and lowered risk of resistance development in microbial populations are promining as a new alternative candidate to conventional antibiotics. Plant-based expression systems can be employed as cost-effective and high scale-up capacity production hosts for recombinant expression of AMPs. LFchimera is a chimerical peptide containing LFcin and LFampin antimicrobial peptides of bovine lactoferrin, and it has stronger bactericidal activity. Here, the LFchimera sequence was codon-optimized for tobacco and fused with endoplasmic reticulum retention signals along with a CaMV 35S promoter and then transferred by agrobacterium-mediated transformation. The integration and expression of the transgene in tobacco plants were confirmed by polymerase chain reaction (PCR) and RT-PCR, respectively. Recombinant production of the peptide was confirmed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE), and peptide functional activity was confirmed by the antibacterial evaluation of total protein extracts against various clinical and phytopathogenic bacteria. Finally, LFchimera peptide was partially purified using an affinity column, and the antibacterial activity was shown. Taken together, these results confirmed that tobacco can be utilized for the recombinant production of antimicrobial peptides such as LFchimera.

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“…In particular, substantial resources are applied to the development of recombinant protein production with P. pastoris (Calik et al 2015), the most widely used system as expression system for heterologous protein production (Gasser et al 2013). Purification of recombinant proteins, including cell lysis is commonly recognized among the most costly parts of entire production processes (Ernst et al 1997).…”

Section: Discussionmentioning

confidence: 99%

Autolysis of Pichia pastoris induced by cold

et al. 2017

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The production of recombinant biopharmaceutical proteins is a multi-billion dollar market. Protein recovery represents a major part of the production costs. Pichia pastoris is one of the microbial systems most used for the production of heterologous proteins. The use of a cold-induced promoter to express lytic enzymes in the yeast after the growth stage could reduce protein recovery costs. This study shows that a cold-shock can be applied to induce lysis of the yeast cells. A strain of P. pastoris was constructed in which the endogenous eng gene encoding a putative endo-β-1,3-glucanase was overexpressed using the cold-shock induced promoter of the cctα gene from Saccharomyces cerevisiae. In the transgenic P. pastoris, the expression of eng increased 3.6-fold after chilling the cells from 30 to 4 °C (cold-shock stage) followed by incubation for 6 h (eng expression stage). The culture was heated to 30 °C for 6 h (ENG synthesis stage) and kept at 37 °C for 24 h (lysis stage). After this procedure the cell morphology changed, spheroplasts were obtained and cellular lysis was observed. Thus, a clone of P. pastoris was obtained, which undergoes autolysis after a cold-shock.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0397-y) contains supplementary material, which is available to authorized users.

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Recombinant protein production in Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter: From carbon source metabolism to bioreactor operation parameters

Cited by 92 publications

References 147 publications

Rationale‐based selection of optimal operating strategies and gene dosage impact on recombinant protein production in Komagataella phaffii (Pichia pastoris)

Rationale‐based selection of optimal operating strategies and gene dosage impact on recombinant protein production in Komagataella phaffii (Pichia pastoris)

Recombinant expression of LFchimera antimicrobial peptide in a plant-based expression system and its antimicrobial activity against clinical and phytopathogenic bacteria

Autolysis of Pichia pastoris induced by cold

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