Increased expression of recombinant chitosanase by co-expression of Hac1p in the yeast Pichia pastoris.

2021 ◽  
Vol 28 ◽  
Author(s):  
Minghai Han ◽  
Weixian Wang ◽  
Xun Gong ◽  
Jianli Zhou ◽  
Cunbin Xu ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Research Effort ◽  
Heterologous Proteins ◽  
Unfolded Protein ◽  
Sds Page ◽  
Sequence Encoding ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
His4 Gene ◽  
Induction Temperature

Background: Pichia pastoris is one of the most popular eukaryotic hosts for producing heterologous proteins, while increasing secretion of target proteins is still a top priority for their application in industrial fields. Recently, the research effort to enhance protein production therein has focused on up-regulating the unfolded protein response (UPR). Objective: We evaluated the effects of activated UPR via Hac1p co-expression with the promoter AOX1 (PAOX1) or GAP (PGAP) on expression of recombinant chitosanase (rCBS) in P. pastoris. Method: The DNA sequence encoding the chitosanase was chemically synthesized and cloned into pPICZαA and the resulted pPICZαA/rCBS was transformed into P. pastoris for expressing rCBS. The P. pastoris HAC1i cDNA was chemically synthesized and cloned into pPIC3.5K to give pPIC3.5K/Hac1p. The HAC1i cDNA was cloned into pGAPZB and then inserted with HIS4 gene from pAO815 to construct the vector pGAPZB/Hac1p/HIS4. For co-expression of Hac1p, the two plasmids pPIC3.5K/Hac1p and pGAPZB/Hac1p/HIS4 were transformed into P. pastoris harboring the CBS gene. The rCBS was assessed based on chitosanase activity and analyzed by SDS-PAGE. The enhanced Kar2p was detected with western blotting to evaluate UPR. Results: Hac1p co-expression with PAOX1 enhanced rCBS secretion by 41% at 28°C. Although the level of UPR resulted from Hac1p co-expression with PAOX1 was equivalent to that with PGAP in terms of the quantity of Kar2p (a hallmark of the UPR), substitution of PGAP for PAOX1 further increased rCBS production by 21%. The methanol-utilizing phenotype of P. pastoris did not affect rCBS secretion with co-expression of Hac1p or not. Finally, Hac1p co-expression with PAOX1 or PGAP promoted rCBS secretion from 22 to 30°C and raised the optimum induction temperature. Conclusion: The study indicated that Hac1p co-expression with PAOX1 or PGAP is an effective strategy to trigger UPR of P. pastoris and a feasible means for improving production of rCBS therein.

scholarly journals The unfolded protein response in Pichia pastoris without external stressing stimuli

2020 ◽  
Vol 20 (7) ◽  
Author(s):  
Yasmin Nabilah Binti Mohd Fauzee ◽  
Naoki Taniguchi ◽  
Yuki Ishiwata-Kimata ◽  
Hiroshi Takagi ◽  
Yukio Kimata
Keyword(s):  
Pichia Pastoris ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Gene Knockout ◽  
Transmembrane Protein ◽  
Dependent Manner ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Stress Dependent ◽  
Protein Response

ABSTRACT Dysfunction or capacity shortage of the endoplasmic reticulum (ER) is cumulatively called ER stress and provokes the unfolded protein response (UPR). In various yeast species, the ER-located transmembrane protein Ire1 is activated upon ER stress and performs the splicing reaction of HAC1 mRNA, the mature form of which is translated into a transcription factor protein that is responsible for the transcriptome change on the UPR. Here we carefully assessed the splicing of HAC1 mRNA in Pichia pastoris (Komagataella phaffii) cells. We found that, inconsistent with previous reports by others, the HAC1 mRNA was substantially, but partially, spliced even without ER-stressing stimuli. Unlike Saccharomyces cerevisiae, growth of P. pastoris was significantly retarded by the IRE1-gene knockout mutation. Moreover, P. pastoris cells seemed to push more abundant proteins into the secretory pathway than S. cerevisiae cells. We also suggest that P. pastoris Ire1 has the ability to control its activity stringently in an ER stress-dependent manner. We thus propose that P. pastoris cells are highly ER-stressed possibly because of the high load of endogenous proteins into the ER.

scholarly journals Engineering of the unfolded protein response pathway in Pichia pastoris: enhancing production of secreted recombinant proteins

2021 ◽  
Author(s):  
Hana Raschmanová ◽  
Astrid Weninger ◽  
Zdeněk Knejzlík ◽  
Karel Melzoch ◽  
Karin Kovar
Keyword(s):  
Pichia Pastoris ◽  
Unfolded Protein Response ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Transcription Activator ◽  
Negative Effects ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Upr Pathway

Abstract Folding and processing of proteins in the endoplasmic reticulum (ER) are major impediments in the production and secretion of proteins from Pichia pastoris (Komagataella sp.). Overexpression of recombinant genes can overwhelm the innate secretory machinery of the P. pastoris cell, and incorrectly folded proteins may accumulate inside the ER. To restore proper protein folding, the cell naturally triggers an unfolded protein response (UPR) pathway, which upregulates the expression of genes coding for chaperones and other folding-assisting proteins (e.g., Kar2p, Pdi1, Ero1p) via the transcription activator Hac1p. Unfolded/misfolded proteins that cannot be repaired are degraded via the ER-associated degradation (ERAD) pathway, which decreases productivity. Co-expression of selected UPR genes, along with the recombinant gene of interest, is a common approach to enhance the production of properly folded, secreted proteins. Such an approach, however, is not always successful and sometimes, protein productivity decreases because of an unbalanced UPR. This review summarizes successful chaperone co-expression strategies in P. pastoris that are specifically related to overproduction of foreign proteins and the UPR. In addition, it illustrates possible negative effects on the cell’s physiology and productivity resulting from genetic engineering of the UPR pathway. We have focused on Pichia’s potential for commercial production of valuable proteins and we aim to optimize molecular designs so that production strains can be tailored to suit a specific heterologous product. Key points • Chaperones co-expressed with recombinant genes affect productivity in P. pastoris. • Enhanced UPR may impair strain physiology and promote protein degradation. • Gene copy number of the target gene and the chaperone determine the secretion rate.

scholarly journals Novel insights into the unfolded protein response using Pichia pastoris specific DNA microarrays

BMC Genomics ◽  
2008 ◽  
Vol 9 (1) ◽  
pp. 390 ◽  
Author(s):  
Alexandra Graf ◽  
Brigitte Gasser ◽  
Martin Dragosits ◽  
Michael Sauer ◽  
Germán G Leparc ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Unfolded Protein Response ◽  
Dna Microarrays ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Oestrogen-driven changes in the bovine uterus are associated with activation of the unfolded protein response

2014 ◽  
Author(s):  
Mohammed A Alfattah ◽  
Paul Anthony McGettigan ◽  
John Arthur Browne ◽  
Khalid M Alkhodair ◽  
Katarzyna Pluta ◽  
...  
Keyword(s):  
Unfolded Protein Response ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Native Ubiquitin Structural Changes Resulting from Complexation with β-methylamino-L-alanine (BMAA)

2020 ◽  
Author(s):  
Katie Mae Wilson ◽  
Aurora Burkus-Matesevac ◽  
Samuel Maddox ◽  
Christopher Chouinard
Keyword(s):  
Structural Changes ◽  
Noncovalent Complex ◽  
Unfolded Protein ◽  
Protein Size ◽  
High Concentrations ◽  
The Unfolded Protein Response ◽  
Critical Binding ◽  
Protein Response ◽  
The Brain ◽  
Lateral Sclerosis

β-methylamino-L-alanine (BMAA) has been linked to the development of neurodegenerative (ND) symptoms following chronic environmental exposure through water and dietary sources. The brains of those affected by this condition, often referred to as amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC), have exhibited the presence of plaques and neurofibrillary tangles (NFTs) from protein aggregation. Although numerous studies have sought to better understand the correlation between BMAA exposure and onset of ND symptoms, no definitive link has been identified. One prevailing hypothesis is that BMAA acts a small molecule ligand, complexing with critical proteins in the brain and reducing their function. The objective of this research was to investigate the effects of BMAA exposure on the native structure of ubiquitin. We hypothesized that formation of a Ubiquitin+BMAA noncovalent complex would alter the protein’s structure and folding and ultimately affect the ubiquitinproteasome system (UPS) and the unfolded protein response (UPR). Ion mobility-mass spectrometry revealed that at sufficiently high concentrations BMAA did in fact form a noncovalent complex with ubiquitin, however similar complexes were identified for a range of additional amino acids. Collision induced unfolding (CIU) was used to interrogate the unfolding dynamics of native ubiquitin and these Ubq-amino acid complexes and it was determined that complexation with BMAA led to a significant alteration in native protein size and conformation, and this complex required considerably more energy to unfold. This indicates that the complex remains more stable under native conditions and this may indicate that BMAA has attached to a critical binding location.

Native Ubiquitin Structural Changes Resulting from Complexation with β-methylamino-L-alanine (BMAA)

2020 ◽  
Author(s):  
Katie Mae Wilson ◽  
Aurora Burkus-Matesevac ◽  
Samuel Maddox ◽  
Christopher Chouinard
Keyword(s):  
Structural Changes ◽  
Noncovalent Complex ◽  
Unfolded Protein ◽  
Protein Size ◽  
High Concentrations ◽  
The Unfolded Protein Response ◽  
Critical Binding ◽  
Protein Response ◽  
The Brain ◽  
Lateral Sclerosis

β-methylamino-L-alanine (BMAA) has been linked to the development of neurodegenerative (ND) symptoms following chronic environmental exposure through water and dietary sources. The brains of those affected by this condition, often referred to as amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC), have exhibited the presence of plaques and neurofibrillary tangles (NFTs) from protein aggregation. Although numerous studies have sought to better understand the correlation between BMAA exposure and onset of ND symptoms, no definitive link has been identified. One prevailing hypothesis is that BMAA acts a small molecule ligand, complexing with critical proteins in the brain and reducing their function. The objective of this research was to investigate the effects of BMAA exposure on the native structure of ubiquitin. We hypothesized that formation of a Ubiquitin+BMAA noncovalent complex would alter the protein’s structure and folding and ultimately affect the ubiquitinproteasome system (UPS) and the unfolded protein response (UPR). Ion mobility-mass spectrometry revealed that at sufficiently high concentrations BMAA did in fact form a noncovalent complex with ubiquitin, however similar complexes were identified for a range of additional amino acids. Collision induced unfolding (CIU) was used to interrogate the unfolding dynamics of native ubiquitin and these Ubq-amino acid complexes and it was determined that complexation with BMAA led to a significant alteration in native protein size and conformation, and this complex required considerably more energy to unfold. This indicates that the complex remains more stable under native conditions and this may indicate that BMAA has attached to a critical binding location.

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