Show/Hide Menu
Hide/Show Apps
Logout
Türkçe
Türkçe
Search
Search
Login
Login
OpenMETU
OpenMETU
About
About
Open Science Policy
Open Science Policy
Open Access Guideline
Open Access Guideline
Postgraduate Thesis Guideline
Postgraduate Thesis Guideline
Communities & Collections
Communities & Collections
Help
Help
Frequently Asked Questions
Frequently Asked Questions
Guides
Guides
Thesis submission
Thesis submission
MS without thesis term project submission
MS without thesis term project submission
Publication submission with DOI
Publication submission with DOI
Publication submission
Publication submission
Supporting Information
Supporting Information
General Information
General Information
Copyright, Embargo and License
Copyright, Embargo and License
Contact us
Contact us
Naturally occurring novel promoters for recombinant protein production around pyruvate branch-point in Pichia pastoris
Date
2018-10-10
Author
Çalık, Pınar
Metadata
Show full item record
This work is licensed under a
Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License
.
Item Usage Stats
218
views
0
downloads
Cite This
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. Promoter of pyruvate kinase (PPYK) has one transcription start-site with a minimum promoter-score of 0.99; while, promoter of pyruvate decarboxylase (PPDC) has two start-sites each having a minimum promoter-score of 0.91. To drive gene expression, putative promoter-regions were replaced with PGAP in parent plasmid pGAPZαA::hGH, harboring human-growth-hormone (hGH) gene. P. pastoris strains carrying single-copy hGH under PPYK, PPDC, and for comparison under PGAP were tested in high-cell-density rhGH fermentations abbreviated, respectively, as BRPYK, BRPDC, and BRGAP. Promoter strength was evaluated by mRNA transcription-copy-number (mTCN) results, rhGH concentration measurements, and calculated flux distributions. PPDC and PPYK performed higher activity compared to PGAP. Maximum rhGH production was obtained in BRPDC as 122 mg dm−3 at t = 15 h, and then in BRPYK as 101 mg dm−3 at t = 12 h, and in BRGAP as 58 mg dm−3 at t = 9 h; while, mTCN of hGH was the highest with PGAP at t = 15 h, and then with PPDC with the maximum at t = 12 h, and lowest with PPYK. Flux distributions demonstrated perturbation effects of the naturally-occurring-novel-promoters in the engineered systems and validated the cross-pathway regulatory interactions.
Subject Keywords
Biotechnology
,
Bioengineering
,
Molecular Biology
,
General Medicine
URI
https://hdl.handle.net/11511/42579
Journal
NEW BIOTECHNOLOGY
DOI
https://doi.org/10.1016/j.nbt.2018.05.184
Collections
Department of Chemical Engineering, Article
Suggestions
OpenMETU
Core
Regulation of crystal protein biosynthesis by Bacillus thuringiensis: I. Effects of mineral elements and pH
ICGEN, Y; Icgen, BÜLENT; Özcengiz, Gülay (Elsevier BV, 2002-11-01)
Crystal protein synthesis by a local isolate of Bacillus thuringiensis was monitored and compared in association with growth and sporulation in media differing in mineral element content. Mg and Cu were the most important metals for the biosynthesis of 135 kDa and 65 kDa toxin components in that the former was essential and the latter was greatly stimulatory at 10(-6) to 10(-7) M concentration. Also the inclusion of Mn favored toxin production at concentrations ranging from 3 x 10(-4) to 10(-5) M. The omiss...
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...
Codon optimization of xylA gene for recombinant glucose isomerase production in Pichia pastoris and fed-batch feeding strategies to fine-tune bioreactor performance
Ata, Ozge; Boy, Erdem; Gunes, Hande; Çalık, Pınar (Springer Science and Business Media LLC, 2015-05-01)
The objectives of this work are the optimization of the codons of xylA gene from Thermus thermophilus to enhance the production of recombinant glucose isomerase (rGI) in P. pastoris and to investigate the effects of feeding strategies on rGI production. Codons of xylA gene from T. thermophilus were optimized, ca. 30 % of the codons were replaced with those with higher frequencies according to the codon usage bias of P. pastoris, codon optimization resulted in a 2.4-fold higher rGI activity. To fine-tune bio...
Regulation of crystal protein biosynthesis by Bacillus thuringiensis: II. Effects of carbon and nitrogen sources.
Içgen, Y; İçgen, Bülent; Özcengiz, Gülay (Elsevier BV, 2002-11-01)
The regulation of crystal protein production in Bacillus thuringiensis 81 by sources of carbon and nitrogen was investigated. The highest titers of toxin were obtained on sucrose, lactose and inulin which also supported sporulation. Whey and molasses were also potential carbon substrates for toxin production. Other carbohydrates including glucose, glycerol, maltose, starch and dextrin yielded lower amounts of toxin. Nitrogen sources were found to exert the most profound controls. Peptone was the best organi...
An alternative supporting electrolyte for enzyme immobilization in conducting polymers
Kiralp, Senem; Balik, Balam; KARATAŞ, SEVİM; Toppare, Levent Kamil; Gungorb, Atilla (Elsevier BV, 2008-03-01)
In this study an alternative supporting electrolyte was used in enzyme immobilization. Invertase was studied to observe the effect of the supporting electrolyte. Sulfonated poly(arylene ether sulfone) was used as the supporting electrolyte during the electrolysis of pyrrole. The results show that the polymeric supporting electrolyte can be used instead of sodium dodecyl sulfate.
Citation Formats
IEEE
ACM
APA
CHICAGO
MLA
BibTeX
P. Çalık, “Naturally occurring novel promoters for recombinant protein production around pyruvate branch-point in Pichia pastoris,”
NEW BIOTECHNOLOGY
, pp. 0–0, 2018, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/42579.