prikaz prve stranice dokumenta Investigation of pyruvate kinase regulation in Pichia pastoris and effects of its overexpression on antibody production
Embargo period 2027-12-21
master's thesis
Investigation of pyruvate kinase regulation in Pichia pastoris and effects of its overexpression on antibody production
Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology, 2022. urn:nbn:hr:159:755118
Jurić, Gabrijela
University of Zagreb
Faculty of Food Technology and Biotechnology
Department of Biochemical Engineering
Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology

Institutional repository: Repository of the Faculty of Food Technology and Biotechnology

Cite this document

Jurić, G. (2022). Investigation of pyruvate kinase regulation in Pichia pastoris and effects of its overexpression on antibody production (Master's thesis). Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology. Retrieved from https://urn.nsk.hr/urn:nbn:hr:159:755118

Jurić, Gabrijela. "Investigation of pyruvate kinase regulation in Pichia pastoris and effects of its overexpression on antibody production." Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2022. https://urn.nsk.hr/urn:nbn:hr:159:755118

Jurić, Gabrijela. "Investigation of pyruvate kinase regulation in Pichia pastoris and effects of its overexpression on antibody production." Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2022. https://urn.nsk.hr/urn:nbn:hr:159:755118

Jurić, G. (2022). 'Investigation of pyruvate kinase regulation in Pichia pastoris and effects of its overexpression on antibody production', Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, accessed 22 November 2024, https://urn.nsk.hr/urn:nbn:hr:159:755118

Jurić G. Investigation of pyruvate kinase regulation in Pichia pastoris and effects of its overexpression on antibody production [Master's thesis]. Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology; 2022 [cited 2024 November 22] Available at: https://urn.nsk.hr/urn:nbn:hr:159:755118

G. Jurić, "Investigation of pyruvate kinase regulation in Pichia pastoris and effects of its overexpression on antibody production", Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, Zagreb, 2022. Available at: https://urn.nsk.hr/urn:nbn:hr:159:755118

Metadata
TitleInvestigation of pyruvate kinase regulation in Pichia pastoris and effects of its overexpression on antibody production
Title (croatian)Istraživanje regulacije piruvat kinaze u kvascu Pichia pastoris i utjecaja njene overekspresije na proizvodnju antitijela
AuthorGabrijela Jurić
MentorMario Novak (mentor)
Committee memberBojan Žunar (predsjednik povjerenstva)
Committee memberAntonija Trontel (član povjerenstva)
Committee memberAndreja Leboš-Pavunc (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Food Technology and Biotechnology
(Department of Biochemical Engineering)
(Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology)
Zagreb
Defense date and country2022-12-20, Croatia
Scientific / art field,
discipline and subdiscipline		BIOTECHNICAL SCIENCES
Biotechnology
Abstract
The goal of the thesis was to investigate whether the main regulatory mechanism of pyruvate kinase (Cdc19) in P. pastoris is allosteric activation by fructose-1,6-bisphosphate (FBP) as in Saccharomyces cerevisiae. To test this hypothesis, the pyruvate kinase gene (CDC19) in P. pastoris genome was substituted with mutant CDC19E397A, and strains were grown in ethanol as a carbon source. In S. cerevisiae, FBP activates pyruvate kinase (Cdc19) by interacting with the glutamate (Glu392). This regulation mechanism is important for Cdc19 inactivation in absence of glucose since it prevents futile cycling and enables gluconeogenic growth in nonfermentable carbon source. The analogous glutamate found in P. pastoris (Glu397) was replaced with alanine in a CRISPR-Cas9-mediated strategy and their growth in ethanol as a sole carbon source was determined. No differences in growth ability were observed, thus it was concluded that allosteric activation by FBP at Glu397 is not the main pyruvate kinase regulation mechanism in P. pastoris and there must be another key regulation mechanism or another interaction site important for allosteric regulation. Effects of CDC19ᴇ₃₉₇ᴀ overexpression in VHH antibody high-producing P. pastoris strain on VHH (Variable domain of the Heavy chain of the Heavychain antibody) yield were also evaluated by screening in glucose-limited conditions and results showed that CDC19ᴇ₃₉₇ᴀ overexpression can improve yield up to 2 fold.
Abstract (croatian)
Cilj ovog rada bio je odrediti je li glavni regulacijski mehanizam piruvat kinaze u P. pastoris alosterička aktivacija pomoću frukoza-1,6-bisfosfata (FBP) kao kod Saccharomyces cerevisiae. Kako bi testirali ovu prepostavku, gen piruvat kinase (CDC19) u genomu P. pastoris je zamijenjen s CDC19E397A te su mutanti uzgajani na etanolu kao izvoru ugljika. U S. cerevisiae, FBP aktivira piruvat kinazu (Cdc19) ulazeći u interakciju s glutamatom (Glu392) te je ovaj regulatorni mehanizam važan za glukoneogeni rast na nefermentabilnim izvorima ugljika jer rezultira inaktivacijom Cdc19 te sprječava uzaludno kruženje metabolita u nedostatku glukoze. Analogni glutamat pronađen u P. pastoris (Glu397) je zamijenjen s alaninom koristeći CRISPR-Cas9 te je određivan rast mutanata na etanolu kao jedinom izvoru ugljika. Rezultati su pokazali jednak rast za mutante u usporedbi s divljim tipom iz čega se može zaljučiti da alosterička regulacija djelovanjem FBP-a na Glu397 nije glavni regulacijski mehanizam piruvat kinaze u P. pastoris te da mora postojati drugi mehanizam regulacije ili je neki drugi aminokiselinski ostatak ključan za alosteričku regulaciju Cdc19. Utjecaj overekspresije CDC19ᴇ₃₉₇ᴀ na prinos VHH antitijela u VHH proizvodnom soju P. pastoris je također ispitan u uvjetima limitacije glukozom. Rezultati su pokazali da overekspresija CDC19ᴇ₃₉₇ᴀ povećava proizvodnju VHH (Variable domain of the Heavy chain of the Heavychain antibody) prinos do 2 puta.
Keywords
Keywords (croatian)
Languageenglish
URN:NBNurn:nbn:hr:159:755118
Study programmeTitle: Bioprocess Engineering
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva (magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva)
Type of resourceText
File originBorn digital
Access conditionsEmbargoed access
Embargo expiration date: 2027-12-21
Terms of useLicence In copyright icon
Created on2024-04-23 12:46:26