Investigation of pyruvate kinase regulation in Pichia pastoris and effects of its overexpression on antibody production

master's thesis

• Title (croatian): Istraživanje regulacije piruvat kinaze u kvascu Pichia pastoris i utjecaja njene overekspresije na proizvodnju antitijela
• Author: Gabrijela Jurić
• Mentor: Mario Novak (mentor)
• Committee member: Bojan Žunar (predsjednik povjerenstva)
• Committee member: Antonija Trontel (član povjerenstva)
• Committee member: Andreja Leboš-Pavunc (član povjerenstva)
• Granter: University 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 country: 2022-12-20, 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: pyruvate kinase; Komagataella phaffii; Pichia pastoris; allosteric regulation; VHH antibody; recombinant protein
• Keywords (croatian): piruvat kinaza; Komagataella phaffii; Pichia pastoris; alosterička regulacija; VHH antitijelo; rekombinantni protein
• Language: english
• URN:NBN: urn:nbn:hr:159:755118
• Study programme: Title: 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 resource: Text
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2027-12-21
• Created on: 2024-04-23 12:46:26