Semi-rational engineering of Adh2 protein for better methanol utilization in yeast Komagataella phaffii

master's thesis

• Title (croatian): Semi-racionalno inženjerstvo proteina Adh2 u svrhu bolje iskoristivosti metanola u kvascu Komagataella phaffii
• Author: Lara Kalogjera
• Mentor: Božidar Šantek (mentor)
• Committee member: Blaženka Kos (predsjednik 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: 2023-03-31, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

The methylotrophic yeast Komagataella phaffii is commonly used as an expression system for recombinant protein production. In a recent study, it was demonstrated that the promiscuous activity of alcohol dehydrogenase 2 (Adh2) is responsible for the majority of methanol oxidation in K. phaffii in the absence of AOX genes. Methanol oxidation by Adh increases the amount of cellular energy (NADH) produced via the dissimilatory pathway. However, in the absence of AOX genes Adh2 is unable to support a growth phenotype on methanol. This thesis aimed to employ semi-rational engineering of the Adh2 protein to produce mutant variants with improved in vivo activity towards methanol. Saturation mutagenesis was performed on target sites (A52, G272, L119, S249, T123, and Y297), and the activity of the mutated ADH2 genes was determined through the in vivo production of eGfp under the FLD1 promoter. The screening results were beneficial for confirming the efficiency of the biosensor strain

Abstract (croatian)

Metilotrofni kvasac Komagataella phaffii učestalo se koristi kao ekspresijski sustav za proizvodnju rekombinantnih proteina. U nedavnom istraživanju, demonstrirano je da je promiskuitetna aktivnost alkohol dehidrogenaze 2 (Adh2) odgovorna za većinu oksidacije metanola u odsutnosti AOX gena. Oksidacija metanola putem alkohol dehidrogenaza povećava količinu stanične energije (NADH) proizvedenu tijekom disimilatornog puta. Unatoč tome, u odsutnosti AOX gena, Adh2 ne može podržati rast kvasca na metanolu. U ovom radu, cilj je bio upotrijebiti semi-racionalno inženjerstvo proteina Adh2 kako bi se proizvele mutirane varijante s poboljšanom in vivo aktivnošću prema metanolu. Usmjerena mutageneza bila je provedena na ciljnim mjestima u genu (A52, G272, L119, S249, T123, and Y297) i aktivnost mutiranih ADH2 gena bila je određena in vivo produkcijom eGFP pod FLD1 promotorom. Rezultati protočne citometrije bili su korisni kako bi se potvrdila efikasnost biosenzora.

• Keywords: Komagataella phaffii; Adh2; semi-rational enzyme engineering; site saturation mutagenesis; flow cytometry
• Keywords (croatian): Komagataella phaffii; Adh2; semi-racionalno inženjerstvo; usmjerena mutageneza; protočna citometrija
• Language: english
• URN:NBN: urn:nbn:hr:159:905585
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2024-05-21 11:23:08