Genetičke modifikacije nekonvencionalnih kvasaca u svrhu proizvodnje 2,3-butandiola i L-laktata

doctoral thesis

• Title (english): Genetic modifications of non-conventional yeasts for production of 2,3-butanediol and L-lactate
• Author: Angela Matanović
• Mentor: Ivan Krešimir Svetec (mentor)
• Committee member: Igor Slivac (predsjednik povjerenstva)
• Committee member: Božidar Šantek (član povjerenstva)
• Committee member: Martina Sudar (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biology and Microbial Genetics); Zagreb
• Defense date and country: 2024, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology
• Universal decimal classification (UDC): 575 - General genetics. General cytogenetics

Abstract

Lignocelulozne sirovine pogodne su za uspostavu održivih bioprocesa budući da su obnovljive i široko dostupne. Kao potencijalni radni mikroorganizmi za održivu proizvodnju biokemikalija ističu se nekonvencionalni kvasci Scheffersomyces stipitis i Kluyveromyces marxianus prvenstveno zahvaljujući sposobnosti fermentacije ksiloze koja čini velik udio suhe tvari lignoceluloznih sirovina. Stoga je cilj ovog rada bio istražiti može li se ciljanim genetičkim modifikacijama ovih kvasaca, koji prirodno ne proizvode L-laktat i 2,3-butandiol, konstruirati producente navedenih biokemikalija. U svrhu proizvodnje L-laktata konstruirani su sojevi kvasaca S. stipitis i K. marxianus s eksprimiranim genima ldhL1 i ldhL2 koji u bakteriji Lactobacillus gasseri kodiraju za L-laktat dehidrogenaze. Za proizvodnju 2,3-butandiola u kvascima su eksprimirani geni alsS i alsD koji u bakteriji Bacillus subtilis kodiraju za α-acetolaktat sintazu i α-acetolaktat dekarboksilazu. Kako bi se povećao prinos L-laktata i 2,3-butandiola, u konstruirane sojeve kvasca K. marxianus unesene su i dodatne modifikacije usmjerene na inhibiciju kompetitivnih metaboličkih puteva. Nizom eksperimenata utvrđeno je da su producenti L-laktata i 2,3-butandiola kvasaca K. marxianus i S. stipitis uspješno konstruirani. Od konstruiranih sojeva, najbolji producent L-laktata je soj kvasca K. marxianus s eksprimiranim genima ldhL1 i ldhL2 te inaktiviranim genom PDC1 koji fermentacijom glukoze proizvodi L-laktat s efikasnošću od 87 %, dok je najbolji producent 2,3- butandiola soj kvasca K. marxianus koji eksprimira gene alsS i alsD te iz glukoze proizvodi 2,3- butandiol s efikasnošću od 62 %.

Abstract (english)

Lignocellulosic raw materials are suitable for establishing sustainable bioprocesses as they are renewable and widely available. Non-conventional yeasts such as Scheffersomyces stipitis and Kluyveromyces marxianus stand out as potential working microorganisms for sustainable biochemical production, primarily due to their ability to ferment xylose, which constitutes a large proportion of the dry matter of lignocellulosic raw materials. Therefore, the aim of this study was to investigate whether it is possible to construct producers of these biochemicals by targeted genetic modifications of these yeasts, which do not naturally produce L-lactate and 2,3-butanediol. For the purpose of producing L- lactate, yeast strains S. stipitis and K. marxianus were constructed with the expressed genes ldhL1 and ldhL2, which code for L-lactate dehydrogenase in the bacterium Lactobacillus gasseri. For the production of 2,3-butanediol, the genes alsS and alsD, which code for α-acetolactate synthase and α- acetolactate decarboxylase in the bacterium Bacillus subtilis, were expressed in yeast. In order to increase the yield of L-lactate and 2,3-butanediol, additional modifications aimed at inhibiting competitive metabolic pathways were introduced into the constructed yeast strains K. marxianus. Through a series of experiments, it was established that the producers of L-lactate and 2,3-butanediol yeasts K. marxianus and S. stipitis were successfully constructed. Of the engineered strains, the best producer of L-lactate is the yeast strain K. marxianus with the expressed ldhL1 and ldhL2 genes and the inactivated PDC1 gene, which produces L-lactate with an efficiency of 87 % through glucose fermentation, while the best producer of 2,3-butanediol is the yeast strain K. marxianus that expresses the alsS and alsD genes and produces 2,3-butanediol from glucose with an efficiency of 62 %.

• Keywords: nekonvencionalni kvasci; Scheffersomyces stipitis; Kluyveromyces marxianus; genetičke modifikacije; L-laktat; 2; 3-butandiol
• Keywords (english): non-conventional yeasts; Scheffersomyces stipitis; Kluyveromyces marxianus; genetic modifications; L-lactate; 2; 3-butanediol
• Language: croatian
• URN:NBN: urn:nbn:hr:159:079520
• Promotion: 2024
• Study programme: Title: Postgraduate PhD Study in Nutrition; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje biotehničkih znanosti, polje nutricionizam (doktor/doktorica znanosti, područje biotehničkih znanosti, polje nutricionizam)
• Type of resource: Text
• Extent: 157 str. 30 cm.
• File origin: Born digital
• Access conditions: Closed access
• Created on: 2024-04-25 08:39:10