doctoral dissertation
Tadej Markuš (Author), Uroš Petrovič (Mentor)

Abstract

Our research focused on developing and engineering a terpenoid-producing Y. lipolytica strain to evaluate the impact of central carbon modifications on the production of cytosolic acetyl-CoA. First, our aim was to establish carnosic acid production in Y. lipolytica; however, our efforts were unsuccessful, and gene expression analysis revealed we were unable to successfully express the ATR1 gene, which was needed for the correct functioning of the cytochrome P450 monooxygenases. On the other hand, we successfully prepared a set of beta-carotene-producing parental strains, each with a different capacity for its production, which we used down the line to evaluate central carbon metabolism modifications. We evaluated two gene combinations for the pyruvate bypass, one for the phosphoketolase bypass, and three for the carnitine shuttle. Upon introducing all bypasses in the beta-carotene-producing parental strains, we discovered that most bypasses negatively impacted beta-carotene production. On the other hand, when we examined lipid production, most of the modifications that negatively impacted beta-carotene production positively impacted lipid production. The highest lipid content we measured was 0.61 ± 0.04 glipids/gDCW, demonstrated by the strains carrying the YlCAT2 gene. At the same time, we observed the highest lipid efficiency improvement for the strains carrying the YlPDC1, YlALD3, and YlACS1 genes of the pyruvate bypass. The inverse correlation between the biosynthesis of lipids and beta-carotene production has not been observed before and hints at possible regulation response at higher acetyl-CoA availability.

Keywords

Yarrowia lipolytica;beta-carotene;lipids;carnosic acid;metabolic bypass;pyruvate bypass;phosphoketolase;phosphoketolase bypass;carnitine;carnitine shuttle;

Data

Language: English
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL BF - Biotechnical Faculty
Publisher: [T. Markuš]
UDC: 602.3:582.282.23:577.182.72:601.4:577.21(043.3)
COBISS: 224641027 Link will open in a new window
Views: 35
Downloads: 4
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Other data

Secondary language: Slovenian
Secondary title: Vpliv izražanja genov piruvatnega in fosfoketolaznega obvoda na proizvodnjo terpenoidov v kvasovki Yarrowia lipolytica
Secondary abstract: Naša raziskava se je osredotočala na razvoj in inženiring sevov kvasovke Y. lipolytica. Za cilj smo si zadali ovrednotenje vpliva modifikacij centralnega metabolizma, ki vodijo do sinteze citosolnega acetil-CoA, na kvasovko Y. lipolytica, ki bo zmožna proizvodnje izbranega terpenoida. Najprej smo se želeli v kvasovki Y. lipolytica vzpostaviti proizvodnjo karnozolne kisline, vendar smo bili pri temu neuspešni. Analize genskega izražanja so pokazale, da nam ni uspelo uspešno izraziti gena ATR1, ki je potreben za pravilno funkcioniranje citokrom P450 monooksigenaz, ki sodelujejo v biosintezni poti karnozolne kisline. Po drugi strani smo uspešno pripravili set starševskih sevov z različnimi kapacitetami za proizvodnjo beta-karotena, ki smo jih nato uporabili v drugem delu naše naloge za poglobljeno ovrednotenje metabolnih obvodov. Za drugi del naše naloge smo pripravili dve genski kaseti z geni povezanimi s piruvatnim obvodom, eno z geni fosfoketolaznega obvoda in tri z geni za prenos acetil-CoA s karnitinom. Vse genske kasete smo integrirali v starševske seve in jih podvrgli seriji gojitvenih eksperimentov. Odkrili smo, da je večina metabolnih obvodov negativno vplivala na sposobnost proizvodnje beta-karotena. Ko pa smo pogledali proizvodnjo lipidov, smo odkrili pozitiven trend – večina modifikacij, ki je negativno vplivala na produkcijo beta-karotena, je pozitivno vplivala na sposobnost transgenih kvasovk na kopičenje lipidov. Najvišjo vsebnost lipidov, 0.61 ± 0.04 glipidi/gDCW smo izmerili pri sevih, ki so vsebovali prekomerno izražen gen karnitinskega obvoda YlCAT2. Najvišjo zvišanje učinkovitosti pri proizvodnji lipidov pa smo izmerili pri sevu, ki je vseboval prekomerno izražene gene piruvatnega obvoda (YlPDC1, YlALD3 in YlACS1). Obraten povezava med vplivom na proizvodnjo lipidov in beta-karotena še ni bila pokazana in namiguje na regulacijo aktivnosti biosinteze lipidov in mevalonatne poti ob povišani prisotnosti acetil-CoA.
Secondary keywords: Yarrowia lipolytica;beta-karoten;lipidi;karnozolna kislina;metabolni obvod;piruvatni obvod;fosfoketolaza;fosfoketolazni obvod;karnitin;karnitinski obvod;
Type (COBISS): Doctoral dissertation
Study programme: 0
Thesis comment: Univ. v Ljubljani, Biotehniška fak.
Pages: 1 spletni vir (1 datoteka PDF (XII, 92 str., [5] str. pril.))
ID: 25814599