Vzpostavitev metode obnovitve fluorescence BODIPY za določanje količine lipidov v oleogeni kvasovki Yarrowia lipolytica in karakterizacija sevov s spremenjenim metabolizmom lipidov

Logonder, Tina (avtor), Gaber, Aljaž (mentor)

Nekonvencionalna oleogena kvasovka Yarrowia lipolytica je zaradi svoje nezahtevnosti gojenja, hitre rasti, robustnosti in sposobnosti kopičenja lipidov pomemben mikroorganizem v industrijski biotehnologiji. Razvoj sevov s spremenjenim kopičenjem lipidov bi lahko povečala izkoristek proizvodnje hidrofobnih spojin. Zato smo vzpostavili metodo obnovitve fluorescence BODIPY (OFB) za visokozmogljivostno analizo kopičenja lipidov, ki temelji na obarvanju znotrajceličnih lipidnih kapljic s fluorescenčnim barvilom BODIPY. Razlikovanje med lipidnimi kapljicami in preostankom citosola nam omogoči kalijev jodid, ki duši fluorescenco BODIPY. BODIPY zato fluorescira v lipidnih kapljicah, drugje v celici pa ne. Jakost signala je sorazmerna s količino lipidov, nakopičenih v lipidnih kapljicah. Rezultati metode OFB, s katero smo analizirali pripravljene seve z izbrisanimi tarčnimi geni, ki so vključeni v metabolizem in kopičenje lipidov, so bili primerljivi z velikostmi lipidnih kapljic, določenih iz mikroskopskih slik. Potrdili smo, da izbris gena TGL4 poveča in izbris genov DGA1 in DGA2 pa zmanjša kopičenje lipidov v celicah kvasovke Yarrowia lipolytica W29. Gen TGL4 namreč zapisuje triacilglicerol lipazo. Ta encim razgrajuje triacilgliceride (TAG), ki so glavni založni lipidi, kopičeni v lipidnih kapljicah. Obratno vlogo imata encima fosfolipid:diacilglicerol aciltransferaza (PDAT) in diacilglicerol aciltransferaza (DGAT), ki sta ključna za sintezo TAG. Prvega zapisuje gen DGA1, drugega pa gen DGA2. Testirali smo tudi vpliv izbrisa genov POX5 in POX6 na kopičenje lipidov. Geni POX1-6 zapisujejo izoencime acil-CoA oksidaza 1-6 (Aox), ki katalizirajo prvi in hitrostno omejujoč korak β-oksidacije. Pri izbrisu le enega od POX genov razlika v kopičenju lipidov v primerjavi z izhodiščnim sevom ni bila signifikantna. Z metodo OFB je bilo torej mogoče oceniti količino znotrajceličnih lipidov in v prihodnje bi metodo lahko uporabili v procesu razvoja novih gensko spremenjenih sevov kvasovke Yarrowia lipolytica, ki bi lahko bistveno povečali učinkovitost proizvodnje hidrofobnih spojin v industrijski biotehnologiji.

• Jezik: Slovenski jezik
• Ključne besede: Yarrowia lipolytica, uravnavanje kopičenja lipidov, določanje vsebnosti lipidov, BODIPY, lipidne kapljice
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-147371
• COBISS.SI-ID: 160214275
• Datum objave v RUL: 03.07.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Establishment of a BODIPY fluorescence recovery assay for lipid determination in the oleaginous yeast Yarrowia lipolytica and characterization of strains with altered lipid metabolism

Izvleček

The nonconventional oleogenous yeast Yarrowia lipolytica is a significant microorganism in industrial biotechnology due to its undemanding cultivation, rapid growth, robustness, and ability to accumulate lipids. The development of strains with altered lipid accumulation could increase the yield of hydrophobic compounds production. Therefore, we established a BODIPY fluorescence recovery (OFB) method for high-throughput lipid accumulation assay based on intracellular lipid droplets staining with the fluorescent dye BODIPY. Potassium iodide, which quenches the fluorescence of BODIPY, allowed us to distinguish between lipid droplets and the rest of the cytosol. The signal intensity is proportional to the amount of lipids accumulated in the lipid droplets The results of the OFB method, which were used to analyze prepared strains with deleted target genes involved in lipid metabolism and accumulation, were comparable to the sizes of lipid droplets determined from microscopic images. We confirmed that deletion of the TGL4 gene increases and deletion of the DGA1 and DGA2 genes decreases lipid accumulation in Yarrowia lipolytica W29. Triacylglycerol lipase, an enzyme that breaks down triacylglycerides (TAG), the primary storage lipids accumulated in lipid droplets, is encoded by the TGL4 gene. The genes DGA1 and DGA2, respectively, encode the enzymes phospholipid:diacylglycerol acyltransferase (PDAT) and diacylglycerol acyltransferase (DGAT), which are essential for TAG synthesis. We also investigated the impact of deleting the POX5 and POX6 genes on lipid accumulation. The POX1-6 genes encode acyl-CoA oxidase 1-6 (Aox) isoenzymes, which catalyze the first and rate-limiting step of beta-oxidation. Only one POX gene deletion did not significantly impact the lipid accumulation in comparison to the wild-type strain. As a result, it was possible to estimate the intracellular lipid content using the OFB method, which could be applied to develop new genetically modified Yarrowia lipolytica yeast strains. In the future, that could substantially improve the productivity of hydrophobic compounds production in the industrial biotechnology.

• Ključne besede: Yarrowia lipolytica, regulating lipid accumulation, determining lipid content, BODIPY, lipid droplets