Optimization of zeaxanthin production in oleaginous yeast Yarrowia lipolytica

Soldat, Mladen (author), Petrovič, Uroš (mentor)

In this study, the oleaginous yeast Yarrowia lipolytica was utilized as a platform for the development of new approaches to optimize carotenoid biosynthesis, focusing primarily on zeaxanthin and β-cryptoxanthin. We based our work on the wild-type strain YB-392, thereby expanding existing knowledge that predominantly focuses on strains from a different lineage, namely Po1. Through genetic engineering, we introduced heterologous pathways for carotenoid biosynthesis, optimized the metabolic flux through the mevalonate pathway, and improved the expression of key enzymes by varying promoter strength and gene copy number. Using bioinformatic tools, we identified 5,590 homologs of the β-carotene hydroxylase enzyme PaCrtZ, selected 21 representatives, and functionally evaluated them in Y. lipolytica. Six homologs demonstrated the ability to synthesize zeaxanthin, and two produced β-cryptoxanthin exclusively. Particular focus was placed on the analysis of the binding and active sites of β-carotene hydroxylase PaCrtZ. Structural analysis of the PaCrtZ active site enabled the identification of three critical catalytic residues (E18, D52, D95) essential for enzymatic activity, and residue E49, involved in substrate positioning and orientation. Biosynthetic efficiency was further enhanced by targeting the PaCrtZ enzyme to the endoplasmic reticulum and peroxisomes, increasing β-carotene accessibility for hydroxylation. Additionally, multienzyme complexes were established through fusion of the enzyme with RIDD and RIAD peptides, leading to improved enzyme co localization and enhanced zeaxanthin production. Our results confirm that rational engineering of metabolic pathways in Y. lipolytica, including enzyme selection, optimization of localization, and/or spatial organization, significantly contributes to increasing the productivity of microbial bioprocesses. This study represents an advancement in the development of sustainable strategies for the biotechnological production of high-value isoprenoids.

• Language: English
• Keywords: Yarrowia lipolytica, carotenoid biosynthesis, zeaxanthin, β-cryptoxanthin, enzyme localization, multienzyme complexes, protein structure-function analysis
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Year: 2025
• PID: 20.500.12556/RUL-174542
• COBISS.SI-ID: 251888131
• Publication date in RUL: 04.10.2025

Secondary language

• Language: Slovenian
• Title: Optimizacija proizvodnje zeaksantina v oljni kvasovki Yarrowia lipolytica

Abstract

V tej raziskavi smo oljno kvasovko Yarrowia lipolytica uporabili kot platformo za razvoj novih pristopov optimizacije biosinteze karotenoidov, predvsem zeaksantina in β-kriptoksantina. Delo je temeljilo na izhodiščnem sevu YB-392, s čimer smo razširili obstoječe znanje, ki je večinoma pridobljeno na sevih iz druge linije, Po1. Z genskim inženiringom smo vstavili heterologne poti za biosintezo karotenoidov, optimizirali pretok metabolitov skozi mevalonatno pot ter izboljšali izražanje ključnih encimov z uporabo različnih promotorjev in števil kopij genov. Poseben poudarek smo namenili analizi β-karoten hidroksilaz. Z bioinformatskimi orodji smo identificirali 5590 homologov encima PaCrtZ, izbrali 21 predstavnikov in ovrednotili njihovo aktivnost v Y. lipolytica. Šest homologov je izkazalo sposobnost sinteze zeaksantina, dva pa ekskluzivno β-kriptoksantina. Strukturna analiza aktivnega mesta PaCrtZ nam je omogočila določitev ključnih katalitičnih ostankov (E18, D52, D95), ki so se izkazali kot bistveni za encimsko aktivnost, ter ostanka E49, ki sodeluje pri orientaciji substrata. Učinkovitost biosinteze smo nadalje izboljšali s ciljnim usmerjanjem encima PaCrtZ v endoplazemski retikulum in peroksisome, kar je povečalo dostopnost β-karotena za hidroksilacijo. Poleg tega smo vzpostavili multiencimske komplekse preko fuzije encima s peptidi RIDD in RIAD, kar je privedlo do kolokalizacije encimov in povečane proizvodnje zeaksantina. Naši rezultati potrjujejo, da racionalno oblikovanje metabolnih poti v Y. lipolytica, vključno z izborom encimov ter optimizacijo njihove lokalizacije in/ali prostorske organizacije, pomembno prispeva k povečanju produktivnosti mikrobnih bioprocesov. Raziskava predstavlja pomemben napredek pri razvoju trajnostnih strategij za biotehnološko proizvodnjo izoprenoidov z visoko vrednostjo.

• Keywords: Yarrowia lipolytica, biosinteza karotenoidov, zeaksantin, β-kriptoksantin, lokalizacija encimov, večenzimski kompleksi, analiza strukture in funkcije proteinov