Optimization of zeaxanthin production in oleaginous yeast Yarrowia lipolyticaSoldat, Mladen (Avtor) Petrovič, Uroš (Mentor) Yarrowia lipolyticacarotenoid biosynthesiszeaxanthinβ-cryptoxanthinenzyme localizationmultienzyme complexesprotein structure-function analysisIn 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.20252025-10-04 07:15:08Doktorsko delo/naloga174542VisID: 267398COBISS_ID: 251888131sl