Application of Single-Molecule Analytical Methods for the Characterization of Multimeric Genetic Elements in Yarrowia lipolytica DSM 3286

A thorough examination via DNA sequencing of multimeric genetic elements or random integration events in production cell lines or in potential host genomes is a critical regulatory component in the assessment of potential health risks associated with therapeutic proteins and gene therapeutics. Current methods are based on short-read sequencing and PCR amplification steps, which are limited in their information content and are often time- and material-consuming. Consequently, new methods must be developed to address the increasing regulatory demand for such analyses. Given that these methods are predominantly oriented towards the analysis of mammalian genomes and that these genomes limit method development due to their considerable size, it is necessary to identify alternative organisms with complete and well-characterized genome sequences that could serve as model organisms to address genomic questions related to mammals.<br /> The oleaginous yeast Yarrowia lipolytica was identified through a comprehensive review of the existing literature as such a potentially suitable model organism. Additionally, the yeast was confirmed as one of the most promising alternative expression systems to established platforms for recombinant protein production. Its genome is notably large and contains a high number of various rDNA clusters situated in the telomeric regions. However, the current genome assemblies of Y. lipolytica are not complete as they lack rDNA clusters and telomeres due to the presence of long, repetitive elements in the sub-telomeric regions. In this study, a nearly complete genome sequence of the biotechnologically important strain DSM 3286 is presented. A hybrid assembly approach that combines Illumina and nanopore sequencing reads to incorporate all six rDNA clusters and telomeric repeats into the genome was developed and utilized. Through fine-tuning of the DNA isolation and library preparation methods, ultra-long reads that not only included multiple mitochondrial genomes but also provided insights into the diversity of rDNA cluster types, both inter- and intra-chromosomally, were generated. Based on single-molecule analysis that revealed the presence of circular dimers, new insights into mitochondrial genome replication were obtained. In addition, the analysis of ultra-long single sequencing reads uncovered ten distinct rDNA units in this strain, which are arranged in a specific order within a cluster. Furthermore, it was shown that the number of rDNA repeats in individual clusters varies among different cells within the same population.<br /> In parallel, RNA-Seq data was successfully employed to refine the annotations of the genome sequence and to discover novel lncRNAs. The TATA box and Inr consensus sequences of Y. lipolytica DSM 3286 were determined, and analysis of the distance between the TATA box location and the TSS revealed a strong correlation between positional conservation and high gene expression levels or regulation. In addition, the TATA box location was found to be more like that of humans compared to other hemiascomycetous yeasts. Overall, the nearly contiguous genome sequence, in combination with the RNA-Seq data, constitutes a novel high-quality level for genome assemblies, and the insights gained into the core promoter features of Y. lipolytica have the potential to be used to design new synthetic promoters or to adapt homologous transcription regulation systems. <br /> The results of this research show that ultra-long sequencing reads cannot only improve the continuity and completeness of genome assemblies but also provide new perspectives at the single-read level, enabling the detailed analysis of individual cells or chromosomes. With the establishment of a nearly complete genome sequence and the discovery of an intra-population variety of multimeric genetic elements by utilizing single-molecule analysis, the foundation has been laid to employ Yarrowia lipolytica as a model organism for the development of new tools for the assessment of multimeric genetic elements and integration events in therapeutically relevant contexts in future research.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bielefeld

Year

2025

Author dc:creator

• Luttermann, Tobias

Repository record source_url

https://pub.uni-bielefeld.de/record/3005806

OAI identifier oai:identifier

oai:pub.uni-bielefeld.de:3005806