Development of \u3cem\u3eCutaneotrichosporon oleaginosus\u3c/em\u3e to Convert Lignin-Derived Phenolics to Oleochemicals

Oleaginous yeasts have long been a target for developing industrial-scale biorefineries due to their ability to accumulate high amounts of lipids, synthesize complex chemicals and proteins, and robustly metabolize diverse feedstocks. In parallel, interest in lignocellulosic biomass as a feedstock has grown. While most processes focus on the carbohydrates from cellulose and hemicellulose, the most energy-dense biopolymer, lignin, remains underutilized. This dissertation describes foundational work describing lignin conversion by Cutaneotrichosporon oleaginosus, a non-model oleaginous yeast known for its metabolism of alternative sugars, including xylose, and tolerance and metabolism toxic lignocellulosic hydrolysate inhibitors such as 5-HMF, furfural, acetic acid. This dissertation is the first to describe robust lipid production by this yeast while utilizing five aromatic substrates as the sole carbon source: phenol, resorcinol, p-hydroxybenzoic acid, p -coumaric acid, and ferulic acid. This yeast can also tolerate an alkaline pretreated lignin hydrolysate and remain oleaginous. The genetic basis of yeast aromatic metabolism is poorly characterized, so a multi-omic approach was applied to improve the existing genome annotation and identify novel gene functions relevant to aromatic catabolism. Genes unique to and common across all six substrates mentioned build a roadmap for future engineering for robust lignin valorization. To this, a small, functional genetic toolkit was developed to improve the genetic accessibility of this non-model yeast. Together, this dissertation demonstrates that C. oleaginosus is poised to become a preferred host for lignocellulosic biomass to oleochemical conversion

(How) is formulaic language universal? Insights from Korean, German and English

The existence of common expressions, also referred to as formulaic language or phraseological units, has been evidenced in a very large number of languages. However, the extent to which languages feature such formulaic material, how formulaicity may be understood across typologically different languages and whether indeed there is a concept of formulaic language that applies across languages, are questions that have been less commonly discussed. Using a novel data set consisting of topically matched corpora in three typologically different languages (Korean, German and English), this study proposes an empirically founded universal concept for formulaic language and discusses what the shape of this concept suggests for the theoretical understanding of formulaic language going forward. In particular, it is argued that the nexus of the concept of formulaic language cannot be fixed at any particular structural level (such as the phrase or the level of polylexicality) and incorporates elements specified at varying levels of abstraction (or schematicity). This means that a cross-linguistic concept of formulaic language fits in well with a constructionist view of linguistic structure

Additional file 5 of Autonomous replication sequences from the Amaranthus palmeri eccDNA replicon enable replication in yeast

Additional file 5: Figure S3. Cloning of the eccDNA autonomous replication sequence in a yeast system. A. Water control (no colonies) B. pRS305 non-replicating plasmid (LEU marker replication − no colonies). C. pRS315 replicating plasmid (LEU marker, CEN6/ARS -lawn of colonies). D. pRS305 + CS-ARS1 + CEN6 (pRS305 + CEN6 + ARS1 − few colonies). E. pRS315 + CS-ARS1 + CEN6 (pRS315ΔARS + ARS1 − few colonies)

Additional file 3 of Autonomous replication sequences from the Amaranthus palmeri eccDNA replicon enable replication in yeast

Additional file 3: Table S1. DNA curvature results of a 256 bp window of sequence extracted from the eccDNA replicon that harbors 2 DNA unwinding elements (DUE) and the extended autonomous consensus sequence (EACS) with homology to yeast

Additional file 2 of Autonomous replication sequences from the Amaranthus palmeri eccDNA replicon enable replication in yeast

Additional file 2: Figure S1. EACs sequence region of the eccDNA replicon. A zoomed in view of the extended autonomous consensus sequence (EACS) highlighted in red. Upstream, highlighted in orange, are the 2 predicted DNA unwinding elements (DUE), with the only conserved sequence within black bars (AATAAA)

Additional file 1 of Autonomous replication sequences from the Amaranthus palmeri eccDNA replicon enable replication in yeast

Additional file 1: Table S2. Yeast plasmids, strains, and primer sequences used to clone and validate the eccDNA ARS sequence

Autonomous replication sequences from the Amaranthus palmeri eccDNA replicon enable replication in yeast

Abstract Objective The objective of the research presented here was to determine whether autonomous replication sequences (ARS) discovered in the eccDNA replicon of glyphosate resistant Amaranthus palmeri enable self-replication in a yeast system. Results Sequence analysis of the eccDNA replicon revealed a region of sharp changes in A + T/G + C content with characteristic bending indicative of an autonomous replication sequence. Further sequence analysis revealed an extended autonomous replication sequence (EACS) in close proximity to multiple DNA unwinding element (DUE) sequences. This region of the eccDNA replicon enabled autonomous replication of an ARS-less yeast plasmid

Additional file 2 of Autonomous replication sequences from the Amaranthus palmeri eccDNA replicon enable replication in yeast

Additional file 2: Figure S1. EACs sequence region of the eccDNA replicon. A zoomed in view of the extended autonomous consensus sequence (EACS) highlighted in red. Upstream, highlighted in orange, are the 2 predicted DNA unwinding elements (DUE), with the only conserved sequence within black bars (AATAAA)

Additional file 4 of Autonomous replication sequences from the Amaranthus palmeri eccDNA replicon enable replication in yeast

Additional file 4: Figure S2. NAC multiple sequence alignment. Multiple sequence alignment of the eccDNA replicon NAC gene that contains the EACS sequence. The EACS region is highlighted in red, and the DNA unwinding elements are highlighted in orange

Additional file 6 of Autonomous replication sequences from the Amaranthus palmeri eccDNA replicon enable replication in yeast

Additional file 6: Figure S4. Summary of the p-values resulting from two-tailed t-tests performed between samples using a 95% confidence level (α = 0.05)