(614b) Cell Factories from Metabolic Engineering of the Extremophile Oleaginous Yeast Debaryomyces Hansenii CBS767

Here, we report the metabolic engineering of the CTG clade yeast D. hansenii CBS 767 after domestication with systems and synthetic biology. D. hansenii has attractive catabolic, anabolic, and tolerance phenotypes. It is osmotolerant and halotolerant, which confers resistance to harsh fermentation conditions and enables growth in saltwater. It naturally grows on the major monosaccharides of lignocellulosic biomass, specifically glucose, xylose, and arabinose. It produces the nutraceutical riboflavin and overproduces lipids. To domesticate D. hansenii, we sequenced the genome with our Prymetime pipeline, performed transcriptomics under stress conditions to understand pathway regulation, determined antibiotic selections, optimized transformation procedures, and developed a modular cloning standard for CTG clade yeasts. This enabled us to test 20 promoter and 20 terminator sequences using automated liquid handling and flow cytometry. The promoters have a range of three orders of magnitude of expression strength, while the terminators alter expression over a range of one order of magnitude. This systems and synthetic biology foundation enables us to perform combinatorial metabolic pathway engineering of the D. hansenii fatty acid pathway. We targeted both acetyl-CoA overproduction and pathways for the production of fatty-acid derived molecules hexadecanol and medium chain alkanes. We report production of these molecules on the major constituents of lignocellulosic biomass – glucose, xylose, and arabinose. Thus, these strains have potential as cell factories for commodity chemicals and drop-in biofuels from lignocellulose. In summary, this work highlights the potential for systems and synthetic biology to uncover novel biology and enable efficient domestication of nonconventional yeasts for the creation of cell factories.