Towards Bioproduction of Yeast-Based Odd-Chain Fatty Acids

Abstract

Odd-chain fatty acids (OCFAs) are high-value lipids with emerging medical and nutritional applications. However, their natural abundance is low, and conventional extraction or chemical synthesis methods are inefficient or unsustainable. Microbial fermentation using oleaginous yeasts offers a promising alternative, particularly when employing short-chain fatty acids digestates rich in propionic acid, the main precursor for OCFA synthesis.In this thesis work yeast-based OCFA production has been explored by improving quantitative analytical methods, identifying suitable yeast cell factories, characterizing their performance, and initiating strain engineering efforts. First, a fatty acid methyl ester protocol was optimized for robust OCFA analysis. Second, eight strains capable of growth on 15 g/L propionic acid and production of OCFA were identified in a screening of nineteen oleaginous yeasts. Third, four yeast species were selected for further study: Cutaneotrichosporon oleaginosus for its high OCFA titers, Rhodotorula toruloides for its high OCFA yield, Yarrowia lipolytica for its high tolerance to propionic acid, and Blastobotrys adeninivorans for its ability to produce OCFAs, grow well in propionic acid and as a relatively underexplored yeast. Cultivation on mixtures of acetic and propionic acids at different carbon-to-nitrogen ratios revealed distinct and interesting performance characteristics for OCFA production in each yeast species. Fourth, a plasmid-based CRISPR-Cas9 metabolic engineering platform was established for B. adeninivorans through the identification of native autonomous replication systems, enabling the generation of mutants with enhanced OCFA content.Overall, this work advances methodologies for fatty acid analysis, identifies promising oleaginous yeasts for OCFA production and characterize them, and establishes a new genetic engineering tool for strain development. These results provide a foundation for the development of efficient yeast cell factories and economically viable bioprocesses for sustainable OCFA production.