Engineering oleaginous yeast Yarrowia lipolytica for production of fatty acid-derived compounds

With a human population estimated to reach 9.7 billion in 2050, activities to fulfil the growing needs for food, energy, and materials will cause increasing environmental damages. Biobased production offers potential solutions by enabling
microorganisms to produce a plethora of compounds. Covering products from alkanes to polyunsaturated fatty acids (PUFAs), production of fatty-acid²derived compounds (FADCs) in microbes could contribute to realizing more sustainable productions. During optimization of FADC production in microbes, metabolic engineering efforts need to address various pathway bottlenecks and cellular limitations. Examples in resolving these challenges are described in the first chapter of this thesis. Yarrowia lipolytica offers the advantage of naturally producing large pool of acetyl-CoA, the main precursor for FADC, compared to the model yeast Saccharomyces cerevisiae. Owing to this, Y. lipolytica is arguably the host-of-choice for production of FADCs. Such potential has motivated the development of well-characterized genetic tools for this yeast, which further encourages the use of Y. lipolytica as bioproduction host. Taking advantage of the increasingly available genetic tools, in this thesis Y. lipolytica was engineered to produce lactones, which are used for giving fruity and milky notes in foods and beverages. Conventional production methods of lactones require hydroxylated fatty acids as starting material. Mainly sourced from plants, series of extraction processes add costs to the production aside from time and land requirements. Through metabolic engineering, production of flavor lactones from more available, non-hydroxylated fatty acids oleic- and linoleic acid was realized and optimized.
PUFAs are arguably the most valuable FADC to date. Dietary requirement of these essential fatty acids is mainly supplied from seed oils and marine fish, both entails environmental concerns on land and in the ocean. PUFAs production in Y.
lipolytica had been reported before. To complement the existing engineering strategies, the last work in this thesis borrowed from acyl-editing mechanism in plants. The work demonstrated improvement of linoleic acid content in the cell upon introducing an acyl-editing enzyme. Since linoleic acid is the precursor of all PUFAs, this new strategy could be employed for production of other PUFAs. Overall, this thesis provides the research community insights to overcome strain engineering challenges in pathways connected to fatty-acid metabolism. The presented research offers a new application of biobased production and a novel approach for metabolic engineering which could inspire future efforts in optimizing
FADC production in Y. lipolytica and other microbes.