Engineering oleaginous yeasts for a bio-based future

Samenvatting

The use of plant-derived oils, especially palm oil, is increasing at an alarming rate, partly because of the demand as a fossil fuel replacement and being a cheap source of useful components. As a result, palm tree groves are replacing the tropical forests, causing deforestation, and contributing to climate change. Therefore, developing a sustainable alternative to palm oil is of utmost interest. Microbial oil-producing yeasts, oleaginous yeasts, are flagged as attractive microbial-cell factories to sustain a bio-based circular economy for industrial implementation. Compared to plant-based oil sources, these yeasts offer several advantages, including independence from arable lands, less affection by venue, season, and climate, and a shorter life cycle. Among the oleaginous yeasts oil from Cutaneotrichosporon oleaginosus and Yarrowia lipolytica have received interest due to having comparable fatty acid composition to that of palm. However, further optimization of yeast lipid production processes is needed to make them economically viable for industrial use. This thesis tackles the challenges of enhancing lipid yields of C. oleaginosus and Y. lipolytica by simultaneously considering biocatalyst, bioprocess, and the interplay between genetic, bioprocess, and environmental factors. In that regard, a streamlined methodology, the Design-Build-Test-Learn approach, was deployed intertwining the process optimization, metabolic modeling, data-driven modeling, and genetic engineering for both oleaginous yeasts. Chapter 2 assesses the lipid accumulation potential of C. oleaginosus and Y. lipolytica under varied C/N ratios of glycerol-containing media and different incubation temperatures. The optimal conditions for lipid production were identified via Response Surface Methodology which guided the work in following chapters. Chapter 3 introduces Comparative Flux Sampling Analysis (CFSA), a tool guiding metabolic engineering strategy by simulating Genome-Scale Metabolic Models (GEMs) under growth and production scenarios. CFSA guided the selection of genetic interventions to enhance lipid production of C. oleaginosus and Y. lipolytica (Chapters 5 and 6). Chapter 4 presents updated genome annotations for C. oleaginosus to improve the accessibility for strain optimization. In Chapter 5, a model-driven engineering approach was employed for C. oleaginosus. Genes and medium supplements were identified for improving lipid production followed by building transformants and experimental validations. The genetic toolbox for C. oleaginosus was expanded by introducing an electroporation-based transformation method, promoters, and terminators. Chapter 7 demonstrates a combinatorial approach to further improve C. oleaginosus lipid production by performing a full factorial design that considers genetic factors and the C/N ratios. Afterward, the best transformant and wild-type were cultivated in 2L bioreactors utilizing a two-stage fermentation process. In Chapter 6, the predictions of CFSA for Y. lipolytica were complemented with previous knowledge obtained for oleaginous yeasts. Selected overexpression targets from lipid synthesis and amino acid synthesis pathway, and knock-out targets from competing pathways revealed their synergistic effect on the lipid accumulation metabolism of Y. lipolytica. In Chapter 8, the benefits of integrating modeling and experimental approach to improve microbial production processes was highlighted. Differences in lipid synthesis mechanisms and fatty acid composition between C. oleaginosus and Y. lipolytica were discussed. The contribution of this work to the development of cost-effective microbial oil production processes was emphasized.