Engineering van Saccharomyces cerevisiae voor de duurzame productie van 3-methyl-1-butanol en 3-methyl-1-butanal

3-Methyl-1-butanol (3MB) and 3-methyl-1-butanal (3MA) are promising platform chemicals for various industries, including flavors, fragrances, solvents and advanced biofuels. These compounds are traditionally derived from petrochemical processes, but increasing demands for sustainable production has created opportunities for their microbial biosynthesis. However, due to interconnected biosynthetic pathways that compete for shared metabolic precursors, conventional metabolic engineering strategies in yeast have typically yielded titers far too low for economic viability. This thesis presents a novel strategy to produce 3MB alongside conventional bioethanol fermentation, leveraging the existing bioethanol industry by valorizing the discarded fusel alcohol byproduct stream. We first identified a robust Saccharomyces cerevisiae chassis strain for bioethanol production from sugarcane molasses, then explored multiple strategies to relieve valine and leucine feedback inhibition and enhance flux toward 3MB. We next tested in silico-predicted gene deletion targets that improved 3MB production in vivo by reducing formation of the byproduct acetate. The final engineered strain achieved a 4.4-fold increase in 3MB yield compared to the wild type (1.5 mg/g sugars), average productivity of 5 mg/L·h, and a 3MB proportion increase from 42% to 71% within the fusel alcohol mix, all while maintaining the same ethanol production as the industrial reference strain Ethanol Red®. For bioethanol industries restricted from using genetically modified (GM) yeasts, we also developed a 3MB-producing bioethanol strain using a non-GM approach that introduced a novel amino acid substitution in Leu4p. This work establishes a new approach for the co-production of 3MB and ethanol in S. cerevisiae, laying the foundation for an economically viable and sustainable strategy to produce 3MB alongside existing bioethanol fermentations. In addition, we investigated 3MA production in S. cerevisiae, as its chemical reactivity makes it an attractive precursor for more energy-efficient bioplasticizer production. We evaluated the deletion of ILV1, a key gene in a competing pathway, which led to increased 3MA accumulation but also caused isoleucine auxotrophy and growth defects. 3MA's toxicity remains a major obstacle that limits further accumulation and hinders the development of high-3MA-producing strains.