Every century brings its own challenges, but the 21st century is the first in which a global transition towards circularity is required to ensure human existence on this planet. Exhaustion of planetary resources, such as oil and rare elements, must be prevented and sustainable circular value chains introduced into our industry and economy. In addition to new challenges, every century also brings new and unique solutions. Today, biotechnology may provide some of the most relevant solutions by providing scientists with the ability to decipher the code of life represented by an organism’s DNA as well as with the tools to edit this code. Especially fast-reproducing microorganisms have a great potential to serve as cell factories, which can convert renewable raw materials into chemicals, materials and food ingredients and thereby support a circular bio-based economy. Recently developed biotechnological tools enable us to rewrite (‘edit’) the blueprint for these microbial cell factories with unprecedented precisions and at unprecedented rates. A myriad of life forms evolved over billions of years to adapt to an incredibly diverse number of habitats on our planet, which led to an immense diversity in survival strategies and metabolic capabilities. Recombining these naturally occurring DNA codes and ‘novel-to-nature’ DNA sequences generated in laboratories offers unique possibilities for development of novel cell factories to address challenges in our century and beyond. Baker’s yeast, Saccharomyces cerevisiae is one of the most intensively studied microorganisms and, as a cell factory, has a long history of successful application in industrial applications. Its story of success began thousands of years ago when processes for production of wine, beer and bread-making were first invented and, over many centuries, improved. Application of yeasts probably started as serendipitous discovery rather than as an invention, when yeast cells from the environment ‘contaminated’ sugar-containing food products and, by accident, turned sugars into ethanol and carbon dioxide, thus yielding the first alcoholic beverages and rising dough. All essential nutrients that yeast require for growth and fermentation were either present in the food product or generated by other microorganisms that inadvertently entered these early fermentation processes. Such a co-existence of multiple microbial species is a natural phenomenon that helps organisms thrive, but in man-made industrial settings such undefined mixed populations are often difficult to control and optimize. When researchers discovered that pure cultures of individual yeast strains were very efficient in producing transport fuels and other interesting chemicals, they therefore developed growth media that contained all essential and non-essential nutrients required for optimal yeast growth, to make these yeast cell factories as productive as possible. For over a century now, yeast cell factories have been under continual development. Classical strain improvement strategies to obtain high-producing strains, later combined with recombinant-DNA technology (genetic engineering) brought microbial production systems to a next level and helped pave the way towards a sustainable bio-based industry. However, while studying and developing product pathways for yeast strains employed in these processes, the specific requirements of these hosts regarding essential nutrients (vitamins) did not always receive attention. Use of generic media, containing excess amounts of vitamins to ensure high productivity, increase overall production costs, complicate down-stream processing and increase contamination risks. The research described in this thesis explores genetic engineering strategies in which heterologous DNA sequences are introduced to improve vitamin synthesis under industrially relevant conditions, with the goal to enable development of fully vitamin-independent (prototrophic) S. cerevisiae strains. The research focusses on a number of compounds that are routinely added to synthetic media for cultivation of S. cerevisiae that, based on their role in human nutrition, are referred to as B-type vitamins. A special focus was laid upon one of the more expensive B vitamins, biotin. The pathway by which some S. cerevisiae strains synthesize biotin is still not completely resolved. By a combination of laboratory evolution, genome analysis and genetic engineering, different strategies were designed and tested to obtain biotin prototrophic and fully vitamin-independent S. cerevisiae strains…