Throughout evolution, mostSaccharomyces cerevisiaestrains have lost their ability to synthesize biotin, an essential cofactor of several carboxylating enzymes. As a result, the essential vitamin or its precursors must be uptaken from the environment and frequently supplemented in fermentations to achieve high cell densities. Engineering of a biotin-independentS. cerevisiaestrain is of interest to eliminate the need for the external biotin supply. Herein, we describe the construction of a biotin-independent yeast strain by engineering a bypass of acetyl-CoA carboxylase, an essential biotin-dependent enzyme in the synthesis of fatty acids. Besides complete rescue of growth in biotin-free media, the resultingS. cerevisiaestrains showed significantly improved growth on malonate compared to biotin. Beyond their industrial relevance, the yeast strains reported here can be valuable in areas of fundamental research, e.g., for developing a new selection marker or increasing the versatility of biotin-streptavidin technologies in living systems.