The increasing demand of sustainably produced chemicals and fuels can be countered by exploitation of acetogenic bacteria in bioprocesses. The acetogen Acetobacterium woodii is a promising candidate due to its ability to convert C1 feedstocks such as CO2, CO, formate and methanol with high carbon and energy efficiency. The well-characterized physiology and metabolism as well as the availability of tools for genetic engineering make this strain an interesting platform organism. Routinely, A. woodii is grown in complex medium with supplemented vitamins. Minimizing or omitting complex components and vitamins in growth media is favored due to reduction in cost and variability of bioprocessing. In this work, an endogenous CRISPR system along with adaptive laboratory evolution was used to generate a strain which is able to grow in synthetic medium without supplemented vitamins. As a first step, the strain was adapted to grow in medium without complex components. Next, all vitamins except pantothenic acid, for which parts of the biosynthesis pathway are missing in the genome, were omitted. The missing genes for pantothenic acid biosynthesis were knocked into the genome and subsequent ALE resulted in a strain that grows robustly in chemically defined medium without vitamins supplemented. Whole genome sequencing was used to reveal changes in the genome across different stages of adaptation. The resulting strain is a powerful basis for further engineering to upgrade C1 substrates such as formate and methanol into chemicals and fuels.