We retrosynthetically designed a pathway along a completely novel set of new-to-nature metabolites for the synthesis of biotin, a high-value-added commercially relevant product currently manufactured by a chemical synthesis. The pathway will be implemented using rational design approaches complemented by advanced high throughput screening methods for the reprogramming of the substrate specificities of known enzymes. The project aims to demonstrate that multi-step biopathway design largely outside of the scope of reactions already known in nature is possible and provides attractive solutions to some of synthetic biology’s currently most intractable synthetic problems. Furthermore, we will demonstrate experimentally that retrosynthetic design is an excellent basis for rational biopathway design. We expect the results to be a milestone in the demonstration of the potential of synthetic biology for the development of chemical syntheses, which hopefully encourages the more frequent application of biosynthesis as an alternative to chemical methods.