Ashbya gossypii has emerged as a promising microbial platform for the sustainable production of high-value biochemicals. This chapter explores the optimization of its natural metabolism through the development and application of an increasingly sophisticated molecular toolbox. Core components of this toolbox include efficient transformation protocols, integrative and plasmid-based expression systems, a library of well-characterized promoters and terminators, scarless genome editing methods, and modular platforms for assembling multigene expression cassettes. Combined with the A. gossypii’s inherent metabolic potential, compact and well-annotated genome, efficient homologous recombination system, and close genetic relationship to Saccharomyces cerevisiae, these tools have enabled the implementation of advanced metabolic engineering and synthetic biology strategies. Such strategies have been employed to rewire endogenous pathways, enhance precursor supply, expand substrate utilization, and boost overall biosynthetic capacity. As a result, A. gossypii has been successfully engineered for the production of a wide array of valuable compounds, including riboflavin, nucleosides, folates, orotic acid, lipids, lactones, monoterpenes, gangliosides, and recombinant proteins. This chapter provides a comprehensive overview of the molecular and metabolic engineering approaches that have transformed A. gossypii into a versatile and industrially relevant chassis for next-generation microbial manufacturing.