Genetically engineered microbes have been intensively investigated as a means for the cost-effective production of isoprenoids. Bacillus subtilis is a promising microbial host for this purpose because of its fast growth rate and GRAS (generally regarded as safe) status. To date, development of this host has been impaired by the lack of genetic tools for modulating the expression of multiple genes. In this study, we present a novel two-promoter system which can be used to independently control the expression levels of two gene cassettes over a large dynamic range. Coupled with protein translation engineering and systematic media optimization, ∼20 mg/L amorphadiene was produced in shake flask scale, a 40-fold improvement over the highest reported isoprenoid product yield in B. subtilis. As the tools and strategies developed here can be extended to the overproduction of other valuable metabolites, this proof-of-concept study lays the foundation for high level heterologous production of isoprenoids in Bacillus. Biotechnol. Bioeng. 2013; 110:2556–2561. © 2013 Wiley Periodicals, Inc.