Creating novel biosynthetic pathways and modulating the synthesis of important compounds are one of the hallmarks of synthetic biology. Understanding the key parameters controlling the flux of chemicals throughout a metabolic pathway is one of the challenges ahead. Isoprenoids are the most functionally and structurally diverse group of natural products from which numerous medicines and relevant fine chemicals are derived. The well-characterized and broadly used production organism Bacillus subtilis forms an ideal background for creating and studying novel synthetic routes. In comparison to other bacteria, B. subtilis emits the volatile compound isoprene, the smallest representative of isoprenoids, in high concentrations and thus represents an interesting starting point for an isoprenoid cell factory. In this study, the effect of systematic overexpression of the genes involved in the methylerythritol phosphate (MEP) pathway on isoprenoid production in B. subtilis was investigated. B. subtilis strains harboring a plasmid containing C30 carotenoid synthetic genes, crtM and crtN, were combined with pHCMC04G plasmids carrying various synthetic operons of the MEP pathway genes. The levels of produced carotenoids, diaponeurosporene and diapolycopene, were used as indication of the role of the various enzymes on the flux of the MEP pathway. It was shown that the production of carotenoids can be increased significantly by overexpressing the MEP pathway enzymes. More broadly, the strains developed in this study can be used as a starting point for various isoprenoid cell factories.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6531-3) contains supplementary material, which is available to authorized users.