Most natural carotenoids have 40-carbon (C-40) backbones, while some bacteria produce carotenoids with C-30 backbones. Carotenoid backbone synthases, the enzyme that catalyze the first committed step in carotenoid biosynthesis, are known to be highly specific. Previously, using C-30 backbone synthase (diapophytoene synthase, CrtM) from Staphylococcus aureus, we reported two size-shifting mutations, F26A and W38A, which confer C-40 synthase activity at the cost of the original C-30 synthase activity. In this study, we performed a directed evolution of the C-40-specialist variant CrtM(F26A) in search of mutations that restore the original C-30 synthase function. Examination of the resultant mutants, together with the site-directed mutagenesis study identified three new mutations (H12A, D27A and I240F) that affect the size specificity of this enzyme. After re-defining the reading frame, we obtained CrtM variants that are highly active in C-30 and C-40 carotenoid synthesis. (c) 2013, The Society for Biotechnology, Japan. All rights reserved.