We show here an efficient synthesis system of isoprenoids from acetoacetate as the main substrate. We expressed in Escherichia coli a Streptomyces mevalonate pathway gene cluster starting from HMG-CoA synthase and including isopentenyl diphosphate isomerase (idi) type 2 gene and the yeast idi type 1 and rat acetoacetate-CoA ligase (Aacl) genes. When the alpha-humulene synthase (ZSS1) gene of shampoo ginger was expressed in this transformant, the resultant E. coli produced 958 mug/mL culture of alpha-humulene with a lithium acetoacetate (LAA) supplement, which was a 13.6-fold increase compared with a control E. coli strain expressing only ZSS1. Next, we investigated if this E. coli strain engineered to utilize acetoacetate can synthesize carotenoids effectively. When the crtE, crtB, and crtI genes required for lycopene synthesis were expressed in the transformant, lycopene amounts reached 12.5 mg/g dry cell weight with addition of LAA, an 11.8-fold increase compared with a control expressing only the three crt genes. As for astaxanthin production with the E. coli transformant, in which the crtE, crtB, crtI, crtY, crtZ, and crtW genes were expressed, the total amount of carotenoids produced (astaxanthin, lycopene, and phytoene) was significantly increased to 7.5 times that of a control expressing only the six crt genes.