(R)-1,3-Butanediol is a valuable chemical extensively used as a key intermediate for the synthesis of pharmaceuticals and several industrial compounds. Despite its high demand, the production has been restricted from multi-step chemical production, petrochemical substrate requirement and a non-existence natural synthesis pathway from renewable biomass. In this study, an artificial synthesis route was genetically engineered in Escherichia coli MG1655 lacI(q) to produce 1,3-butanediol from glucose. The selection of heterologous genes from several organisms and activity level of their corresponding gene products were demonstrated to be the key element for the product formation. Improvement of fermentation aeration and the application of fed-batch system significantly enhanced 1,3-butanediol production. Under the optimized conditions, 1,3-butanediol was produced up to 9.05 g/l (100.4 mM) with 98.5 +/- 0.2% enantiomeric excess (% ee) of (R)-1,3-butanediol. This is the highest yield of 1,3-butanediol produced from glucose with the highest optical purity by the recombinant strain reported thus far. (c) 2012, The Society for Biotechnology, Japan. All rights reserved.