Lactate is an important industrial material with numerous potential applications, and its production from carbon dioxide is very attractive. D-Lactate is an essential monomer for production of thermostable polylactide. The photoautotrophic prokaryote cyanobacterium Synechocystis sp. FCC 6803 represents a promising host for biosynthesis of D-lactate from CO2 as it only contains D-lactate dehydrogenase. The production of D-lactate from CO2 by an engineered strain of Synechocystis sp. FCC 6803 with overexpressing D-lactate dehydrogenase and a soluble transhydrogenase has been reported recently. Here, we report an alternative engineering strategy to produce o-lactate from CO2. This strategy involves blocking two competitive pathways, the native poly-3-hydroxybutyrate and acetate pathways from the acetylCoA node, and introducing a more efficient o-lactate dehydrogenase into Synechocystis sp. FCC 6803. The engineered strain of Synechocystis sp. FCC 6803 was capable of producing 1.06 g/L of D-lactate from CO2. This alternative strategy for the production of optically pure D-lactate could also be used to produce other acetyl-CoA-derived chemicals from CO2 by using engineered cyanobacteria. (C) 2014 Elsevier Ltd. All rights reserved.