In this study, two response regulator (RR) encoding genes slr1037 as well as sll0039 were co-overexpressed in Synechocystis sp. PCC 6803 by metabolic engineering and the 1-butanol tolerance was successfully improved by 133%. Aiming to explore the possible mechanisms for the enhancing 1-butanol tolerance, a quantitative iTRAQ-LC-MS/MS proteomics approach was then employed, identifying 216 up-regulated and 99 down-regulated proteins compared to wild type after 1-butanol treatment. This study mapped the potential target genes regulated by Slr1037 and Sll0039 and demonstrated the feasibility of engineering response regulators for modifying the biofuel tolerance in cyanobacteria. (C) 2017 Elsevier Ltd. All rights reserved.