3-Hydroxypropionate (3HP) is an important platform chemical, and malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus can convert malonyl-CoA, a common metabolic intermediate in Escherichia coli, into 3HP.In previous study, MCR was dissected into two functional distinct fragments, and malonyl-CoA was reduced with NADPH by MCR-C into malonate semialdehyde, which was further reduced into 3HP by MCR-N.In this study, it is discovered that the 3HP production via malonyl-CoA pathway was severely depressed by low activity of MCR-C and metabolic burden caused by excessive overexpression of MCR-N and MCR-C.To increase 3HP productivity, the enzymatic activity of MCR-C was improved by directed evolution,and a series of promoters was tested to achieve balance between enzyme activity required by 3HP synthesis and metabolic burden caused by heterologous protein expression.Finally, our engineered E.coli strain accumulated 40.6 g/L 3HP using glucose as sole carbon source under fed-batch fermentation conditions, about 270 times higher than that of our original strain and 90 times higher than the previously reported 3HP production via malonyl-CoA pathway by recombinant Saccharomyces cerevisiae.