3-Hydroxypropionic acid (3-HP) can be converted into derivatives such as acrylic acid, a source for producing super absorbent polymers. Although Escherichia coli has often been used for 3-HP production, it exhibits low tolerance to 3-HP. To circumvent this problem, we selected the fission yeast Schizosaccharomyces pombe as this microorganism has higher tolerance to 3-HP than E. coll. Therefore, we constructed S. pombe transformants overexpressing two genes, one encoding the S. pombe acetyl-CoA carboxylase (Cut6p) and the other encoding the malonyl-CoA reductase derived from Chloroflexus aurantiacus (CaMCR). To prevent the degradation of these expressed proteins, we employed an S. pombe protease-deficient strain. Moreover, to increase the cytosolic concentration of acetyl-CoA, we supplemented acetate to the medium, which improved 3-HP production. To further produce 3-HP by overexpressing Cut6p and CaMCR, we exploited the highly expressing S. pombe hsp9 promoter. Finally, culturing in high-density reached 3-HP production to 7.6 g/L at 31 h. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.