Rice straw and sugar cane industrial waste are a plentiful source of lignocellulosic biomass with a high polysaccharide content, that is hydrolyzed into sugar for microbial growth and their metabolites. 3-Hydroxypropionic acid (3-HP) is a promising chemical building block that can be produced from renewable resources. The malonyl-CoA pathway is one of the biosynthetic routes for 3-HP production by expressing the malonyl-CoA reductase gene (). However, the problem of the activity imbalance between the C and N-terminal causes a low conversion rate of malonyl-CoA to 3-hydroxypropionic acid. This study aimed to balance the bi-functional MCR enzyme by dissecting MCR into two fragments and enhancing the supply of intermediates to increase the production of 3-HP. The recombinant strain harboring the dissected gene showed a 21-fold increase in 3-HP titer compared to the strain carrying the full-length gene. The addition of cerulenin and acetate to the fermented medium enhanced 3-HP yield by 8 times, in which recombinant yeast produced 3-HP up to 10 g/L (0.201 g/g). The results of using rice straw hydrolysate as a carbon source indicated that S2 produced 3-HP of 4.02 g/L, which was 0.074 g/g in the diluted hydrolysate. These findings provide an alternative and sustainable strategy for utilizing lignocellulosic biomass for future 3-HP production at an industrial scale.