3-Hydroxypropionic acid (3-HP) is an important platform chemical, and biological production of 3-HP fromglycerol as a carbon source using glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) has beenrevealed to be effective because it involves a relatively simple metabolic pathway and exhibits higher yield andproductivity than other biosynthetic pathways. Despite the successful attempts of 3-HP production from glycerol,the biological process suffers from problems arising from low activity and inactivation of the two enzymes.To apply the directed evolutionary approach to engineer the 3-HP production system, we constructed a syntheticselection device using a 3-HP-responsive transcription factor and developed a selection approach for screening 3-HP-producing microorganisms. The method was applied to an ALDH library, specifically aldehyde-binding sitelibrary of alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH). Only two serial cultures resulted in enrichmentof strains showing increased 3-HP production, and an isolated KGSADH variant enzyme exhibited a2.79-fold higher catalytic efficiency toward its aldehyde substrate than the wild-type one. This approach willprovide the simple and efficient tool to engineer the pathway enzymes in metabolic engineering.

3-Hydroxypropionic acid (3-HP) is an important platform chemical, and biological production of 3-HP fromglycerol as a carbon source using glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) has beenrevealed to be effective because it involves a relatively simple metabolic pathway and exhibits higher yield andproductivity than other biosynthetic pathways. Despite the successful attempts of 3-HP production from glycerol, the biological process suffers from problems arising from low activity and inactivation of the two enzymes. To apply the directed evolutionary approach to engineer the 3-HP production system, we constructed a syntheticselection device using a 3-HP-responsive transcription factor and developed a selection approach for screening 3-HP-producing microorganisms. The method was applied to an ALDH library, specifically aldehyde-binding sitelibrary of alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH). Only two serial cultures resulted in enrichmentof strains showing increased 3-HP production, and an isolated KGSADH variant enzyme exhibited a2.79-fold higher catalytic efficiency toward its aldehyde substrate than the wild-type one. This approach will provide the simple and efficient tool to engineer the pathway enzymes in metabolic engineering.