3. Enzyme Engineering

Directed evolution of the 3-hydroxypropionic acid production pathway byengineering aldehyde dehydrogenase using a synthetic selection device

Metabolic Engineering, 2018, 47, 113-120.
Metabolic%20Engineering_2018


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.

Engineering an aldehyde dehydrogenase toward its substrates, 3-hydroxypropanal and NAD+, for enhancing the production of 3-hydroxypropionic acid

Scientific Reports, 2017, 7, 17155
KGSADH_Scientific%20reports_2017


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.

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Synthetic Protein Engineering Laboratory

Department of Molecular Science and Technology, Ajou University

206 Worldcup-ro, Yeongtong-gu, Suwon, 16499 Korea