Engineering of the Substrate Pocket of α-ketoglutaric Semialdehyde Dehydrogenase for Improving the Activity toward 3-hydroxypropanal

• Published in: Biotechnology and bioprocess engineering Vol. 28; no. 6; pp. 1015 - 1024
• Main Authors: Park, Ye Seop, Nasir, Abdul, Nguyen-Vo, Thuan Phu, Ryu, Huichang, Seok, Joo Yeon, Jung, Gyoo Yeol, Park, Sunghoon, Yoo, Tae Hyeon
• Format: Journal Article
• Language: English
• Published: Seoul The Korean Society for Biotechnology and Bioengineering 01.12.2023; Springer Nature B.V; 한국생물공학회
• Subjects: 3-Hydroxypropionaldehyde; Aldehyde dehydrogenase; Aldehydes; Azospirillum brasilense; Biotechnology; Catalytic activity; Chemical synthesis; Chemistry; Chemistry and Materials Science; Dehydration; Dehydrogenase; Dehydrogenases; Enzymes; Glycerol; Glycerol dehydratase; Industrial and Production Engineering; Molecular dynamics; Oxidation; Pseudomonas denitrificans; Research Paper; Substrates; transcription factors; 생물공학; 3-Hydroxypropanal; enzyme engineering; aldehyde dehydrogenase; 3-Hydroxypropionic acid; molecular dynamics simulation
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-021-0335-3

3-Hydroxypropionic acid (3-HP) is a key building block for value-added chemicals. A biological route for synthesizing this molecule is two-step enzymatic reactions; dehydration of glycerol to 3-hydroxypropanal (3-HPA) by glycerol dehydratase and then oxidation of 3-HPA to 3-HP by aldehyde dehydrogenase. Here, we report an aldehyde dehydrogenase, an engineered α-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense. The variant, named 2C10, was obtained by applying a large KGSADH library to a selection method based on a 3-HP-responsive transcription factor and then a screening method for observing the activities of individual clones. 2C10 exhibited a 4.65-fold higher catalytic activity (k cat /K m : 100 ± 7.1 s −1 mM −1 ) toward 3-HPA than the wild-type enzyme. The flask culture of Pseudomonas denitrificans with 2C10 resulted in an approximately 30% increase in 3-HP titer (43.2 mM) compared with that obtained using wild-type KGSADH (33.1 mM). Molecular dynamics simulations suggested that compared to the wild-type enzyme, 2C10 has a less flexible and smaller binding pocket for aldehyde substrates.