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

• Published in: Scientific reports Vol. 7; no. 1; pp. 17155 - 12
• Main Authors: Park, Ye Seop, Choi, Un Jong, Nam, Nguyen Hoai, Choi, Sang Jin, Nasir, Abdul, Lee, Sun-Gu, Kim, Kyung Jin, Jung, Gyoo Yeol, Choi, Sangdun, Shim, Jeung Yeop, Park, Sunghoon, Yoo, Tae Hyeon
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 07.12.2017; Nature Publishing Group
• Subjects: 3-Hydroxypropionaldehyde; 42/70; 631/45/603; 631/61/318; 82/47; 82/80; 82/83; Aldehyde dehydrogenase; Aldehyde Oxidoreductases - chemistry; Aldehyde Oxidoreductases - metabolism; Azospirillum brasilense - enzymology; Bacterial Proteins - metabolism; Binding sites; Cell culture; Dehydration; Dehydrogenases; Enzymes; Glyceraldehyde - analogs & derivatives; Glyceraldehyde - metabolism; Glycerol; Glycerol - metabolism; Humanities and Social Sciences; Ketoglutaric acid; Lactic Acid - analogs & derivatives; Lactic Acid - metabolism; multidisciplinary; NAD; NAD - metabolism; NADH; Oxidation; Propane - metabolism; Protein Conformation; Protein Engineering - methods; Science; Science (multidisciplinary); Substrate Specificity; Toxicity
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-017-15400-x

3-Hydroxypropionic acid (3-HP) can be produced via the biological route involving two enzymatic reactions: dehydration of glycerol to 3-hydroxypropanal (3-HPA) and then oxidation to 3-HP. However, commercial production of 3-HP using recombinant microorganisms has been hampered with several problems, some of which are associated with the toxicity of 3-HPA and the efficiency of NAD + regeneration. We engineered α-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense for the second reaction to address these issues. The residues in the binding sites for the substrates, 3-HPA and NAD + , were randomized, and the resulting libraries were screened for higher activity. Isolated KGSADH variants had significantly lower K m values for both the substrates. The enzymes also showed higher substrate specificities for aldehyde and NAD + , less inhibition by NADH, and greater resistance to inactivation by 3-HPA than the wild-type enzyme. A recombinant Pseudomonas denitrificans strain with one of the engineered KGSADH variants exhibited less accumulation of 3-HPA, decreased levels of inactivation of the enzymes, and higher cell growth than that with the wild-type KGSADH. The flask culture of the P. denitrificans strain with the mutant KGSADH resulted in about 40% increase of 3-HP titer (53 mM) compared with that using the wild-type enzyme (37 mM).