Engineering an aldehyde dehydrogenase toward its substrates, 3-hydroxypropanal and NAD(+), for enhancing the production of 3-hydroxypropionic acid
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- Engineering an aldehyde dehydrogenase toward its substrates, 3-hydroxypropanal and NAD(+), for enhancing the production of 3-hydroxypropionic acid
- Park, Ye Seop; Choi, Un Jong; Nguyen Hoai Nam; Choi, Sang Jin; Nasir, Abdul; Lee, Sun-Gu; Kim, Kyung Jin; Jung, Gyoo Yeol; Choi, Sangdun; Shim, Jeung Yeop; Park, Sunghoon; Yoo, Tae Hyeon
- Issue Date
- NATURE PUBLISHING GROUP
- SCIENTIFIC REPORTS, v.7, no., pp.17155 -
- 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 a-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 Km 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).
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