title: NAD(+)-independent aldehyde oxidase catalyzes cofactor balanced 3-hydroxypropionic acid production in Klebsiella pneumoniae.
abstract: The limiting step for biosynthesis of 3-hydroxypropionic acid (3-HP) in Klebsiella pneumoniae is the conversion of 3-hydroxypropionaldehyde (3-HPA) to 3-HP. This reaction is catalyzed by aldehyde dehydrogenase (ALDH) with NAD(+) as a cofactor. Although NAD(+)-dependent ALDH overexpression facilitates 3-HP biosynthesis, ALDH activity decreases and 3-HP stops accumulation when NAD(+) is exhausted. Here, we show that an NAD(+)-independent aldehyde oxidase (AOX) from Pseudomonas sp. AIU 362 holds promise for cofactor-balanced 3-HP production in K. pneumoniae. The AOX coding gene, alod, was heterologously expressed in E. coli and K. pneumoniae, and their respective crude cell extracts showed 38.1 U/mg and 16.6 U/mg activities toward propionaldehyde. The recombinant K. pneumoniae expressing alod showed 13.7 U/mg activity toward 3-HPA; K m and V max were 6.7 mM and 42 μM/min/mg, respectively. In shake-flask cultures, the recombinant K. pneumoniae strain produced 0.89 g 3-HP/l, twice that of the control. Moreover, it produced 3 g 3-HP/l during 24 h fed-batch cultivation in a 5 l bioreactor. The results indicate that AOX can efficiently convert 3-HPA into 3-HP.
authors: Beijing Key Lab of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, People's Republic of China.
authors: Ying
authors: Y
authors: Li
authors: Luo
authors: L
authors: Liu
authors: Pingfang
authors: P
authors: Tian
mesh_terms: D000444:Aldehyde Dehydrogenase; D042931:Aldehyde Oxidase / Q000235:genetics / Q000378:metabolism*; D018533:Biomass; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism*; D019344:Lactic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D009243:NAD; D019344:Lactic Acid; C031601:hydracrylic acid; D000444:Aldehyde Dehydrogenase; D042931:Aldehyde Oxidase