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NAD+-independent aldehyde oxidase catalyzes cofactor balanced 3-hydroxypropionic acid production in Klebsiella pneumoniae


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.

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This work was supported by Grants from National Natural Science Foundation of China (No. 21276014) and National Basic Research Program of China (973 Program) (2012CB725200).

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Correspondence to Pingfang Tian.

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Ying Li and Luo Liu have contributed equally to this work.

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Li, Y., Liu, L. & Tian, P. NAD+-independent aldehyde oxidase catalyzes cofactor balanced 3-hydroxypropionic acid production in Klebsiella pneumoniae . Biotechnol Lett 36, 2215–2221 (2014).

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  • Aldehyde dehydrogenase
  • Aldehyde oxidase
  • Glycerol
  • 3-Hydroxypropionic acid
  • Klebsiella pneumoniae
  • NAD+