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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.

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Acknowledgments

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).

Author information

Correspondence to Pingfang Tian.

Additional information

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). https://doi.org/10.1007/s10529-014-1590-6

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Keywords

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