Although the de novo biosynthetic mechanism of 3-hydroxypropionic acid (3-HP) in glycerol-fermenting microorganisms is still unclear, the propanediol utilization protein (PduP) of Lactobacillus species has been suggested to be a key enzyme in this regard. To verify this hypothesis, a pduP gene from Lactobacillus reuteri was cloned and expressed, and the encoded protein was characterized. Recombinant L. reuteri PduP exhibited broad substrate specificity including 3-hydroxypropionaldehyde and utilized both NAD+ and NADP+ as a cofactor. Among various aldehyde substrates tested, the specific activity was highest for propionaldehyde, at pH 7.8 and 37 °C. The K m and V max values for propionaldehyde in the presence of NAD+ were 1.18 mM and 0.35 U mg−1, respectively. When L. reuteri pduP was overexpressed in Klebsiella pneumoniae, 3-HP production remarkably increased as compared to the wild-type strain (from 0.18 g L−1 to 0.72 g L−1) under shake-flask culture conditions, and the highest titer (1.38 g L−1 3-HP) was produced by the recombinant strain under batch fermentation conditions in a bioreactor. This is the first report stating the enzymatic properties of PduP protein and the probable role in biosynthesis of 3-HP in glycerol fermentation.
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The authors are very thankful to Prof. Sunghoon Park for generously supplying 3-hydroxypropionaldehyde. This work was supported by the Korea Research Foundation (KRF) grant funded by Korea Government (MEST) (Basic Researcher Promotion Program 2010-0015871).
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Schematic representation of the strategy used for construction of the recombinant plasmids pET-pduP (a) and pVOTLp (b). Abbreviations for restriction enzymes: B BamHI, N NcoI, Xb XbaI, X XhoI. (PPT 54 kb)
Amino acid alignment of L. reuteri PduP with respect to the enzymes of L. collinoides (CAD83152), Salmonella enterica (NP460996), and Klebsiella pneumoniae (YP001336844). The putative active sites cysteine residues are indicated in bold. (PPT 69 kb)
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Luo, L.H., Seo, JW., Baek, JO. et al. Identification and characterization of the propanediol utilization protein PduP of Lactobacillus reuteri for 3-hydroxypropionic acid production from glycerol. Appl Microbiol Biotechnol 89, 697–703 (2011). https://doi.org/10.1007/s00253-010-2887-6
- 3-Hydroxypropionic acid
- Klebsiella pneumoniae
- Lactobacillus reuteri
- Propanediol utilization protein PduP