Identification and characterization of the propanediol utilization protein PduP of Lactobacillus reuteri for 3-hydroxypropionic acid production from glycerol

• Published in: Applied microbiology and biotechnology 2011-02, Vol.89 (3), p.697-703
• Main Authors: Luo, Lian Hua, Seo, Jeong-Woo, Baek, Jin-Oh, Oh, Baek-Rock, Heo, Sun-Yeon, Hong, Won-Kyung, Kim, Dae-Hyuk, Kim, Chul Ho
• Format: Article
• Language: English
• Subjects: 3-Hydroxypropionaldehyde; 3-Hydroxypropionic acid; Acid production; Acids; Aldehydes - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological and medical sciences; Biomass; Biomedical and Life Sciences; Bioreactors; Biosynthesis; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Cloning; Cloning, Molecular; Coenzymes - metabolism; Dehydrogenases; E coli; Enzyme kinetics; Enzymes; Enzymes - genetics; Enzymes - metabolism; Fermentation; Fundamental and applied biological sciences. Psychology; Gene Expression; Glycerol; Glycerol - metabolism; Kinases; Kinetics; Klebsiella pneumoniae; Klebsiella pneumoniae - enzymology; Klebsiella pneumoniae - genetics; Lactic Acid - analogs & derivatives; Lactic Acid - metabolism; Lactobacillus reuteri; Lactobacillus reuteri - enzymology; Lactobacillus reuteri - metabolism; Life Sciences; Metabolism; Microbial Genetics and Genomics; Microbiology; Microorganisms; NAD - metabolism; NADP - metabolism; Plasmids; Propanediol utilization protein PduP; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Studies; Substrate Specificity
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-010-2887-6

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.