Identification and characterization of the propanediol utilization protein PduP of Lactobacillus reuteri for 3-hydroxypropionic acid production from glycerol.
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.
Luo
Lian Hua
LH
Microbe-based Fusion Technology Research Center, Jeonbuk Branch Institute, KRIBB, Jeongeup, South Korea.
Seo
Jeong-Woo
JW
Baek
Jin-Oh
JO
Oh
Baek-Rock
BR
Heo
Sun-Yeon
SY
Hong
Won-Kyung
WK
Kim
Dae-Hyuk
DH
Kim
Chul Ho
CH
D000447:Aldehydes / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D004798:Enzymes / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005990:Glycerol / Q000378:metabolism*; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics; D019344:Lactic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D052200:Limosilactobacillus reuteri / Q000201:enzymology* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D000447:Aldehydes; D001426:Bacterial Proteins; D003067:Coenzymes; D004798:Enzymes; D011994:Recombinant Proteins; D009243:NAD; D019344:Lactic Acid; D009249:NADP; C005556:propionaldehyde; C031601:hydracrylic acid; D005990:Glycerol