Identification and characterization of the propanediol utilization protein PduP of Lactobacillus reuteri for 3-hydroxypropionic acid production from glycerol.
ABSTRACT 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.
- ChemSusChem 01/2009; 2(1):57-8. · 7.48 Impact Factor
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ABSTRACT: 3-Hydroxypropionic acid (3-HP) is an important platform chemical from which several commodity and specialty chemicals can be generated. The present investigation focuses on the construction and evaluation of a recombinant strain Escherichia coli SH254 that produces 3-HP from glycerol. The strain was developed by cloning two genes, dhaB of Klebsiella pneumoniae DSM 2026 encoding glycerol dehydratase and aldH of E. coli K-12 MG1655 encoding aldehyde dehydrogenase, respectively. In vitro assays of crude enzyme extract of glycerol dehydratase (DhaB) showed 37.0 U mg−1 protein on glycerol with coenzyme B12, and partially purified aldehyde dehydrogenase (AldH) exhibited 22.8 U mg−1 protein on 3-hydroxypropionaldehyde (3-HPA) with NAD+ as a cofactor. When cultivated aerobically on a glycerol medium containing yeast extract, the recombinant E. coli SH254 produced 3-HP at a maximum of 6.5 mmol l−1 (0.58 g l−1). The highest specific rate and yield of 3-HP production were estimated as 6.6 mmol g−1 cdw h−1 and 0.48 mol mol−1 glycerol, respectively. Although not optimized extensively, this study is encouraging for further development of a bioprocess to produce 3-HP from glycerol.Process Biochemistry. 04/2008;
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ABSTRACT: 3-Hydroxypropionic acid (3-HP) is a commercially valuable chemical with the potential to be a key building block for deriving many industrially important chemicals. However, its biological production has not been well documented. Our previous study demonstrated the feasibility of producing 3-HP from glycerol using the recombinant Escherichia coli SH254 expressing glycerol dehydratase (DhaB) and aldehyde dehydrogenase (AldH), and reported that an "imbalance between the two enzymes" and the "instability of the first enzyme DhaB" were the major factors limiting 3-HP production. In this study, the efficiency of the recombinant strain(s) was improved by expressing DhaB and AldH in two compatible isopropyl-thio-beta-galactoside (IPTG) inducible plasmids along with glycerol dehydratase reactivase (GDR). The expression levels of the two proteins were measured. It was found that the changes in protein expression were associated with their enzymatic activity and balance. While cloning an alternate aldehyde dehydrogenase (ALDH), alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH), instead of AldH, the recombinant E. coli SH-BGK1 showed the highest level of 3-HP production (2.8 g/L) under shake-flask conditions. When an aerobic fed-batch process was carried out under bioreactor conditions at pH 7.0, the recombinant SH-BGK1 produced 38.7 g 3-HP/L with an average yield of 35%. This article reports the highest level of 3-HP production from glycerol thus far.Biotechnology and Bioengineering 07/2009; 104(4):729-39. · 3.65 Impact Factor