Characterization of 1,3-propanediol oxidoreductase (DhaT) from Klebsiella pneumoniae J2B

Lama, Suman; Ro, Su Moon; Seol, Eunhee; Sekar, Balaji Sundara; Ainala, Satish Kumar; Thangappan, Jayaraman; Song, Hyohak; Seung, Doyoung; Park, Sunghoon

doi:10.1007/s12257-015-0635-6

Cited by 18 publications

(7 citation statements)

References 27 publications

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“…Accession: WP_004224052.1) of K. pneumoniae were selected (Fig. 3a ) 49 , 51 , 52 . In the enzyme-screening process, we first compared the efficiency of the selected 1,3-propanediol oxidoreductase encoding genes in combination with aldD Hb (plasmids p90 and p95).…”

Section: Resultsmentioning

confidence: 99%

“…The oxidative activity (reverse reaction) of AdhP, DhaT Pp , and DhaT Kp were measured as follows: The reaction mixture containing 100 mM potassium carbonate buffer (pH 9.0), 30 mM ammonium sulfate, and 100 nM enzyme was incubated for 2 min at 37 °C. The reaction was initiated by the addition of 2 mM of NAD + and 150 mM 1,3-propanediol 51 . The activity of the aldehyde dehydrogenase was determined by measuring the level of NAD + reduction to NADH at 340 nm.…”

Section: Methodsmentioning

confidence: 99%

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Hyperproduction of 3-hydroxypropionate by Halomonas bluephagenesis

Jiang

Yan

et al. 2021

Nat Commun

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Abstract3-Hydroxypropionic acid (3HP), an important three carbon (C3) chemical, is designated as one of the top platform chemicals with an urgent need for improved industrial production. Halomonas bluephagenesis shows the potential as a chassis for competitive bioproduction of various chemicals due to its ability to grow under an open, unsterile and continuous process. Here, we report the strategy for producing 3HP and its copolymer poly(3-hydroxybutyrate-co-3-hydroxypropionate) (P3HB3HP) by the development of H. bluephagenesis. The transcriptome analysis reveals its 3HP degradation and synthesis pathways involving endogenous synthetic enzymes from 1,3-propanediol. Combing the optimized expression of aldehyde dehydrogenase (AldDHb), an engineered H. bluephagenesis strain of whose 3HP degradation pathway is deleted and that overexpresses alcohol dehydrogenases (AdhP) on its genome under a balanced redox state, is constructed with an enhanced 1.3-propanediol-dependent 3HP biosynthetic pathway to produce 154 g L−1 of 3HP with a yield and productivity of 0.93 g g−1 1,3-propanediol and 2.4 g L−1 h−1, respectively. Moreover, the strain could also accumulate 60% poly(3-hydroxybutyrate-co-32–45% 3-hydroxypropionate) in the dry cell mass, demonstrating to be a suitable chassis for hyperproduction of 3HP and P3HB3HP.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Hyperproduction of 3-hydroxypropionate by Halomonas bluephagenesis

Jiang

Yan

et al. 2021

Nat Commun

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“…The color-coding scheme is used throughout the study to identify the protein modules and enzymes. dehydratase (dhaB1) and its activating protein (dhaB2) and (2) the hydrogenation of 3-HPA to 1,3 PDO with a 1,3-PDO dehydrogenase (dhaT) (Figure 1) (O'Brien et al, 2004;Lama et al, 2015). All enzymes considered in this study were selected from C. butyricum.…”

Section: Gene Selection and Construction Of Self-assembling Pathway Enzymes For The Conversion Of Glycerol To 13-pdomentioning

confidence: 99%

Self-Assembling Metabolon Enables the Cell Free Conversion of Glycerol to 1,3-Propanediol

Alahuhta

Hewitt

et al. 2021

Front. Energy Res.

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Cell free biocatalysis is showing promise as a replacement or complement to conventional microbial biocatalysts due to the potential for achieving high yields, titers, and productivities. However, there exist several challenges that need to be addressed before its broader industrial adoption is achieved. New paradigms and innovative solutions are needed to overcome these challenges. In this study we demonstrate high levels of glycerol conversion to 1,3-propanediol using a self-assembling metabolic pathway leveraging the arraying strategy (protein scaffolds) used by thermophilic cellulolytic bacteria to assemble their biomass degrading enzymes. These synthetic metabolons were capable of producing 1,3-PDO at a yield more than 95% at lower glycerol concentration and close to 70% at higher concentrations at a higher productivity rate than the equivalent microbial strain. One of the benefits of our approach is the fact that no enzyme purification is required, and that the assembly of the complex is accomplished in vivo before immobilization, while product formation is conducted in vitro. We also report the recovery of enzymatic activity upon fusion enzymes binding to these protein scaffolds, which could have broader applications when assembling arrayed protein complexes.

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“…One unit of 1,3-propanediol oxidoreductase activity was defined as the amount of enzyme needed to oxidize 1 μmol of NADH to NAD + in one minute. The oxidative activity (reverse reaction) was measured using 1,3-propanediol as the substrate following the method described by Lama et al [13]. The reduction of NAD + to NADH was monitored at 340 nm.…”

Section: Determination Of Enzyme Activity and Kinetic Parametersmentioning

confidence: 99%

“…Despite its importance to 1,3-PD production, however, DhaT's kinetic properties have not been well studied. Only recently, our group fully characterized DhaT of K. pneumoniae J2B (Kp) for the forward and reverse reactions using 3-HPA and propionaldehyde and their corresponding alcohols, 1,3-PD and 1-propanol [13]. One important finding was that DhaT is seriously inactivated by its physiological substrate, 3-HPA, even at a low concentration of 5 mM.…”

Section: Introductionmentioning

confidence: 99%

Improvement of 1,3-propanediol oxidoreductase (DhaT) stability against 3-hydroxypropionaldehyde by substitution of cysteine residues

Sekar

et al. 2016

Biotechnol Bioproc E

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1,3-propanediol oxidoreductase (DhaT), which catalyzes the conversion of 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) with the oxidation of NADH to NAD + , is a key enzyme in the production of 1,3-PD from glycerol. DhaT is known to be severely inactivated by its physiological substrate, 3-HPA, due to the reaction of 3-HPA with the thiol group of the cysteine residues. In this study, using site-directed mutagenesis, four cysteine residues in Klebsiella pneumoniae J2B DhaT were substituted to alanine, the amino acid commonly found in cysteine's positions in other DhaT, individually and in combination. Among the total of 15 mutants developed, a double mutant (C28A_C107A) and a triple mutant (C28A_C93A_C107A) exhibited approximately 50 and 16% higher activity than the wild-type counterpart, respectively, after 1 h incubation with 10 mM 3-HPA. According to detailed kinetic studies, the double mutant had slightly better kinetic properties (V max , K cat , and K m for both 3-HPA and NADH) than wild-type DhaT. This study shows that DhaT stability against 3-HPA can be increased by cysteine-residue removal, albeit to a limited extent.

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Characterization of 1,3-propanediol oxidoreductase (DhaT) from Klebsiella pneumoniae J2B

Cited by 18 publications

References 27 publications

Hyperproduction of 3-hydroxypropionate by Halomonas bluephagenesis

Hyperproduction of 3-hydroxypropionate by Halomonas bluephagenesis

Self-Assembling Metabolon Enables the Cell Free Conversion of Glycerol to 1,3-Propanediol

Improvement of 1,3-propanediol oxidoreductase (DhaT) stability against 3-hydroxypropionaldehyde by substitution of cysteine residues

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