Fermentation of glycerol to 1,3-propanediol in continuous cultures of Citrobacter freundii

Boenigk, Rainer; Bowien, Susanne; Gottschalk, Gerhard

doi:10.1007/bf00242936

Cited by 144 publications

(89 citation statements)

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“…Assuming that this lysine residue can be autophosphorylated on sensing the environmental signal, it is tempting to speculate that after transfer of the phosphoryl group from the sensor protein to the receiver domain of the response regulator, the latter binds to DNA and, like most members of the AraC͞XylS family (40,48), acts as a transcriptional activator. Interestingly, a DNA region showing strong homology to the consensus sequence for AraC binding (40), i.e., AGCN 7 TCCATA is found overlapping the Ϫ35 region of the experimentally determined promoter of the 1,3-PD operon. This would suggest direct interactions of the response regulator with C. butyricum RNA polymerase, as indicated for other AraC͞ XylS family members, for which the few target sequences characterized to date have been located adjacent to or overlapping the Ϫ35 region of the regulated promoters (40).…”

Section: Discussionmentioning

confidence: 99%

“…E. coli ⌬adhE (pSPD5) cell extracts were prepared by sonication by using the anaerobic procedure of Vasconcelos et al (16). 1,3-PD dehydrogenase activity was determined spectrophotometrically by the procedure of Boenigk et al (7). The assay mixture contained in a 1-ml final volume, 30 mM ammonium sulfate, 100 mM potassium carbonate (pH 9.0), 2 mM DTT, 2 mM NAD ϩ , and 100 mM 1,3-PD.…”

Section: Methodsmentioning

confidence: 99%

“…In all of the natural 1,3-PD producers characterized to date, i.e., K. pneumoniae, Citrobacter freundii, and Clostridium pasteurianum, glycerol conversion to 1,3-PD involves a coenzyme B12-dependent glycerol dehydratase (5)(6)(7)(8)(9)(10). We recently reported that the glycerol dehydratase of Clostridium butyricum VPI1718, extracted from 1,3-PD-producing cells, was not stimulated by coenzyme B12 and was extremely oxygen sensitive, which suggested it might be a coenzyme B12-independent enzyme (11).…”

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Molecular characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum

Raynaud

Sarçabal

Meynial-Salles

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

207

160

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The genes encoding the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum VPI1718 were characterized from a molecular and a biochemical point of view. This operon is composed of three genes, dhaB1, dhaB2, and dhaT. When grown in a vitamin B12-free mineral medium with glycerol as carbon source, Escherichia coli expressing dhaB1, dhaB2, and dhaT produces 1,3-PD and high glycerol dehydratase and 1,3-PD dehydrogenase activities. dhaB1 and dhaB2 encode, respectively, a new type of glycerol dehydratase and its activator protein. The deduced proteins DhaB1 and DhaB2, with calculated molecular masses of 88,074 and 34,149 Da, respectively, showed no homology with the known glycerol dehydratases that are all B12 dependent but significant similarity with the pyruvate formate lyases and pyruvate formate lyases activating enzymes and their homologues. The 1,158-bp dhaT gene codes for a 1,3-PD dehydrogenase with a calculated molecular mass of 41,558 Da, revealing a high level of identity with other DhaT proteins from natural 1,3-PD producers. The expression of the 1,3-PD operon in C. butyricum is regulated at the transcriptional level, and this regulation seems to involve a two-component signal transduction system DhaAS͞DhaA, which may have a similar function to DhaR, a transcriptional regulator found in other natural 1,3-PD producers. The discovery of a glycerol dehydratase, coenzyme B12 independent, should significantly influence the development of an economical vitamin B12-free biological process for the production of 1,3-PD from renewable resources.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Molecular characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum

Raynaud

Sarçabal

Meynial-Salles

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

207

160

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“…freundii was grown in continuous culture under anaerobic conditions at 37ЊC as described previously (3). Cells of the effluent were harvested by centrifugation at 6,000 ϫ g for 20 min, washed twice with 50 mM imidazole buffer (pH 7.0) supplemented with 2 mM FeSO 4 ⅐ 7H 2 O and 2 mM reduced glutathione (GSH), and resuspended in the same buffer.…”

Section: Methodsmentioning

confidence: 99%

Purification of 1,3-propanediol dehydrogenase from Citrobacter freundii and cloning, sequencing, and overexpression of the corresponding gene in Escherichia coli

1995

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1,3-Propanediol dehydrogenase (EC 1.1.1.202) was purified to homogeneity from Citrobacter freundii grown anaerobically on glycerol in continuous culture. The enzyme is an octamer of a polypeptide of 43,400 Da. When tested as a dehydrogenase, the enzyme was most active with substrates containing two primary alcohol groups separated by one or two carbon atoms. In the physiological direction, 3-hydroxypropionaldehyde was the preferred substrate. The apparent K m values of the enzyme for 3-hydroxypropionaldehyde and NADH were 140 and 33 M, respectively. The enzyme was inhibited by chelators of divalent cations but could be reactivated by the addition of Fe 2؉ . The dhaT gene, encoding the 1,3-propanediol dehydrogenase, was cloned, and its nucleotide sequence (1,164 bp) was determined. The deduced dhaT gene product (387 amino acids, 41,324 Da) showed a high level of similarity to a novel family (type III) of alcohol dehydrogenases. The dhaT gene was overexpressed in Escherichia coli 274-fold by using the T7 RNA polymerase/promoter system. Microorganisms such asCitrobacter freundii or Klebsiella pneumoniae are able to grow anaerobically on glycerol as the sole carbon and energy source (17). In the absence of an external oxidant, glycerol is fermented by a dismutation process involving two pathways. Through one pathway glycerol is dehydrogenated by an NAD ϩ -linked glycerol dehydrogenase to dihydroxyacetone, which is then phosphorylated and funneled to glycolysis by dihydroxyacetone kinase (18). Through the other pathway, glycerol is dehydrated by the coenzyme B 12 -dependent glycerol dehydratase to form 3-hydroxypropionaldehyde, which is reduced to the major fermentation product 1,3-propanediol by the NADH-linked 1,3-propanediol dehydrogenase, thereby regenerating NAD ϩ (13). The four key enzymes of this pathway are encoded by the dha regulon, the expression of which is induced when dihydroxyacetone or glycerol is present (13, 27).Recently we have cloned and expressed the dha regulon of C. freundii in Escherichia coli (8). The coding region of the whole regulon is located on the 40-kb recombinant cosmid pRD1. In this report, we describe the purification of 1,3-propanediol dehydrogenase (EC 1.1.1.202) from C. freundii and the subcloning, sequencing, and overexpression of the corresponding gene in E. coli. MATERIALS AND METHODSMaterials. Q-Sepharose Fast Flow and Blue Sepharose CL-6B were obtained from Pharmacia LKB GmbH, Freiburg, Germany. Tris, EDTA, and sodium dodecyl sulfate were from Serva, Heidelberg, Germany. 1,3-Propanediol and 1,2-propanediol were purchased from Merck, Darmstadt, Germany. 3-Hydroxypropionaldehyde was synthesized by the method of Durrwachter et al. (11). All other reagents used were commercial products of the highest grade available.Bacterial strains, plasmids, and growth conditions. C. freundii DSM 30040 was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany. The gene coding for 1,3-propanediol dehydrogenase was isolated from the recombinant cos...

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“…The microbial conversion of glycerol to 1,3-PD is particularly attractive because the process uses renewable feedstock and does not generate toxic byproducts (Biebl et al, 1999;Saxena et al, 2009). Several bacterial groups such as Klebsiella (Huang et al, 2002;Wang et al, 2011), Citrobacter (Boenigk et al, 1993) and Clostridium (Gungormusler et al, 2010;Saint-Amans et al, 2001) could convert glycerol to 1,3-PD in significant quantities. However, most researches have been conducted using pure glycerol as substrate.…”

Section: Introductionmentioning

confidence: 99%

Production of 1,3-propanediol by Klebsiella pneumoniae using raw glycerol from Zygosacharomyces rouxii

Ma¹,

Shentu²,

Bian³

et al. 2012

Afr. J. Biotechnol.

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The conversion of glycerol to 1,3-propanediol (1,3-PD) is an environmentally friendly biological process and has attracted great interests worldwide. Efficient utilization of raw glycerol could bring significant economic benefits for 1,3-PD production. In the present study, the glycerol broth from strain Zygosacharomyces rouxii JL2011 was first used as the sole carbon source for 1,3-PD production by Klebsiella pneumoniae. The result indicates that the glycerol broth affected K. pneumoniae growth and 1,3-PD yield dramatically. Therefore, the raw glycerol was further prepared by extraction and purification, and the effect of raw glycerol on 1,3-PD production was investigated. The results obtained showed that the purified glycerol could be directly utilized in shake-flask culture by K. pneumoniae with a result comparable with that attained from pure glycerol. In addition, the fed-batch culture using raw glycerol as the substrate was conducted giving 56.1 g⋅ ⋅ ⋅ ⋅L -1 with a yield of 0.40 g⋅ ⋅ ⋅ ⋅g -1 glycerol and productivity of 1.12 g⋅ ⋅ ⋅ ⋅L

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Fermentation of glycerol to 1,3-propanediol in continuous cultures of Citrobacter freundii

Cited by 144 publications

References 14 publications

Molecular characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum

Molecular characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum

Purification of 1,3-propanediol dehydrogenase from Citrobacter freundii and cloning, sequencing, and overexpression of the corresponding gene in Escherichia coli

Production of 1,3-propanediol by Klebsiella pneumoniae using raw glycerol from Zygosacharomyces rouxii

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