Dataset Information

Application of a propionyl coenzyme A synthetase for poly(3-hydroxypropionate-co-3-hydroxybutyrate) accumulation in recombinant Escherichia coli.

ABSTRACT: The genetic operon for propionic acid degradation in Salmonella enterica serovar Typhimurium contains an open reading frame designated prpE which encodes a propionyl coenzyme A (propionyl-CoA) synthetase (A. R. Horswill and J. C. Escalante-Semerena, Microbiology 145:1381-1388, 1999). In this paper we report the cloning of prpE by PCR, its overexpression in Escherichia coli, and the substrate specificity of the enzyme. When propionate was utilized as the substrate for PrpE, a K(m) of 50 microM and a specific activity of 120 micromol. min(-1). mg(-1) were found at the saturating substrate concentration. PrpE also activated acetate, 3-hydroxypropionate (3HP), and butyrate to their corresponding coenzyme A esters but did so much less efficiently than propionate. When prpE was coexpressed with the polyhydroxyalkanoate (PHA) biosynthetic genes from Ralstonia eutropha in recombinant E. coli, a PHA copolymer containing 3HP units accumulated when 3HP was supplied with the growth medium. To compare the utility of acyl-CoA synthetases to that of an acyl-CoA transferase for PHA production, PHA-producing recombinant strains were constructed to coexpress the PHA biosynthetic genes with prpE, with acoE (an acetyl-CoA synthetase gene from R. eutropha [H. Priefert and A. Steinbüchel, J. Bacteriol. 174:6590-6599, 1992]), or with orfZ (an acetyl-CoA:4-hydroxybutyrate-CoA transferase gene from Clostridium propionicum [H. E. Valentin, S. Reiser, and K. J. Gruys, Biotechnol. Bioeng. 67:291-299, 2000]). Of the three enzymes, PrpE and OrfZ enabled similar levels of 3HP incorporation into PHA, whereas AcoE was significantly less effective in this capacity.

SUBMITTER: Valentin HE

PROVIDER: S-EPMC92453 | biostudies-literature | 2000 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Application of a propionyl coenzyme A synthetase for poly(3-hydroxypropionate-co-3-hydroxybutyrate) accumulation in recombinant Escherichia coli.

Valentin H E HE Mitsky T A TA Mahadeo D A DA Tran M M Gruys K J KJ

Applied and environmental microbiology 20001201 12

The genetic operon for propionic acid degradation in Salmonella enterica serovar Typhimurium contains an open reading frame designated prpE which encodes a propionyl coenzyme A (propionyl-CoA) synthetase (A. R. Horswill and J. C. Escalante-Semerena, Microbiology 145:1381-1388, 1999). In this paper we report the cloning of prpE by PCR, its overexpression in Escherichia coli, and the substrate specificity of the enzyme. When propionate was utilized as the substrate for PrpE, a K(m) of 50 microM an ...[more]

PMID: 11097899

Similar Datasets

Project description:The model organism for polyhydroxybutyrate (PHB) biosynthesis, Ralstonia eutropha H16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinant Escherichia coli BL21(DE3) strains were used to study the impact of selected PHB depolymerases of R. eutropha H16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinant E. coli BL21(DE3) strains were constructed, which harbored a plasmid carrying the phaCAB operon from R. eutropha H16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase from R. eutropha H16. It is shown in this study that the growth behavior of the respective recombinant E. coli strains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboring phaZ7 reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed if phaZ1 was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay of these enzymes.

Project description:Biosynthesis of poly-3-hydroxybutyrate (PHB) as a fermentation product enables the coupling of growth and product generation. Moreover, the reduction of oxygen supply should reduce operative cost and increase product yield. Generation of PHB as a fermentation product depends on the in vivo activity of an NADH-preferring acetoacetyl-CoA reductase. Proof of this concept requires (i) quantification of the cofactor preference, in physiologically relevant conditions, of a putative NADH-preferring acetoacetyl-CoA reductase and (ii) verification of PHB accumulation using an NADH-preferring acetoacetyl-CoA reductase in a species naturally incapable of doing so, for example, Escherichia coli. This dataset contains kinetic data obtained by spectrophotometry and data from a continuous culture of an engineered E. coli strain accumulating PHB under oxygen-limiting conditions. In this dataset it is possible to find (1) enzyme stability assays; (2) initial rates and progress curves from reactions catalyzed by two acetoacetyl-CoA reductases; (3) estimations of the relative use of NADH and NADPH by two acetoacetyl-CoA reductases; (4) estimations of the flux capacity of the reaction catalyzed by an acetoacetyl-CoA reductase; (5) biomass composition of an engineered E. coli strain transformed with a plasmid; (6) calculation of reconciled specific rates of this engineered strain growing on sucrose as the sole carbon source under oxygen limitation and (7) metabolic fluxes distributions during the continuous growth of this engineered strain. Because a relatively small number of acetoacetyl-CoA reductases have been kinetically characterized, data and scripts here provided could be useful for further kinetic characterizations. Moreover, the procedure described to estimate biomass composition could be interesting to estimate plasmid and protein burden in other strains. Application of data reconciliation to fermentations should help to obtain specific rates consistent with the principle of mass and electron conservation. All the required data and scripts to perform these analyses are deposited in a Mendeley Data repository. This article was co-submitted with the manuscript entitled "An NADH preferring acetoacetyl-CoA reductase is engaged in poly-3-hydroxybutyrate accumulation in Escherichiasia. coli".

Dataset Information

Application of a propionyl coenzyme A synthetase for poly(3-hydroxypropionate-co-3-hydroxybutyrate) accumulation in recombinant Escherichia coli.

Publications

Application of a propionyl coenzyme A synthetase for poly(3-hydroxypropionate-co-3-hydroxybutyrate) accumulation in recombinant Escherichia coli.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets