Fermentative production of (R)-(−)-3-hydroxybutyrate using 3-hydroxybutyrate dehydrogenase null mutant of Ralstonia eutropha and recombinant Escherichia coli

Shiraki, Mari; Endo, Takakazu; Saito, Terumi

doi:10.1263/jbb.102.529

Cited by 39 publications

(33 citation statements)

References 19 publications

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“…Because the ratio of the glucose-derived (R)-3HB was approximately 70%, the carbon yield from glucose to (R)-3HB was estimated to be 61%. To further increase the concentration of (R)-3HB in the medium, a strategy of feeding glucose and acetate using a jar-fermentor would be effective, based on the previous studies (Liu et al 2007, Shiraki et al 2006.…”

Section: Discussionmentioning

confidence: 99%

“…The PhaZ pathway has reportedly achieved up to 7.3 g L -1 (R)-3HB production in a shake flask culture (Shiraki et al 2006). This method has an advantage over (R)-3HB synthesis by the acidic hydrolysis of P(3HB) that generates acid derivatives (de Roo et al 2002, Shiraki et al 2006. However, the PhaZ pathway requires a two step fermentation, accumulation of the P(3HB) polymer and also its degradation.…”

Section: Introductionmentioning

confidence: 99%

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Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase

Matsumoto

Okei

Honma

et al. 2012

Appl Microbiol Biotechnol

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(R)-3HB-CoA is hydrolyzed into (R)-3HB by modifying enzymes or undergoes 2 degradation of the polymerized product. In the present study, we constructed a new (R)-3HB-generating pathway from glucose by using propionyl-CoA transferase (PCT).This pathway was designed to excrete (R)-3HB by means of a PCT-catalyzed reaction coupled with regeneration of acetyl-CoA, the starting substance for synthesizing (R)-3HB-CoA. Considering the equilibrium reaction of PCT, the PCT-catalyzed (R)-3HB production would be expected to be facilitated by the addition of acetate since it acts as an acceptor of CoA. As expected, the engineered Escherichia coli harboring the phaAB and pct genes produced 1.0 g L -1 (R)-3HB from glucose, and with the addition of acetate into the medium, the concentration was increased up to 5.2 g L -1 , with a productivity of 0.22 g L -1 h -1 . The effectiveness of the extracellularly added acetate was evaluated by monitoring the conversion of 13 C carbonyl carbon labeled acetate into (R)-3HB using gas chromatography/mass spectrometry. The enantiopurity of (R)-3HB was determined to be 99.2% using chiral liquid chromatography. These results demonstrate that the PCT pathway achieved a rapid co-conversion of glucose and acetate into (R)-3HB.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase

Matsumoto

Okei

Honma

et al. 2012

Appl Microbiol Biotechnol

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“…The importance of the proteinaceous PHB surface layer to the susceptibility of PHB granules for in vitro hydrolysis has been described for a PHB depolymerase purified from Rhodospirillum rubrum (9-11, 26, 27). The intracellular expression of the PHB depolymerase PhaZ7 of Paucimonas lemoignei, an extracellular PHB depolymerase with unusual specificity for proteinaceous and amorphous nPHB granules (7), in a PHBaccumulating background resulted in an increase of secreted 3HB (37). Therefore, the high depolymerase activity of cautiously isolated nPHB granules could be indicative of a physiological function of the respective protein.…”

Section: Discussionmentioning

confidence: 99%

Poly(3-Hydroxybutyrate) (PHB) Depolymerase PhaZa1 Is Involved in Mobilization of Accumulated PHB in Ralstonia eutropha H16

Uchino

Saito

Jendrossek

2008

Appl Environ Microbiol

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The recently finished genome sequence of Ralstonia eutropha H16 harbors nine genes that are thought to encode functions for intracellular depolymerization (mobilization) of storage poly(3-hydroxybutyrate) (PHB). Based on amino acid similarities, the gene products belong to four classes (PhaZa1 to PhaZa5, PhaZb, PhaZc, and PhaZd1/PhaZd2). However, convincing direct evidence for the in vivo roles of the gene products is poor. In this study, we selected four candidate genes (phaZa1, phaZb, phaZc, and phaZd1) representing the four classes and investigated the physiological function of the gene products (i) with recombinant Escherichia coli strains and (ii) with R. eutropha null mutants. Evidence for weak but significant PHB depolymerase activity was obtained only for PhaZa1. The physiological roles of the other potential PHB depolymerases remain uncertain.Polyhydroxyalkanoates (PHA) are typical storage compounds of carbon and energy and are found widely in prokaryotes. The most common PHA is poly(3-hydroxybutyrate) (PHB), and this polymer can accumulate up to 90% of the cellular dry weight of some bacteria. PHA are thermoplasts, and despite their relatively high production costs, biologically produced PHA have entered the industrial market.PHA can be degraded extracellularly by many types of bacteria that are able to secrete specific extracellular PHA depolymerases into the environment or by the intracellular mobilization of PHA in the accumulating strain itself. Considerable knowledge of the biochemical properties of the respective extracellular PHA depolymerases has accumulated (14,17,18). Intracellular mobilization of PHA differs from extracellular degradation because of the differences between the biophysical conformations of extracellular (denatured) PHA and those of intracellular (native) PHA. (For definitions of the terms "denatured" and "native PHA," see references 15, 25, and 26.) Several groups have reported on the identification of potential intracellular PHB depolymerases (1,5,8,19,20,33,40,43; for an overview see references 15, 17, and 34). These data, together with the genome sequence of Ralstonia eutropha, suggested that R. eutropha H16 might have as many as nine PHB depolymerases/oligomer hydrolases (29). Five putative PHB depolymerase isoenzymes (PhaZa1 to PhaZa5), two 3-hydroxybutyric acid (3HB) oligomer hydrolases (PhaZb and PhaZc [PhaY1 and PhaY2]), and two isoenzymes of a recently found new type of putative intracellular PHB depolymerase (PhaZd1 and PhaZd2) have been described (1). The last two have amino acid sequences that are significantly similar to those of the catalytic domain of extracellular PHB depolymerases (16, 36). We were surprised to find evidence for so many PHB depolymerases in one organism, and we wondered whether all of these proteins were physiologically important for intracellular PHB mobilization. Clarification of this point appeared necessary because independent evidence for the function of the above-mentioned proteins such as the physiological PHB depolymerases exists only...

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“…In contrast, enzyme-catalyzed reactions are highly stereoselective and can be performed in aqueous solutions under mild conditions (21). As a result, the use of biological processes for chiral molecule production has been extensively investigated (4,28,32,36). One example of such a process is the biosynthesis of 3-hydroxybutyric acid (3HB), a versatile chiral molecule containing one hydroxyl group and one carboxyl group, used as a building block for the synthesis of optically active fine chemicals, such as vitamins, antibiotics, pheromones, and flavor compounds (5,6,18,27).…”

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confidence: 99%

Metabolic Engineering of Escherichia coli for Enhanced Production of ( R )- and ( S )-3-Hydroxybutyrate

Tseng¹,

Martin

Nielsen³

et al. 2009

Appl Environ Microbiol

108

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Synthetic metabolic pathways have been constructed for the production of enantiopure (R)-and (S)-3-hydroxybutyrate (3HB) from glucose in recombinant Escherichia coli strains. To promote maximal activity, we profiled three thiolase homologs (BktB, Thl, and PhaA) and two coenzyme A (CoA) removal mechanisms (Ptb-Buk and TesB). Two enantioselective 3HB-CoA dehydrogenases, PhaB, producing the (R)-enantiomer, and Hbd, producing the (S)-enantiomer, were utilized to control the 3HB chirality across two E. coli backgrounds, BL21Star(DE3) and MG1655(DE3), representing E. coli B-and K-12-derived strains, respectively. MG1655(DE3) was found to be superior for the production of each 3HB stereoisomer, although the recombinant enzymes exhibited lower in vitro specific activities than BL21Star(DE3). Hbd in vitro activity was significantly higher than PhaB activity in both strains. The engineered strains achieved titers of enantiopure (R)-3HB and (S)-3HB as high as 2.92 g liter ؊1 and 2.08 g liter ؊1 , respectively, in shake flask cultures within 2 days. The NADPH/NADP ؉ ratio was found to be two-to three-fold higher than the NADH/NAD ؉ ratio under the culture conditions examined, presumably affecting in vivo activities of PhaB and Hbd and resulting in greater production of (R)-3HB than (S)-3HB. To the best of our knowledge, this study reports the highest (S)-3HB titer achieved in shake flask E. coli cultures to date.

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Fermentative production of (R)-(−)-3-hydroxybutyrate using 3-hydroxybutyrate dehydrogenase null mutant of Ralstonia eutropha and recombinant Escherichia coli

Cited by 39 publications

References 19 publications

Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase

Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase

Poly(3-Hydroxybutyrate) (PHB) Depolymerase PhaZa1 Is Involved in Mobilization of Accumulated PHB in Ralstonia eutropha H16

Metabolic Engineering of Escherichia coli for Enhanced Production of ( R )- and ( S )-3-Hydroxybutyrate

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