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

(R)-3-hydroxybutyrate [(R)-3HB] is a useful precursor in the synthesis of value-added chiral compounds such as antibiotics and vitamins. Typically, (R)-3HB has been microbially produced from sugars via modified (R)-3HB-polymer-synthesizing pathways in which acetyl CoA is converted into (R)-3-hydroxybutyryl-coenzyme A [(R)-3HB-CoA] by β-ketothiolase (PhaA) and acetoacetyl CoA reductase (PhaB). (R)-3HB-CoA is hydrolyzed into (R)-3HB by modifying enzymes or undergoes 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 ¹³C 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.     

Preparation of alkyl (R)-(-)-3-hydroxybutyrate by acidic alcoholysis of poly-(R)-(-)-3-hydroxybutyrate

An efficient method for the preparation of optically active alkyl (R)-(-)-3-hydroxybutyrates by chemical depolymerization of biopolymer, poly-(R)-(-)-(3-hydroxybutyrate), was established. This method consists of simple recovery of poly-(R)-(-)-(3-hydroxybutyrate) from bacterial cells followed by acidic alcoholysis. When poly-(R)-(-)-(3-hydroxybutyrate) was purified by a simple digestion method that used 0.2 N sodium hydroxide, alkyl (R)-(-)-hydroxybutyrates were most efficiently produced by alcoholysis with anhydrous hydrochloric acid.     

Efficient synthesis of the ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer

The ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer were prepared in a short, operationally simple synthetic sequence from racemic β-butyrolactone. Enantioselective hydrolysis of β-butyrolactone with immobilized Candida antarctica lipase-B (CAL-B) results in (R)-β-butyrolactone and (S)-β-hydroxybutyric acid, which are easily converted to (R) or (S)-ethyl-3-hydroxybutyrate and reduced to (R) or (S)-1,3 butanediol. Either enantiomer of ethyl-3-hydroxybutyrate and 1,3 butanediol are then coupled, again using CAL-B, to produce the ketone body ester product. This is an efficient, scalable, atom-economic, chromatography-free, and low cost synthetic method to produce the ketone body esters.     

A two-step enzymatic resolution process for large-scale production of (S)- and (R)-ethyl-3-hydroxybutyrate

An efficient two-step enzymatic process for production of (R)- and (S)-ethyl-3-hydroxybutyrate (HEB), two important chiral intermediates for the pharmaceutical market, was developed and scaled-up to a multikilogram scale. Both enantiomers were obtained at 99% chemical purity and over 96% enantiomeric excess, with a total process yield of 73%. The first reaction involved a solvent-free acetylation of racemic HEB with vinylacetate for the production of (S)-HEB. In the second reaction, (R)-enriched ethyl-3-acetoxybutyrate (AEB) was subjected to alcoholysis with ethanol to derive optically pure (R)-HEB. Immobilized Candida antarctica lipase B (CALB) was employed in both stages, with high productivity and selectivity. The type of butyric acid ester influenced the enantioselectivity of the enzyme. Thus, extending the ester alkyl chain from ethyl to octyl resulted in a decrease in enantiomeric excess, whereas using bulky groups such as benzyl or t-butyl, improved the enantioselectivity of the enzyme. A stirred reactor was found unsuitable for large-scale production due to attrition of the enzyme particles and, therefore, a batchwise loop reactor system was used for bench-scale production. The immobilized enzyme was confined to a column and the reactants were circulated through the enzyme bed until the targeted conversion was reached. The desired products were separated from the reaction mixture in each of the two stages by fractional distillation. The main features of the process are the exclusion of solvent (thus ensuring high process throughput), and the use of the same enzyme for both the acetylation and the alcoholysis steps. Kilogram quantities of (S)-HEB and (R)-HEB were effectively prepared using this unit, which can be easily scaled-up to produce industrial quantities.     

Poly(3-hydroxybutyrate) production by Cupriavidus necator using waste glycerol

Polyhydroxyalkanoates (PHAs) have been recognized as good substitutes for the non-biodegradable petrochemically produced polymers. However, their high (real or estimated) current production cost limits their industrial applications. This work exploits two strategies to enhance PHAs substitution potential: the increase in PHA volumetric productivity in high density cultures and the use of waste glycerol (GRP), a by-product from the biodiesel industry, as primary carbon source for cell growth and polymer synthesis. Cupriavidus necator DSM 545 was used to accumulate poly(3-hydroxybutyrate) (P(3HB)) from GRP and from commercial glycerol (PG) as control substrate. On PG, productivities between 0.6 g_PHB L^?1 h^?1 and 1.5 g_PHB L^?1 h^?1 were attained. The maximum cell DW was 82.5 g_DW L^?1, the P(3HB) content being 62%. When GRP was used, 68.8 g_DW L^?1 with a P(3HB) accumulation of 38% resulting in a final productivity of 0.84 g_PHB L^?1 h^?1 was obtained. By decreasing the biomass concentration at which accumulation was triggered, a productivity of 1.1 g_PHB L^?1 h^?1 (50% P(3HB), w/w) was attained using GRP. P(3HB) molecular weights (M_w) ranged from 7.9 × 10⁵ to 9.6 × 10⁵ Da.     

(R)-3-hydroxybutyrate dehydrogenase: selective phosphatidylcholine binding by the C-terminal domain

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme that has a specific requirement of phosphatidylcholine (PC) for function. The C-terminal domain (CTBDH) of human heart BDH (residues 195-297) has now been expressed in Escherichia coli as a chimera with a soluble protein, glutathione S-transferase (GST), yielding GST-CTBDH, a novel fusion protein that has been purified and shown to selectively bind to PC vesicles. Both recombinant human heart BDH (HH-Histag-BDH) and GST-CTBDH (but not GST) form well-defined protein-lipid complexes with either PC or phosphatidylethanolamine (PE)/diphosphatidylglycerol (DPG) vesicles (but not with digalactosyl diglyceride vesicles) as demonstrated by flotation in sucrose gradients. The protein-PC complexes are stable to 0.5 M NaCl, but complexes of either HH-Histag-BDH or GST-CTBDH with PE/DPG vesicles are dissociated by salt treatment. Thrombin cleavage of GST-CTBDH, either before or after reconstitution with PC vesicles, yields CTBDH (12 111 Da by MALDI mass spectrometry) which retains lipid binding without attached GST. The BDH activator, 1-palmitoyl-2-(1-pyrenyl)decanoyl-PC (pyrenyl-PC), at <2.5% of total phospholipid in vesicles, efficiently quenches a fraction (0.36 and 0.47, respectively) of the tryptophan fluorescence of both HH-Histag-BDH and GST-CTBDH with effective Stern-Volmer quenching constants, (K(Q))(eff), of 11 and 9.3 (%)(-)(1), respectively (half-maximal quenching at approximately 0.1% pyrenyl-PC). Maximal quenching by pyrenyl-PC obtains at approximately stoichiometric pyrenyl-PC to protein ratios, reflecting high-affinity interaction of pyrenyl-PC with both HH-Histag-BDH and GST-CTBDH. The analogous pyrenyl-PE effects a similar maximal quenching of tryptophan fluorescence for both proteins but with approximately 15-fold lower (K(Q))(eff) (half-maximal quenching at approximately 1.5% pyrenyl-PE) referable to nonspecific interaction of pyrenyl-PE with HH-Histag-BDH or GST-CTBDH. Thus, the 103-residue CTBDH constitutes a PC-selective lipid binding domain of the PC-requiring BDH.     

Transmembrane ion transport by polyphosphate/poly-(R)-3-hydroxybutyrate complexes

Transmembrane ion transport, a critical process in providing energy for cell functions, is carried out by pore-forming macromolecules capable of discriminating among very similar ions and responding to changes in membrane potential. It is widely regarded that ion channels are exclusively proteins, relatively late arrivals in cell evolution. Here we discuss the formation of ion-selective, voltage-activated channels by complexes of two simple homopolymers, namely, inorganic polyphosphates (polyPs) and poly-(R)-3-hydroxybutyrates (PHBs), derived from phosphate and acetate, respectively. Each has unique molecular characteristics that facilitate ion selection, solvation, and transport. Complexes of the two polymers, isolated from bacterial plasma membranes or prepared from the synthetic polymers, form voltage-dependent, Ca2+-selective channels in planar lipid bilayers that are selective for divalent over monovalent cations, permeant to Ca2+, Sr2+, and Ba2+, and blocked by transition metal cations in a concentration-dependent manner. Recently, both polyP and PHB have been found to be components of ion-conducting proteins: namely, the human erythrocyte Ca2+-ATPase pump and the Streptomyces lividans potassium channel. The contribution of polyP and PHB to ion selection and/or transport in these proteins is yet unknown, but their presence gives rise to the hypothesis that these and other ion transporters are supramolecular structures in which proteins, polyP, and PHB cooperate in forming well-regulated and specific cation transfer systems.     

Isolated poly(3-hydroxybutyrate) (PHB) granules are complex bacterial organelles catalyzing formation of PHB from acetyl coenzyme A (CoA) and degradation of PHB to acetyl-CoA

Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eutropha catalyzed formation of PHB from ¹⁴C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (protein-free) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD⁺, indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentrations of key metabolites (CoA, acetyl-CoA, 3HB-CoA, NAD⁺/NADH) determine whether a cell accumulates or degrades PHB. Since the degradation product of PHB is 3HB-CoA, the cells do not waste energy by synthesis and degradation of PHB. Thus, our results explain the frequent finding of simultaneous synthesis and breakdown of PHB.     

Synthesis of ethyl (R)-4-chloro-3-hydroxybutyrate by immobilized cells using amino acid-modified magnetic nanoparticles

Fe₃O₄-Arg was selected as the optimal carrier due to its high activity recovery of immobilized cells in the preparation of Fe₃O₄-Arg-Cells. The optimal immobilization conditions for the preparation of Fe₃O₄-Arg-Cells were 30 °C, 4 h, pH 7, and 3 g dry yeast. The activity recovery of immobilized cells reached 76.8 %. For a batch reduction in a shaker in an alternating magnetic field, Fe₃O₄-Arg-Cells were used as a catalyst to gain ethyl (R)-4-chloro-3-hydroxybutyrate ((R)-CHBE). For further improvement in reduction productivity, a continuous reduction in the magnetic fluidized bed reactor system (MFBRS) was completed. Under their optimal transformation conditions, it took 24 h for Fe₃O₄-Arg-Cells to complete the conversion of ethyl 4-chloro-3-oxobutanoate (COBE) (0.8553 mol/L) in the shaker and only 8 h for the batch reduction in an alternating magnetic field. Continuous reduction in MFBRS provided new ideas for the efficient production of (R)-CHBE; 1.5882 mol/L (10 mL) of COBE can be completely converted in 6 h. The conversion and enantiomeric excess (e.e.) of (R)-CHBE were 100 % and above 99.9 % respectively, in the three reaction systems mentioned above.     

The role of acetyl coenzyme A: butyrate coenzyme A in the transferase uptake of butyrate by isolated membrane vesicles of Escherichia coli

The acetyl CoA:butyrate CoA transferase catalyzes the translocation of butyrate in membrane vesicles prepared from a strain of Escherichia coli which is depressed for the acetoacetate degradation operon. Butyrate accumulated in the membranes as butyryl CoA. The role of the transferase in uptake is supported by the following observations: (i) uptake is stimulated by acetyl CoA; (ii) the solubilized CoA transferase and uptake exhibit K_mS for butyrate, pH optima and levels inhibition by N-ethylmaleimide that are virtually identical; (iii) significant amounts of the CoA transferase are found associated with the membranes and uptake is rapidly inhibited by butyryl CoA and acetate, the products of the CoA transferase-catalyzed reaction. The fact that butyrate uptake did not exhibit saturation kinetics with increasing concentrations of acetyl CoA suggested that the transferase is not localized on the outer surface of the membrane. The level of free butyrate in the vesicles, the fact that butyrate uptake exhibited saturation kinetics with increasing concentrations of butyrate, and the observation that radioactivity was not rapidly lost from the vesicles following addition of butyryl CoA or acetate to incubation mixtures indicated that butyrate is translocated rather than trapped by the CoA transferase.     

Identification of an acetoacetyl coenzyme A synthetase-dependent pathway for utilization of L-(+)-3-hydroxybutyrate in Sinorhizobium meliloti

D-(-)-3-Hydroxybutyrate (DHB), the immediate depolymerization product of the intracellular carbon store poly-3-hydroxybutyrate (PHB), is oxidized by the enzyme 3-hydroxybutyrate dehydrogenase to acetoacetate (AA) in the PHB degradation pathway. Externally supplied DHB can serve as a sole source of carbon and energy to support the growth of Sinorhizobium meliloti. In contrast, wild-type S. meliloti is not able to utilize the L-(+) isomer of 3-hydroxybutyrate (LHB) as a sole source of carbon and energy. In this study, we show that overexpression of the S. meliloti acsA2 gene, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase, confers LHB utilization ability, and this is accompanied by novel LHB-CoA synthetase activity. Kinetics studies with the purified AcsA2 protein confirmed its ability to utilize both AA and LHB as substrates and showed that the affinity of the enzyme for LHB was clearly lower than that for AA. These results thus provide direct evidence for the LHB-CoA synthetase activity of the AcsA2 protein and demonstrate that the LHB utilization pathway in S. meliloti is AcsA2 dependent.     

Poly(3-hydroxybutyrate) production from whey using recombinant Escherichia coli

Recombinant Escherichia colistrains harboring the genes from Alcaligenes eutrophusfor polyhydroxyalkanoate biosyn-thesis were constructed and compared for their ability to synthesize poly(3-hydroxybutyrate) in a defined medium with whey as the sole carbon source. The highest PHB concentration and PHB content obtained were 5.2 g/L and 81% of dry cell weight, respectively.     

Sorting signal of Escherichia coli OmpA is modified by oligo-(R)-3-hydroxybutyrate

Escherichia coli outer membrane protein A (OmpA) is a well-established model for the study of membrane assembly. Previous studies have shown that the essential sequence for outer membrane localization, known as the sorting signal, is contained in a segment of the eighth beta-strand, residues 163-171. Sequential digestion of OmpA, purified from outer membranes or inclusion bodies with cyanogen bromide and Staphylococcus aureus GluC, yielded peptides 162-174(LSLGVSYRFGQGE). Western blot and chemical assays indicated that the peptide was covalently modified by oligo-(R)-3-hydroxybutyrate (cOHB), a flexible, amphipathic oligoester. MALDI/MS was consistent with modification of peptides 162-174 by up to ten R-3-hydroxybutyrate (HB) residues. Western blot analysis of mutants of the peptide, using anti-OHB IgG, indicated that cOHB modification was not inhibited by the single mutations S163G, S167G, Y168F, R169N or R169D; however, cOHB was not detected on peptides containing the double mutations S163G:S167G S163G:V166G, L162G:S167G, and L164G:S167G. MALDI/MS/MS of double mutant S163G:S167G confirmed the absence of cOHB-modification. The results suggest that cOHB may be attached to one or both serines, and point to the importance of the flanking hydrophobic residues. Modification by cOHB may play a role in outer membrane targeting and assembly of OmpA.     

Efficient production of (R)-3-hydroxycarboxylic acids by biotechnological conversion of polyhydroxyalkanoates and their purification

An efficient method to prepare enantiomerically pure (R)-3-hydroxycarboxylic acids from bacterial polyhydroxyalkanoates (PHAs) accumulated by Pseudomonas putida GPo1 is reported in this study. (R)-3-Hydroxycarboxylic acids from whole cells were obtained when conditions were provided to promote in vivo depolymerization of intracellular PHA. The monomers were secreted into the extracellular environment. They were separated and purified by acidic precipitation, preparative reversed-phase column chromatography, and subsequent solvent extraction. Eight (R)-3-hydroxycarboxylic acids were isolated: (R)-3-hydroxyoctanoic acid, (R)-3-hydroxyhexanoic acid, (R)-3-hydroxy-10-undecenoic acid, (R)-3-hydroxy-8-nonenoic acid, (R)-3-hydroxy-6-heptenoic acid, (R)-3-hydroxyundecanoic acid, (R)-3-hydroxynonanoic acid, and (R)-3-hydroxyheptanoic acid. The overall yield based on released monomers was around 78 wt % for (R)-3-hydroxyoctanoic acid. All obtained monomers had a purity of over 95 wt %. The physical properties of the purified monomers and their antimicrobial activities were also investigated.     

Characterization of (R)-2-hydroxyisocaproate dehydrogenase and a family III coenzyme A transferase involved in reduction of L-leucine to isocaproate by Clostridium difficile

The strictly anaerobic pathogenic bacterium Clostridium difficile occurs in the human gut and is able to thrive from fermentation of leucine. Thereby the amino acid is both oxidized to isovalerate plus CO(2) and reduced to isocaproate. In the reductive branch of this pathway, the dehydration of (R)-2-hydroxyisocaproyl-coenzyme A (CoA) to (E)-2-isocaprenoyl-CoA is probably catalyzed via radical intermediates. The dehydratase requires activation by an ATP-dependent one-electron transfer (J. Kim, D. Darley, and W. Buckel, FEBS J. 272:550-561, 2005). Prior to the dehydration, a dehydrogenase and a CoA transferase are supposed to be involved in the formation of (R)-2-hydroxyisocaproyl-CoA. Deduced amino acid sequences of ldhA and hadA from the genome of C. difficile showed high identities to d-lactate dehydrogenase and family III CoA transferase, respectively. Both putative genes encoding the dehydrogenase and CoA transferase were cloned and overexpressed in Escherichia coli; the recombinant Strep tag II fusion proteins were purified to homogeneity and characterized. The substrate specificity of the monomeric LdhA (36.5 kDa) indicated that 2-oxoisocaproate (K(m) = 68 muM, k(cat) = 31 s(-1)) and NADH were the native substrates. For the reverse reaction, the enzyme accepted (R)- but not (S)-2-hydroxyisocaproate and therefore was named (R)-2-hydroxyisocaproate dehydrogenase. HadA showed CoA transferase activity with (R)-2-hydroxyisocaproyl-CoA as a donor and isocaproate or (E)-2-isocaprenoate as an acceptor. By site-directed mutagenesis, the conserved D171 was identified as an essential catalytic residue probably involved in the formation of a mixed anhydride with the acyl group of the thioester substrate. However, neither hydroxylamine nor sodium borohydride, both of which are inactivators of the CoA transferase, modified this residue. The dehydrogenase and the CoA transferase fit well into the proposed pathway of leucine reduction to isocaproate.     

Poly(3-hydroxybutyrate) production from xylose by recombinant Escherichia coli

Several recombinant Escherichia coli strains harboring the Alcaligenes eutrophus polyhydroxyalkanoate biosynthesis genes were used to produce poly(3-hydroxybutyrate), PHB, from xylose. By flask culture of TG1 (pSYL107) in a defined medium containing 20?g/l xylose, PHB concentration of 1.7?g/l was obtained. Supplementation of a small amount of cotton seed hydrolysate or soybean hydrolysate could enhance PHB production by more than two fold. The PHB concentration, PHB content, and PHB yield on xylose obtained by supplementing soybean hydrolysate were 4.4?g/l, 73.9%, and 0.226?g PHB/g xylose, respectively.     

Inhibition of d(-)-3-hydroxybutyrate dehydrogenase by malonate analoges

(1) d(-)-3-Hydroxybutyrate dehydrogenase activity from guinea pig, rat, and bovine heart and from guinea pig liver is inhibited by malonate and tartronate, and more potently by the analogs methylmalonate, bromomalonate, chloromalonate, and mesoxalate. Little or no inhibitory effect was found for aminomalonate, ethylmalonate, dimethylmalonate, succinate, glutarate, oxaloacetate, malate, propionate, pyruvate, d- and l-lactate, n-butyrate, isobutyrate, and cyclopropanecarboxylate. (2) In initial velocity kinetics at pH 8.1 with a soluble enzyme preparation from bovine heart, the inhibition by the active malonate derivatives is competitive with respect to 3-hydroxybutyrate and uncompetitive with respect to acetoacetate, NAD⁺ or NADH. With d-3-hydroxybutyrate as the variable reactant (K_{m app} = 0.26 mM) the inhibition constant of methylmalonate (K_is) was 0.09 mm. (3) The rate of utilization of d-3-hydroxybutyrate (78 μm) by coupled rat heart mitochondria in the presence of ADP was inhibited 50% by 150 μm methylmalonate. (4) With coupled guinea pig liver mitochondria oxidizing n-octanoate in the absence of added ADP, methylmalonate (1–3 mm) depressed 3-hydroxybutyrate formation substantially more than total ketone production. However, the intramitochondrial NADH (or NADPH) levels were unchanged by the addition of methylmalonate, indicating that the changes in ratios of accumulated 3-hydroxybutyrate and acetoacetate were caused by direct inhibition of 3-hydroxybutyrate dehydrogenase. Methylmalonate had the same effect on 3-hydroxybutyrate/acetoacetate ratios and ketone body formation with pyruvate or acetate as the source of acetyl groups. Similar results were obtained with malonate (10 mm) although the inhibition of total ketone formation from octanoate was more severe.