Microsoft Word - cet-01.docx CHEMICAL ENGINEERING TRANSACTIONS VOL. 46, 2015 A publication of The Italian Association of Chemical Engineering Online at www.aidic.it/cet Guest Editors: Peiyu Ren, Yancang Li, Huiping Song Copyright © 2015, AIDIC Servizi S.r.l., ISBN978-88-95608-37-2; ISSN 2283-9216 Production and Cytophysiology Applications of 3- hydroxyalkanoic Acid Monomers Xianghui Zou*a, Lina W ua, Yunying W ua, Guangcai Zhaa, Yuanyou Lib a Hanshan Normal University, Dongshan Road, Xiangqiao District, Chaozhou City, Guangdong Province, China b Shantou University, Shantou, Guangdong, China zxh11043@126.com Polyhydroxyalkanoates (PHAs) have been developed for use as polymeric materials to make bioplastics, fine chemicals, implant biomaterials, medicine, and biofuels. PHAs are produced by microorganisms grown under restricted conditions and they are comprised of structurally diverse monomers. Various 3-hydroxyalkanoic acids (3-HA) can be prepared by chemical synthesis, as well as by depolymerizing PHAs and microbial metabolic engineering. PHAs derived from 3-HA monomers are compatible with alcohol and carboxylic acids due to their easily modified hydroxyl and carboxyl functional groups. Therefore, an important application for 3 - HA monomers is as a starting material for fine chemical synthesis, such as production of antibiotics, vitamins, aromatics and pheromones. 3-HA also has different physiological uses such and as such can be used for drug manufacture. Here, we focus on the production of 3-HA monomers, their relationship with the promotion of cell proliferation inhibition of apoptosis and suppression of oxidative and nitrosative stress. These discussions will provide a foundation for understanding the properties of 3-HA to optimize their applications in cytophysiology. 1. Introduction Polyhydroxyalkanoates (PHAs) are microbial stored polyesters consisting of more than 150 types of 3-HA monomers. Common forms of 3-HA monomers are 3-hydroxybutyrate (3-HB), 3-hydroxyvalerate (3-HV), 3- hydroxyhexanoate (3-HHx), 3-hydroxyoctanoate (3-HO), 3-hydroxydecanoate (3-HD), and 3- hydroxydodecanoate (3-HDD) (Chen and Wu 2005 a). 3-HHx, 3-HO, 3-HD, and 3-HDD monomers are of medium chain length PHA (Zhang et al. 2009) and 3-HB is a short-chain PHA and a degradation product of PHB as well as the chief plasma and peripheral tissue ketone body in mammals, produced h epatically by long chain fatty acids (Massieu et al. 2003). Previously, diabetic ketoacidosis was considered to be a worrisome biomarker, especially when they reached 25 mM, as this depleted blood bicarbonate and led to hypovolemia due to urinary water, sodium, and potassium excretion from ketonuria (Gerhardt 1865). However, mild ketosis is now known to be therapeutic in various diseases (Chen and Wu 2005 b, Henderson 2008). In fact, 3-HB can be antimicrobial, insecticidal, and antiviral under certain conditions (Shiraki et al. 2006). Here we summarize the production and cytophysiology applications of 3-HA monomers, emphasizing 3-HB with respect to thepromotion of cell proliferation and inhibition of apoptosis and suppression of oxidative and nitrosative stress. 2. Production of 3-hydroxybutyric acid monomers 2.1 Chemical synthesis (S)-3-hydroxytetradecanoic acid can be synthesized from (S)-epichlorohydrin (27% yield) as follows: (1) region- and chemo-selective epoxide opening of (S)-epichlorohydrin with a Grignard reagent under catalysis by Cu(I) followed by consecutive epoxide formation; (2) regioselective epoxide opening of (S)-1,2- epoxytridecane with cyanide anion under pH-controlled conditions followed by consecutive nitrile hydrolysis with alkaline H2O2 (to yield crude (S)-3-hydroxytetradecanoic acid); and (3) purification with N,N- dicyclohexylammonium salt (Matsuyama and Ikunaka 1999). Optically pure R and S enantiomers of 3 - hydroxytetradecanoic acid and its methyl esters were synthesized by porcine pancreas lipase-catalyzed hydrolysis of racemic methyl 3-hydroxytetradecanoate in an aqueous medium (Kücük and Yusufoğlu 2013). A DOI: 10.3303/CET1546006 Please cite this article as: Zou X.H., Wu L.N., Wu Y.Y., Zha G.C., Li Y.Y., 2015, Production and cytophysiology applications of 3- hydroxyalkanoic acid monomers, Chemical Engineering Transactions, 46, 31-36 DOI:10.3303/CET1546006 31 versatile route for modular synthesis of 3-HA had been described (Jaipuri et al. 2004) and within this method, steps included a microwave-assisted catalytic transfer hydrogenation and a facile microwave-assisted hydrolysis of N-methoxy-N-methyl (Weinreb) amide, which enhanced the practicality of this protecting group for carboxylic acids. Moreover, efficient synthesis of (R)-3-hydroxytetradecanoic acid, a key component of bacterial endotoxins, included (R)-oxirane acetic acid ethyl ester as chiral source (Huang and Hollingsworth 1998). Various β-hydroxyalkanoic acid derivatives were prepared via palladium catalyzed aerobic oxidative- carbonylations of terminal olefins under normal carbon monoxide and oxygen mixed gas pressure (Urata 1988). Production of optically active 3-HB is possible by chemical synthesis but this is complex, expensive and inefficient and few reports describe chemosynthesis of optically pure 3-HB. 2.2 Production of 3-HB via depolymerization of PHA An efficient technique for producing pure 3-HA and 3-HA methyl esters is acidic or basic hydrolysis of PHA isolated via solvent recovery and hydrolyzed by acid methanolysis. The obtained 3-HA methylester mixture is then distilled into several fractions (overall yield of 96.6% w/w) (De Roo et al. 2002). Abiotic hydrolysis of PHB has been investigated in acid and base media using native amorphous granules, precipitates and solvent-cast films of PHB matrix as raw materials, and the formation of two monomeric hydrolytic products, 3 -HB and crotonic acid (CA) were depicted. Monomeric products were not released from PHB specimens in acidic solutions (0.1–4 N H+), but were measured as the major hydrolytic products from alkaline hydrolysis (0.1–4 N OH−) (Yu et al. 2005). 3-Hydroxybutyrate methyl ester (HBME) was prepared from hydrolysis of bacterial PHB using methanol as an esterification agent in the presence of sulfuric acid (Wang et al. 2010). Another efficient method for pure 3-HA production is in vitro and in vivo enzymatic hydrolysis of PHA. PHB is degraded by various specific hydrolytic enzymes from microorganisms that can be broadly classified as intracellular and extracellular depolymerases (Calabia and Tokiwa 2004). An example is thermophilic Streptomyces sp. MG which has strong hydrolytic activity for depolymerization of PHB to produce 3 -HB monomer. An advantage of this strain is high-temperature (50°C) stability which minimizes contamination not only during fermentation, but also during enzymatic degradation of PHB (Tokiwa and Ugwu 2007). 3 -HB can be produced at a 96% yield in 30 min by in vivodepolymerization of PHB when cells have high intracellular PHA depolymerase activity and low (R)-(−)-3-hydroxybutyric acid dehydrogenase activity under suitable environmental conditions (Lee et al. 1999). Similar approaches have been used by other researchers to produce PHA monomers with microbial intracellular depolymerase (Saito and Saegusa 1994). 2.3 Microbial metabolic engineering More efficient systems for 3-HA production have been developed by emplying PHA-producing bacteria and recombinant E. coli. To produce 3-HA, pathways of PHA production and degradation must be exploited. Taking PHB as an example, metabolic pathways and possible options for 3-HA production are shown in Fig. 1. Figure 1: Metabolic pathway for microbial production of 3-HA from carbohydrates. phbA, phbB, phbC, phbZ, ptb, buk, AACS, BDH, phaG, and tesB represent genes of -ketothiolase, acetoacetyl-CoA reductase, PHB polymerase, PHB depolymerase, phosphotransbutyrylase, butyrate kinase, acetoacetyl -CoA synthetase, 3- hydroxybutyrate dehydrogenase, (R)-3-hydroxydecanoyl-ACP: CoA transacylase, and thioesterase II, respectively. Recently, a metabolic pathway for production and in vivo hydrolysis of PHB to release 3-HB in culture supernatant was investigated (Shirakiet al. 2006). High yields of 3-HB can be produced in PHA-producing bacteria by providing environmental conditions for high intracellular depolymeraseactivity (Lee et al. 1999). However, a significant drawback with this procedure is that the depolymerized products could be further cellularly metabolized to acetoacetate by 3-HB dehydrogenase (BDH). To overcome this obstacle, metabolically engineered E. coli strains (without the BDH gene) harboring heterologous PHA synthesis and 32 degradation pathways were established (Lee and Lee 2003). W. eutropha PHA biosynthesis genes were integrated into E. coli chromosomes to disrupte the pta (phosphotrans-acetylase) gene. This stable recombinant E. coli strain was constructed and when the W. Eutropha intracellular depolymerase gene was expressed, 3-HB was efficiently produced (Park et al. 2004). New pathways for enhanced 3-HB production were constructed by simultaneous expression of genes for phbA, phbB, ptb, buk in E. coli DH5α (Liu and Steinbüchel 2000 a and b, Gao et al. 2002). With an isogenic tesB-negative knockout strain, E. coli CH01, expression of tesB and phaG were reported to up-regulate one other. In addition, 3-HD was synthesized from glucose or fructose by recombinant E. coli harboringphaG and tesB(Zheng et al. 2004 a). This study supports the hypothesis that the physiologic al role of tesB in E. coli is to prevent abnormal accumulation of intracellular acyl-CoA (Zheng et al. 2004 b). Recombinant P. putida harboring tesB also allowed the production of extracellular 3-HD from carbohydrates (Zheng et al. 2004 c). Zhao and the colleagues reported that addition of acrylic acid significantly increased production of 3 -HB and mcl-3-HA consisting of 3-HB and 3-HD at a ratio of 1: 3 (Zhao et al. 2003). E. coli BW25113 (pSPB01) harboring phbA, phbB, and tesB genes produced approximately 4 g/L 3-HB in shake flask culture within 24 h with a glucose carbon source. Under anaerobic growth conditions, 3-HB production was more effective and 0.47 g 3-HB/g glucose was produced compared with 0.32 g 3-HB/g glucose obtained from an aerobic process (Liu et al. 2007). Researchers have invested effort into identifying engineered organisms produce 3 -HA. For example, engineered cyanobacteria (PCC 6803) photosynthetically produced (S)- and (R)-3-HB directly from sunlight and CO2. Both 3-HB molecule types were produced and readily secreted from Synechocystis cells without transporter over-expression. Additional inactivation of the competing pathway by deleting slr1829 and slr1830 (encoding PHB polymerase) from the Synechocystis genome also promoted 3-HB production (Wang et al. 2013). 3. Promotion of cell proliferation and inhibition of apoptosis To investigate cytotoxicity of oligo (3-hydroxyalkanoic acids; OHAs) to mouse fibroblast L929 cells, liposomes were employed to encapsulate OHAs and facilitate cytosolic transfer (Sun et al. 2007). OHAs (less than 20 mg/L) did not significantly affect cell viability, whereas OHAs exceeding 40 mg/L reduced cell viability as evidenced by apoptosis, cell cycle delays and reduced cell proliferation. Cytotoxicity decreased with increasing OHA side-chain length and the 3-HB monomer, a degradation product released from biopolymer PHA, is speculated to contribute to tissue regeneration. 3-HB stimulated cell cycle progression mediated by a calcium-dependent signaling pathway (Cheng et al. 2005) and 0.005–0.10 g/L 3-HB promoted cell proliferation of murine fibroblast L929 cells, human umbilical vein endothelial cells, and rabbit articular cartilage. In L929 cells, 0.02 g/L 3-HB stimulated a rapid increase cytosolic calcium and 3-HB promoted proliferation of L929 cells in high-density (1×105 cells/well) cultures (but not low density cultures) by preventing apoptotic and necrotic cell death induced by serum withdrawal (Cheng et al. 2006). Effects of 3-HB on murine osteoblast MC3T3-E1 cell differentiation in vitro and on anti-osteoporosis in vivo were evaluated as well (Zhao et al. 2007). The intensity of in vitro cell differentiation directly proportional to the concentration of 3-HB when it was less than 0.01 g/L. Calcium deposition, a strong indication of cell differentiation, also increased with increasing 3-HB concentration (from 0 to 0.1 g/L). RT-PCR indicated higher expression of osteocalcin (OCN) mRNA in MC3T3-E1 cells after 3-HB administration. In vivo work indicated that 3-HB enhanced femur maximal load and bone deformation resistance and improved trabecular bone volume. Animal experiments suggest that 3-HB increased serum ALP activity and calcium deposition, decreased serum OCN, and prevented bone mineral density reductions in ovariectomized animals. In vitro effects of 3-HB and 3-HBME on cell apoptosis and cytosolic Ca2+ in mouse glial cells were evaluated and 3-HB derivatives were found to inhibit cell apoptosis mediated by signaling pathways related to cytosolic Ca2+elevation (Xiao et al. 2007). Cells undergoing apoptosis decreased significantly in the presence of 3 -HB and 3-HBME and 3-HB derivatives dramatically elevated cytosolic Ca2+. The effect of 3-HBME on cytosolic Ca2+ was reduced by nifedipine, an L-type voltage-dependent calcium channel antagonist. In comparison, 3- HBME permeated cells better than D-3-HB and DL-3-HB. Next, 3-HB, 3-HBME and 3-HBEE (3- hydroxybutyrate ethyl ester) were evaluated for their ability to stimulate metabolic activity of neuroglial cells (Zou et al. 2009). After 1–3 days in culture, 3-HB (0.5–10 mg/L) stimulated neuroglial cell metabolism significantly more than controls and maximal stimulation was observed after three days of treatment with 5 mg/L 3-HBEE, and after two days of treatment with 5 mg/L 3-HB or 3-HBME. 4. Suppression of oxidative and nitrosative stress Oxidative stress is involved in neuron apoptosis, specifically via damage by reactive oxygen species (ROS) such as H2O2, superoxide, and free radicals. Oxidative stress is implicated in neurodegenerative diseases and cerebral ischemic injury. 3-HB was studied (Cheng et al. 2013) in PC12 cells exposed to different 33 concentrations of H2O2 over different periods after 3-HB pretreatment and 3-HB was shown to slow loss of cell viability induced by H2O2. Also 3-HB decreased apoptosis induced by H2O2. Changes in intracellular ROS, total glutathione (GSH), mitochondrial membrane potential (MMP) and caspase-3 activity due to H2O2 exposure were partially reversed in PC12 cells with 3-HB. Thus, 3-HB inhibited oxidative stress in PC12 cells. 3-HB is an endogenous and specific inhibitor of class I histone deacetylases (HDACs). Inhibition of HDAC by 3-HB was correlated with global changes in transcription, including that of genes encoding oxidative stress resistance factors FOXO3A and MT2 (Shimazu et al. 2013). Treatment of cells with 3 -HB increased histone acetylation at the Foxo3a and Mt2 promoters and both genes were activated by selective depletion of HDAC1 and HDAC2. Data show that treatment of mice with 3-HB was accompanied by increased FOXO3A and MT2 activity, and that this conferred substantial protection against oxidative stress. The in vitro effect of normal (0.01 to 1 mM) and subketotic (1 to 2.5 mM) doses of 3-HB on respiratory burst activity of bovine polymorphonuclear leucocytes (PMNL) was studied with chemiluminescence (CL) (Hoeben et al. 1997). In a cell-free assay, consisting of sonicated cells and H2O2, no activity changes were observed. Myeloperoxidase activity was not significantly altered as shown by an ortho-dianisidine-oxidation assay. Also, O2- production was not affected by different doses of 3-HB which did not scavenge hypochlorite. Subketotic concentrations of 3-HB significantly inhibited CL, and decreased production of H2O2. This inhibitory effect on respiratory burst activity in PMNL suggests that elevated 3-HB after parturition in high yielding cows may be partly responsible for greater susceptibility to local and systemic infections during the postpartum period and during subclinical and clinical ketosis (Klucinski et al. 1988). Recent work indicates that 3-HB and acetoacetate (1 mM each) decreased cell death in acutely dissociated rat neocortical neurons subjected to 10 μM glutamate excitotoxicity (Maalouf et al. 2007). These compounds also prevented changes in neuronal membrane properties induced by glutamate. Ketones significantly decreased mitochondrial production of ROS and associated excitotoxic changes by increasing NADH oxidation in the mitochondrial respiratory chain. However, neither compound changed endogenous antioxidant GSH. Data show that ketones reduce glutamate-induced free radical formation by increasing the NAD+/NADH ratio and enhancing mitochondrial respiration in oxidatively stressed neocortical neurons. Other studies confirm that 3-HB prevented hippocampal neuron death after exposure to Aβ1–42, and protected cultured mesencephalic dopaminergic neurons from toxic effects of 1-methyl-4-phenylpyridinium (MPP+, an inhibitor of NADH dehydrogenase that increases oxygen radical formation), and reduced brain injury in rodents (Suzuki et al. 2002). 3-HB and Vitamin E significantly reduced striatal lesions and lipid peroxidation, suggesting that glycolytic impairment favors a pro-oxidant condition, and that oxidative damage is an important mediator of in vivo induced excitotoxicity. Data indicate a neuroprotective potential for 3-HB against in vivo excitotoxic oxidative damage (Mejía-Toiber et al. 1979). Ketone bodies can oxidize coenzyme Q and a major source of mitochondrial free radicals is the half -reduced semiquinone of coenzyme Q (Chance et al. 1979). Q semiquinone reacts directly with O2 to form superoxide radical O2-. By decreasing reduced coenzyme Q forms, mitochondrial production of free radicals is decreased. Next, ketone body metabolism can reduce mitochondrial NAD and cytoplasmic free NADP, favoring GSH reduction, which is in near equilibrium through the action of GSH reductase (Krebs and Veech 1969). This then favors destruction of H2O2 by GSH peroxidase reactions (Veech et al. 2001). Mitochondrial dysfunction leading to increased ROS is associated with neurodegenerative disorders. Rotenone, a mitochondrial stressor, induces caspase-9 and -3 activation and leads to proteolytic cleavage of substrate nuclear poly (ADP-ribose) polymerase (PARP). Cleavage of PARP is directly related to apoptotic cell death (Kabiraj et al. 2012). Na-D-β-hydroxybutyrate (NaβHB) markedly reduces the incidence of synphilin- 1 (a rotenone-induced parkinsonin-onset biomarker) aggregation. Furthermore, a metabolic byproduct of NaβHB also prevents rotenone-induced caspase-activated apoptotic cell death in dopaminergic SH-SY5Y cells. These data suggest that NaβHB is neuroprotective, attenuates effects originating from mitochondrial insult, and can serve as a scaffold for the design and development of s poradic neuropathies. Acknowledgments This work was supported by the Fund of the Natural Science Foundation of Guangdong Province (S2013010013433), the Science and Technological Innovation Projects of Department of Education of Guangdong Province (2013KJCX0126), the Food Processing and Safety Control Engineering Technology Development Center of East Guangdong (GCZX-A1415) and the Open Fund of Guangdong Provincial Key Laboratory of Marine Biotechnology (GPKLMB201402). References Calabia BP, Tokiwa Y (2004). Microbial degradation of poly (d-3-hydroxybutyrate) by a new thermophilic Streptomyces isolate. Biotechnol Lett 26: 15–19, DOI: 10.1023/B:BILE.0000009453.81444.51 34 Chance B, Sies H, Boveris A (1979) Hydroperoxide metabolism in mammalian organs. Physiol Rev 59: 527– 605. Chen GQ, W u Q (2005 a). Microbial production and applications of chiral hydroxyalkanoates. Appl Microbiol Biotechnol 67: 592–599, DOI: 10.1007/s00253-005-1917-2 Chen GQ, Wu Q (2005 b) The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials 26:6565–6578, DOI: 10.1016/j.biomaterials.2005.04.036. Cheng B, Lu H, Bai B, Chen J (2013). d-β-Hydroxybutyrate inhibited the apoptosis of PC12 cells induced by H2O2 via inhibiting oxidative stress. Neurochem Int 62:620–625, DOI: 10.1016/j.neuint.2012.09.011 Cheng S, Wu Q, Yang F, Xu M, Leski M, Chen GQ (2005). Influence of DL-beta-hydroxybutyric acid on cell proliferation and calcium influx. Biomacromolecules 6: 593–597. Cheng S, Chen GQ, Leski M, Zou B, Wang Y, W u Q (2006). The effect of D,L-beta-hydroxybutyric acid on cell death and proliferation in L929 cells. Biomaterials 27: 3758–3765. De Roo G, Kellerhals MB, Ren Q, Witholt B, Kessler B (2002). Production of chiral R-3-hydroxyalkanoic acids and R-3-hydroxyalkanoic acid methylesters via hydrolytic degradation of polyhydroxyalkanoate synthesized by Pseudomonads. BiotechnolBioeng 77: 717–722. Gao HJ, Wu Q, Chen GQ (2002). Enhanced production of d-(−)-3-hydroxybutyric acid by recombinant Escherichia coli. FEMS microbiol Lett 213: 59–65. Gerhardt J (1865). Diabetes mellitus und aceton. Wiener Medizinische Presse 6: 672. Henderson ST (2008). Ketone bodies as a therapeutic for Alzheimer’s disease. Neurotherapeutics 5: 470–80. Hoeben D, Heyneman R, Burvenich C (1997). Elevated levels of beta-hydroxybutyric acid in periparturient cows and in vitro effect on respiratory burst activity of bovine neutrophils. Vet Immunol Immunopathol 58: 165–170. Huang G, Hollingsworth RI (1998). An efficient synthesis of (R)-hydroxytetradecanoic acid. Tetrahedron Asymmetr 9:4113–4115, DOI: 10.1016/S0957-4166(98)00441-8 Jaipuri FA, Jofre MF, Schwarz KA, Pohl NL (2004). Microwave assisted cleavage of Weinreb amide for carboxylate protection in the synthesis of a (R)-3-hydroxyalkanoic acid. Tetrahedron Lett 45: 4149–4152, DOI: 10.1016/j.tetlet.2004.03.148 Kabiraj P, Pal R, Varela-Ramirez A, Miranda M, Narayan M (2012). Nitrosative stress mediated misfolded protein aggregation mitigated by Na-D-β-hydroxybutyrate intervention. Biochem Biophys Res Commun 426: 438–444, DOI: 10.1016/j.bbrc.2012.08.121 Klucinski W, Degorski A, Miemik-Degorska E, Targowski S, Winnicka A (1988). Effect of ketone bodies on the phagocytic activity of bovine milk macrophages and polymorphonuclear leukocytes. J Vet Med 35: 632– 639. Krebs HA, Veech RL (1969). Pyridine nucleotide interrelations in Papa S, Tager JM, Quagliariello E, Slate EC eds. The Energy and Metabolic Control in Mitochondria. Bari Adriatica Editrice pp: 329–382. Kücük HB, Yusufoğlu A (2013). Enantioselective synthesis of 3-hydroxytetradecanoic acid and its methyl ester enantiomers as new antioxidants and enzyme inhibitors. MonatsheftefürChemie - Chemical Monthly 144: 1087–1091. Lee SY, Lee Y (2003). Metabolic engineering of Escherichia coli for production of enantiomerically pure (R)- (−)-hydroxycarboxylic acids. Appl Environ Microbiol 69: 3421–3426, DOI: 10.1128/AEM.69.6.3421- 3426.2003 Lee SY, Lee Y, W ang F (1999). Chiral compounds from bacterial polyesters: sugars to plastics to fine chemicals. Biotechnol Bioeng 65: 363–368, DOI: 10.1002/(SICI)1097-0290(19991105)65: 3%3C363::AID- BIT15%3E3.0.CO;2-1 Liu Q, Ouyang SP, Chung A, Wu Q, Chen GQ (2007). Microbial production of R-3-hydroxybutyric acid by recombinant E. coli harboring genes of phbA,phbB, and tesB. Appl Microbiol Biotechnol 76: 811–818, DOI: 10.1007/s00253-007-1063-0 Liu SJ, Steinbüchel A (2000). A novel genetically engineered pathway for synthesis of poly (hydroxyalkanoic acids) in Escherichia coli. Appl Environ Microbiol 66: 739–743. Maalouf M, Sullivan PG, Davis L, Kim DY, Rho JM (2007). Ketones inhibit mitochondrial production of reactive oxygen species production following glutamate excitotoxicity by increasing NADH oxidation. Neuroscience 145: 256–264. Massieu L, Haces ML, Montiel T, Hernandez-Fonseca K (2003). Acetoacetate protects hippocampal neurons against glutamatemediated neuronal damage during glycolysis inhibition. Neuroscience 120: 365–378. Matsuyama K, Ikunaka M (1999). A practical enantioselective synthesis of (S)-3-hydroxytetradecanoic acid. Tetrahedron: Asymmetry 10:2945–2950, DOI: 10.1016/S0957-4166(99)00290-6 Mejía-Toiber J, Montiel T, Massieu L (2006). D-beta-hydroxybutyrate prevents glutamate-mediated lipoperoxidation and neuronal damage elicitedduring glycolysis inhibition in vivo. Neurochem Res 31: 1399–1408. 35 Park SJ, Lee SY, Lee Y (2004). Biosynthesis of (R)-3-hydroxyalkanoic acids by metabolically engineered Escherichia coli.ApplBiochemBiotechnol 113: 373–379, DOI: 10.1385/ABAB: 114:1-3:373 Saito M, Saegusa H (1994). PHA intracellular degrading enzyme. Japanese patent 6,086,681A2. Shimazu T, Hirschey MD, Newman J, He W, Shirakawa K, Le Moan N, Grueter CA, Lim H, Saunders LR, Stevens RD, Newgard CB, Farese RV Jr, de Cabo R, Ulrich S, Akassoglou K, Verdin E (2013). Suppression of oxidative stress by β-hydroxybutyrate, an endogenous histone deacetylase inhibitor. Science 339:211–214, DOI: 10.1126/science.1227166 Shiraki M, Endo T, Saito T (2006). Fermentative production of (R)-(−)-3-hydroxybutyrate using 3- hydroxybutyrate dehydrogenase null mutant of Ralstoniaeutrophaand recombinant Escherichia coli. J Biosci Bioeng 102: 529–534. Sun J, Dai Z, Zhao Y, Chen GQ (2007). In vitro effect of oligo-hydroxyalkanoates on the growth of mouse fibroblast cell line L929. Biomaterials 28: 3896–3903, DOI: j.biomaterials.2007.05.011 Suzuki M, Kitamura Y, Mori S, Sato K, Dohi S, Sato T, Matsuura A, Hiraide A (2002). Beta-hydroxybutyrate, a cerebral function improving agent, protects rat brain against ischemic damage caused by permanent and transient focal cerebral ischemia. Jpn J Pharmacol 89: 36–43. Tokiwa Y, Ugwu CU (2007). Biotechnological production of (R)-3-hydroxybutyric acid monomer. J Biotechnol 132: 264–272, DOI: 10.1016/j.jbiotec.2007.03.015 Urata H, Fujita A, Fuchikami T (1988). Aerobic oxidative-carbonylation of olefins. A convenient preparation of β-hydroxyalkanoic acid derivatives from olefins Original Research Article. Tetrahedron Letters 1988: 4435– 4436. Veech RL, Chance B, Kashiwaya Y, Lardy HA, Cahill GF Jr (2001). Ketone bodies, potential therapeutic uses. IUBMB Life 51: 241–247. Wang B, Pugh S, Nielsen DR, Zhang W , Meldrum DR (2013). Engineering cyanobacteria for photosynthetic production of 3-hydroxybutyrate directly from CO2. Metab Eng 16C: 68–77, DOI: 10.1016/j.ymben.2013.01.001 Wang SY, Wang Z, Liu MM, Xu Y, Zhang XJ, Chen GQ (2010). Properties of a new gasoline oxygenate blend component: 3-Hydroxybutyrate methyl ester produced from bacterial poly-3-hydroxybutyrate. Biomass and bioenergy 34: 1216–1222, DOI: 10.1016/j.biombioe.2010.03.020 Xiao XQ, Zhao Y, Chen GQ (2007). The effect of 3-hydroxybutyrate and its derivatives on the growth of glial cells. Biomaterials 28: 3608–3616, DOI: 10.1016/j.biomaterials.2007.04.046 Yu J, Plackett D, Chen XL (2005). Kinetics and mechanism of the monomeric products from abiotic hydrolysis of poly [(R)-3-hydroxybutyrate] under acidic and alkaline conditions. Polymer Degradation and Stability 89: 289–299, DOI: 10.1016/j.polymdegradstab.2004.12.026 Zhang XJ, Luo RC, Wang Z, Deng Y, Chen GQ (2009). Application of (R)-3-hydroxyalkanoate methyl esters derived from microbial polyhydroxyalkanoates as novel biofuels. Biomacromolecules 10: 707–711, DOI: 10.1021/bm801424e Zhao K, Tian G, Zheng Z, Chen JC, Chen GQ (2003). Production of D-(−)-3-hydroxyalkanoic acid by recombinant Escherichia coli. FEMS Microbiol Lett 218: 59–64, DOI: 10.1016/S0378-1097(02)01108-4 Zhao Y, Zou B, Shi Z, W u Q, Chen GQ (2007). The effect of 3-hydroxybutyrate on the in vitro differentiation of murine osteoblast MC3T3-E1 and invivo bone formation in ovariectomized rats. Biomaterials 28: 3063– 3073. Zheng Z, Zhang MJ, Zhang G, Chen GQ (2004 a). Production of 3-hydroxydecanoic acid by recombinant Escherichia coli HB101 harboring phaG gene.Antonie Van Leeuwenhoek 85: 93–101, DOI: 10.1023/B:ANTO.0000020275.23140.ca Zheng Z, Gong Q, Liu T, Deng Y, Chen JC, Chen GQ (2004 b). Thioesterase II of Escherichia coil plays an important role in 3-hydroxydecanoic acid production. Appl Environ Microbiol 70: 3807–3813. Zheng Z, Gong Q, Chen GQ (2004 c). A novel method for production of 3-hydroxydecanoic acid by recombinant Escherichia coli and Pseudomonas putida. Chin J Chem Eng 12: 550–555. Zou XH, Li HM, Wang S, Leski M, Yao YC, Yang XD, Huang QJ, Chen GQ (2009). The effect of 3- hydroxybutyrate methyl ester on learning and memory in mice. Biomaterials 30: 1532–1541. 36