Correction of a pathogenic gene mutation in human embryos.

Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.

Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability.

Little is known about the genetics of nonsyndromic intellectual disability (NSID). We hypothesized that de novo mutations (DNMs) in synaptic genes explain an important fraction of sporadic NSID cases. In order to investigate this possibility, we sequenced 197 genes encoding glutamate receptors and a large subset of their known interacting proteins in 95 sporadic cases of NSID. We found 11 DNMs, including ten potentially deleterious mutations (three nonsense, two splicing, one frameshift, four missense) and one neutral mutation (silent) in eight different genes. Calculation of point-substitution DNM rates per functional and neutral site showed significant excess of functional DNMs compared to neutral ones. De novo truncating and/or splicing mutations in SYNGAP1, STXBP1, and SHANK3 were found in six patients and are likely to be pathogenic. De novo missense mutations were found in KIF1A, GRIN1, CACNG2, and EPB41L1. Functional studies showed that all these missense mutations affect protein function in cell culture systems, suggesting that they may be pathogenic. Sequencing these four genes in 50 additional sporadic cases of NSID identified a second DNM in GRIN1 (c.1679_1681dup/p.Ser560dup). This mutation also affects protein function, consistent with structural predictions. None of these mutations or any other DNMs were identified in these genes in 285 healthy controls. This study highlights the importance of the glutamate receptor complexes in NSID and further supports the role of DNMs in this disorder.

Regulation of dendritic arborization by BCR Rac1 GTPase-activating protein, a substrate of PTPRT.

Dendritic arborization is important for neuronal development as well as the formation of neural circuits. Rac1 is a member of the Rho GTPase family that serve as regulators of neuronal development. Breakpoint cluster region protein (BCR) is a Rac1 GTPase-activating protein that is abundantly expressed in the central nervous system. Here, we show that BCR plays a key role in neuronal development. Dendritic arborization and actin polymerization were attenuated by overexpression of BCR in hippocampal neurons. Knockdown of BCR using specific shRNAs increased the dendritic arborization as well as actin polymerization. The number of dendrites in null mutant BCR(-/-) mice was considerably increased compared with that in wild-type mice. We found that the function of the BCR GTPase-activating domain could be modulated by protein tyrosine phosphatase receptor T (PTPRT), which is expressed principally in the brain. We demonstrate that tyrosine 177 of BCR was the main target of PTPRT and the BCR mutant mimicking dephosphorylation of tyrosine 177 alleviated the attenuation of dendritic arborization. Additionally the attenuated dendritic arborization found upon BCR overexpression was relieved upon co-expression of PTPRT. When PTPRT was knocked down by a specific shRNA, the dendritic arborization was significantly reduced. The activity of the BCR GTPase-activating domain was modulated by means of conversions between the intra- and inter-molecular interactions, which are finely regulated through the dephosphorylation of a specific tyrosine residue by PTPRT. We thus show conclusively that BCR is a novel substrate of PTPRT and that BCR is involved in the regulation of neuronal development via control of the BCR GTPase-activating domain function by PTPRT.

Structural characteristics and biological performance of silk fibroin nanofiber containing microalgae Spirulina extract.

Silk fibroin (SF) nanofiber scaffold containing microalgae Spirulina extract were prepared by electrospinning and the performance and functionality of the scaffold were evaluated. The viscosity and conductivity of the dope solution of Spirulina containing SF were examined for electrospinability and we found that the morphological structure of SF nanofiber is affected by the concentration of Spirulina extract added. The platelet adhesion and coagulation time test confirmed that the Spirulina containing SF nanofiber scaffold had excellent ability to prevent blood clotting or antithrombogenicity that is comparable to heparin. Low cytotoxicity and excellent cell adhesion and proliferation were also observed for Sprulina containing SF nanofiber scaffold by methylthiazolyldiphenyl-tetrazolium bromide assay and confocal fluorescence microscope using fibroblast and human umbilical vein endothelial cells. Based on these results, we believe SF nanofiber scaffold containing Spirulina extract has the potential to be used as tissue engineering scaffold that requires high hemocompatibility.

Dual effect of exogenous nitric oxide on neuronal excitability in rat substantia gelatinosa neurons.

Nitric oxide (NO) is an important signaling molecule involved in nociceptive transmission. It can induce analgesic and hyperalgesic effects in the central nervous system. In this study, patch-clamp recording was used to investigate the effect of NO on neuronal excitability in substantia gelatinosa (SG) neurons of the spinal cord. Different concentrations of sodium nitroprusside (SNP; NO donor) induced a dual effect on the excitability of neuronal membrane: 1 mM of SNP evoked membrane hyperpolarization and an outward current, whereas 10 µM induced depolarization of the membrane and an inward current. These effects were prevented by hemoglobin and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (c-PTIO) (NO scavengers), phenyl N-tert-butylnitrone (PBN; nonspecific reactive oxygen species scavenger), and through inhibition of soluble guanylyl cyclase (sGC). Pretreatment with n-ethylmaleimide (NEM; thiol-alkylating agent) also decreased effects of both 1 mM and 10 µM SNP, suggesting that these responses were mediated by direct S-nitrosylation. Charybdotoxin (CTX) and tetraethylammonium (TEA) (large-conductance Ca(2+)-activated K(+) channel blockers) and glybenclamide (ATP-sensitive K(+) channel blocker) decreased SNP-induced hyperpolarization. La(3+) (nonspecific cation channel blocker), but not Cs(+) (hyperpolarization-activated K(+) channel blocker), blocked SNP-induced membrane depolarization. In conclusion, NO dually affects neuronal excitability in a concentration-dependent manner via modification of various K(+) channels.

Prediction of the minimum amount of anti-adhesive agent required for entire intra-abdominal cavity using fluorescent dye.

PURPOSE: Studies on the appropriate amount of anti-adhesive agents for preventing postoperative adhesion are lacking. This animal study aimed to investigate the distribution of an anti-adhesive agent in the abdominal cavity and estimate the necessary amount to cover the entire cavity. METHODS: Fluorescent dye Flamma-552 was conjugated to Guardix-sol to create Guardix-Flamma, which was laparoscopically applied to the abdominal cavity of two 10-kg pigs in different amounts: 15 mL for G1 and 35 mL for G2. After 24 hours, the distribution of Guardix-Flamma was examined under the near-infrared mode of the laparoscope, and the thickness was measured in tissues from the omentum, small, and large intestine by immunohistochemistry. RESULTS: The average area of the abdominal cavity in 10 kg pigs was 2,755 cm2. Guardix-Flamma fluorescence was detected in the greater omentum, ascites in the pelvis, and right quadrant area in G1, whereas in G2, it was detected everywhere. On average, the total thickness of G1 and G2 were 12.68 ± 9.80 µm and 18.16 ± 15.57 µm, respectively. Guardix-Flamma thickness applied to the omentum, small, and large intestines of G2 were 1.31-, 1.45-, and 1.49-times thicker than those of G1, respectively, and were all statistically significant (P < 0.05). CONCLUSION: The entire abdominal cavity of the 10 kg pig was not evenly covered with 15 mL of Guardix. Although 35 mL of Guardix is sufficient to cover the same area with an average thickness of 18 µm, further studies should evaluate the minimum thickness required for an effective anti-adhesive function.

Metabolic engineering of Ralstonia eutropha for the biosynthesis of 2-hydroxyacid-containing polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) are bio-based and biodegradable polyesters synthesized by numerous microorganisms. PHAs containing 2-hydroxyacids as monomer units have attracted much attention, but their production has not been efficient. Here, we metabolically engineered Ralstonia eutropha strains for the in vivo synthesis of PHAs containing 2-hydroxyacids as monomers. This was accomplished by replacing the R. eutropha phaC gene in the chromosome with either the R. eutropha phaC S506G A510K gene, which contains two point mutations, or the Pseudomonas sp. MBEL 6-19 phaC1437 gene. In addition, the R. eutropha phaAB genes in the chromosome were replaced with the Clostridium propionicum pct540 gene. All of the engineered R. eutropha strains produced PHAs containing 2-hydroxyacid monomers, including lactate and 2-hydroxybutyrate (2HB), along with 3-hydroxybutyrate (3HB) and/or 3-hydroxyvalerate (3HV), when they were cultured in nitrogen-free medium containing 5 g/L lactate or 4 g/L 2HB and 20 g/L glucose as carbon sources. Expression of the Escherichia coli ldhA gene in engineered R. eutropha strains allowed production of poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] from glucose as the sole carbon source. This is the first report on the production of 2-hydroxyacid-containing PHAs by metabolically engineered R. eutropha.

Axl acts as a tumor suppressor by regulating LIGHT expression in T lymphoma.

Axl is an oncogenic receptor tyrosine kinase that plays a role in many cancers. LIGHT (Lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells) is a ligand that induces robust anti-tumor immunity by enhancing the recruitment and activation of effector immune cells at tumor sites. We observed that mouse EL4 and human Jurkat T lymphoma cells that stably overexpressed Axl also showed high expression of LIGHT. When Jurkat-Axl cells were treated with Gas6, a ligand for Axl, LIGHT expression was upregulated through activation of the PI3K/AKT signaling pathway and transcriptional induction by Sp1. The lytic activity of cytotoxic T lymphocytes and natural killer cells was enhanced by EL4-Axl cells. In addition, tumor volume and growth were markedly reduced due to enhanced apoptotic cell death in EL4-Axl tumor-bearing mice as compared to control mice. We also observed upregulated expression of CCL5 and its receptor, CCR5, and enhanced intratumoral infiltration of cytotoxic T lymphocytes and natural killer cells in EL4-Axl-bearing mice as compared to mock controls. These data strongly suggested that Axl exerts novel tumor suppressor effects by inducing upregulation of LIGHT in the tumor microenvironment of T lymphoma.

Ag Nanoparticle/Polydopamine-Coated Inverse Opals as Highly Efficient Catalytic Membranes.

Polymeric three-dimensional inverse-opal (IO) structures provide unique structural properties useful for various applications ranging from optics to separation technologies. Despite vast needs for IO functionalization to impart additional chemical properties, this task has been seriously challenged by the intrinsic limitation of polymeric porous materials that do not allow for the easy penetration of waterborne moieties or precursors. To overcome this restriction, we present a robust and straightforward method of employing a dipping-based surface modification with polydopamine (PDA) inside the IO structures, and demonstrate their application to catalytic membranes via synthetic incorporation of Ag nanoparticles. The PDA coating offers simultaneous advantages of achieving the improved hydrophilicity required for the facilitated infiltration of aqueous precursors and successful creation of nucleation sites for a reduction of growth of the Ag nanoparticles. The resulting Ag nanoparticle-incorporated IO structures are utilized as catalytic membranes for the reduction of 4-nitrophenol to its amino derivatives in the presence of NaBH4. Synergistically combined characteristics of high reactivity of Ag nanoparticles along with a greatly enhanced internal surface area of IO structures enable the implementation of remarkably improved catalytic performance, exhibiting a good conversion efficiency greater than 99% while minimizing loss in the membrane permeability.

Biosynthesis of poly(2-hydroxyisovalerate-co-lactate) by metabolically engineered Escherichia coli.

Polyhydroxyalkanoates (PHAs) containing 2-hydroxyacids such as lactate (LA) and 2-hydroxybutyrate (2HB) have recently been produced by metabolically engineered microorganisms. Here, we further expanded 2-hydroxyacid monomer spectrum of PHAs by engineering Escherichia coli to produce PHAs containing 2-hydroxyisovalerate (2HIV). To generate 2HIV in vivo, feedback resistant ilvBNmut genes encoding acetohydroxyacid synthase and ilvCD genes encoding ketol-acid reductoisomerase and dihydroxyacid dehydratase, respectively, and panE gene encoding d-2-hydroxyacid dehydrogenase are overexpressed. Also, pct540 gene encoding evolved propionyl-CoA transferase and phaC1437 gene encoding evolved PHA synthase are overexpressed along with ilvBNmut, ilvCD, and panE genes in E. coli strain for in vivo synthesis of 2HIV containing PHAs. E. coli strain expressing all of these genes can produce poly(13.2 mol% 2HIV-co-7.5 mol% 2HB-co-42.5 mol% 3HB-co-36.8 mol% LA) when it is cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 3-hydroxybutyrate (3HB). To produce PHA containing only 2HIV and LA monomers, poxB, pflB, adhE and frdB genes encoding enzymes involved in competing pathways for pyruvate are deleted so that cells can generate more 2HIV and LA. When this engineered E. coli strain expressing ilvBNmut, ilvCD, panE, pct540 and phaC1437 genes is cultured in the medium containing 20 g/L of glucose and 2 mM l-isoleucine, which can inhibit l-threonine dehydratase responsible for in vivo 2HB generation, poly(20 mol% 2HIV-co-80 mol% LA) can be produced to the polymer content of 9.6% w/w. These results suggest that novel PHAs containing 2HIV can be produced by engineering branched-chain amino acid metabolism.

Delayed allergic reaction to secondary administrated epidural hyaluronidase.

We are reporting a rare case of a delayed hypersensitivity reaction caused by hyaluronidase allergy following a lumbar transforaminal epidural block. Using an intradermal skin test, we have provided evidence that the systemic allergic reaction resulted from hypersensitivity to hyaluronidase. To our knowledge, this is a rare case of a delayed hypersensitivity reaction to epidural hyaluronidase, comprised of an initial exposure to hyaluronidase with no subsequent allergic response in prior block followed by a subsequent delayed reaction to hyaluronidase during a second epidural block.

Si/Ti2O3/Reduced Graphene Oxide Nanocomposite Anodes for Lithium-Ion Batteries with Highly Enhanced Cyclic Stability.

Silicon (Si) has attracted tremendous attention as a high-capacity anode material for next generation Li-ion batteries (LIBs); unfortunately, it suffers from poor cyclic stability due to excessive volume expansion and reduced electrical conductivity after repeated cycles. To circumvent these issues, we propose that Si can be complexed with electrically conductive Ti2O3 to significantly enhance the reversible capacity and cyclic stability of Si-based anodes. We prepared a ternary nanocomposite of Si/Ti2O3/reduced graphene oxide (rGO) using mechanical blending and subsequent thermal reduction of the Si, TiO2 nanoparticles, and rGO nanosheets. As a result, the obtained ternary nanocomposite exhibited a specific capacity of 985 mAh/g and a Coulombic efficiency of 98.4% after 100 cycles at a current density of 100 mA/g. Furthermore, these ternary nanocomposite anodes exhibited outstanding rate capability characteristics, even with an increased current density of 10 A/g. This excellent electrochemical performance can be ascribed to the improved electron and ion transport provided by the Ti2O3 phase within the Si domains and the structurally reinforced conductive framework comprised of the rGO nanosheets. Therefore, it is expected that our approach can also be applied to other anode materials to enable large reversible capacity, excellent cyclic stability, and good rate capability for high-performance LIBs.

Si-Mn/reduced graphene oxide nanocomposite anodes with enhanced capacity and stability for lithium-ion batteries.

Although Si is a promising high-capacity anode material for Li-ion batteries (LIB), it suffers from capacity fading due to excessively large volumetric changes upon Li insertion. Nanocarbon materials have been used to enhance the cyclic stability of LIB anodes, but they have an inherently low specific capacity. To address these issues, we present a novel ternary nanocomposite of Si, Mn, and reduced graphene oxide (rGO) for LIB anodes, in which the Si-Mn alloy offers high capacity characteristics and embedded rGO nanosheets confer structural stability. Si-Mn/rGO ternary nanocomposites were synthesized by mechanical complexation and subsequent thermal reduction of mixtures of Si nanoparticles, MnO2 nanorods, and rGO nanosheets. Resulting ternary nanocomposite anodes displayed a specific capacity of 600 mAh/g with ∼90% capacity retention after 50 cycles at a current density of 100 mA/g. The enhanced performance is attributed to facilitated Li-ion reactions with the MnSi alloy phase and the formation of a structurally reinforced electroconductive matrix of rGO nanosheets. The ternary nanocomposite design paradigm presented in this study can be exploited for the development of high-capacity and long-life anode materials for versatile LIB applications.

Multiscale porous interconnected nanocolander network with tunable transport properties.

A nanocolander network is developed by embedding mesoporous block copolymers inside the structural frame of a macroporous inverse-opal structure. Spontaneously formed macroconduits interconnecting the macropores are utilized as internal bypasses for enhancing the bulk transport properties. A demonstrative application for the membrane of the nanocolander network is of perfect size-selectivity for nanoparticle separation without compromising the high permeability of the transporting medium.

Synergistic anti-allodynic effect between intraperitoneal thalidomide and morphine on rat spinal nerve ligation-induced neuropathic pain.

BACKGROUND: Thalidomide has been recognized as having an anti-allodynic effect against neuropathic pain induced by spinal nerve ligation. Its clinical beneficial effects are mainly derived from its immune-modulating property, which is known to influence the analgesic action of morphine. The possible characteristics of systemic interactions between thalidomide and morphine in the context of spinal nerve ligation-induced neuropathic pain were examined in rats. METHODS: Neuropathic pain was induced by ligation of the L5/6 spinal nerves in male Sprague-Dawley rats and mechanical allodynia was assessed using von Frey filaments. The ED50 was calculated for thalidomide and for morphine, and the mixture of both drugs was intraperitoneally administered at different doses of ED50 of each drug (1/8, 1/4, 1/2, 1/1 of ED50) to obtain the experimental ED50 value for the combination of thalidomide and morphine. Isobolographic analysis was used to evaluate the characteristics of drug interactions between morphine and thalidomide. RESULTS: The ED50 of thalidomide was three-fold higher than that of morphine. The experimental ED50 value of the mixture of thalidomide and morphine was significantly lower than the calculated theoretical ED50 value. Isobolographic analysis revealed a synergistic interaction for anti-allodynic effect after intraperitoneal delivery of the thalidomide-morphine mixture. CONCLUSIONS: These results suggest that thalidomide acts synergistically with morphine to produce an anti-allodynic effect in neuropathic pain induced by spinal nerve ligation in rats. Thus, the combination of thalidomide with morphine may be one of the useful strategies in the management of neuropathic pain.

Neuronal synapse formation induced by microglia and interleukin 10.

Recently, it was found that microglia regulated synaptic remodeling of the developing brain, but their mechanisms have not been well understood. In this study, the action of microglia on neuronal synapse formation was investigated, and the primary target of microglial processes was discovered. When the developing microglia were applied to cultured hippocampal neurons without direct contact, the numbers of dendritic spines and excitatory and inhibitory synapses significantly increased. In order to find out the main factor for synaptic formation, the effects of cytokines released from microglia were examined. When recombinant proteins of cytokines were applied to neuronal culture media, interleukin 10 increased the numbers of dendritic spines in addition to excitatory and inhibitory synapses. Interestingly, without external stimuli, the amount of interleukin 10 released from the intact microglia appeared to be sufficient for the induction of synaptic formation. The neutralizing antibodies of interleukin 10 receptors attenuated the induction of the synaptic formation by microglia. The expression of interleukin 10 receptor was newly found in the hippocampal neurons of early developmental stage. When interleukin 10 receptors on the hippocampal neurons were knocked down with specific shRNA, the induction of synaptic formation by microglia and interleukin 10 disappeared. Pretreatment with lipopolysaccharide inhibited microglia from inducing synaptic formation, and interleukin 1ß antagonized the induction of synaptic formation by interleukin 10. In conclusion, the developing microglia regulated synaptic functions and neuronal development through the interactions of the interleukin 10 released from the microglia with interleukin 10 receptors expressed on the hippocampal neurons.

Single-step solvothermal synthesis of mesoporous Ag-TiO2-reduced graphene oxide ternary composites with enhanced photocatalytic activity.

With growing interest in the photocatalytic performance of TiO2-graphene composite systems, the ternary phase of TiO2, graphene, and Ag is expected to exhibit improved photocatalytic characteristics because of the improved recombination rate of photogenerated charge carriers and potential contribution of the generation of localized surface plasmon resonance at Ag sites on a surface of the TiO2-graphene binary matrix. In this work, Ag-TiO2-reduced graphene oxide ternary nanocomposites were successfully synthesized by a simple solvothermal process. In a single-step synthetic procedure, the reduction of AgNO3 and graphene oxide and the hydrolysis of titanium tetraisopropoxide were spontaneously performed in a mixed solvent system of ethylene glycol, N,N-dimethylformamide and a stoichiometric amount of water without resorting to the use of typical reducing agents. The nanocomposites were characterized by X-ray diffraction, X-ray photoelectron spectroscopy, along with different microscopic and spectroscopic techniques, enabling us to confirm the successful reduction of AgNO3 and graphite oxide to metallic Ag and reduced graphene oxide, respectively. Due to the highly facilitated electron transport of well distributed Ag nanoparticles, the synthesized ternary nanocomposite showed enhanced photocatalytic activity for degradation of rhodamine B dye under visible light irradiation.

Antinociceptive effect of intrathecal nefopam and interaction with morphine in formalin-induced pain of rats.

BACKGROUND: Nefopam, a non-opiate analgesic, has been regarded as a substance that reduces the requirement for morphine, but conflicting results have also been reported. The inhibition of monoamine reuptake is a mechanism of action for the analgesia of nefopam. The spinal cord is an important site for the action of monoamines however, the antinociceptive effect of intrathecal nefopam was not clear. This study was performed to examine the antinociceptive effect of intrathecal (i.t.) nefopam and the pattern of pharmacologic interaction with i.t. morphine in the formalin test. METHODS: Male Sprague-Dawley rats were implanted with an i.t. catheter, and were randomly treated with a vehicle, nefopam, or morphine. Formalin was injected into the hind-paw 10 min. after an i.t. injection of the above experiment drugs. After obtaining antinociceptive ED(50) of nefopam and morphine, the mixture of nefopam and morphine was tested for the antinociceptive effect in the formalin test at a dose of 1/8, 1/4, 1/2 of ED(50), or ED(50) of each drug followed by an isobolographic analysis. RESULTS: Intrathecal nefopam significantly reduced the flinching responses in both phases of the formalin test in a dose-dependent manner. Its effect, however, peaked at a dose of 30 µg in phase 1 (39.8% of control) and 10 µg during phase 2 (37.6% of control). The isobolograhic analysis indicated an additive interaction of nefopam and morphine during phase 2, and a synergy effect in antinociception during phase 1. CONCLUSIONS: This study demonstrated that i.t. nefopam produces an antinociceptive effect in formalin induced pain behavior during both phases of the formalin test, while interacting differently with i.t. morphine, synergistically during phase 1, and additively during phase 2.

Propionyl-CoA dependent biosynthesis of 2-hydroxybutyrate containing polyhydroxyalkanoates in metabolically engineered Escherichia coli.

We have previously reported in vivo biosynthesis of 2-hydroxyacid containing polyesters including polylactic acid (PLA), poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)], and poly(3-hydroxybutyrate-co-2-hydroxybutyrate-co-lactate) [P(3HB-co-2HB-co-LA)] employing metabolically engineered Escherichia coli strains by the introduction of evolved Clostridium propionicum propionyl-CoA transferase (Pct(Cp)) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1(Ps6-19)). In this study, we further engineered in vivo PLA biosynthesis system in E. coli to synthesize 2HB-containing PHA, in which propionyl-CoA was used as precursor for 2-ketobutyrate that was converted into 2HB-CoA by the sequential actions of Lactococcus lactis (D)-2-hydroxybutyrate dehydrogenase (PanE) and Pct(Cp) and then 2HB-CoA was polymerized by PhaC1(Ps6-19). The recombinant E. coli XL1-blue expressing the phaC1437 gene, the pct540 gene, and the Ralstonia eutropha prpE gene together with the panE gene could be grown to 0.66 g/L and successfully produced P(70 mol%3HB-co-18 mol%2HB-co-12 mol%LA) up to the PHA content of 66 wt% from 20 g/L of glucose, 2 g/L of 3HB and 1 g/L of sodium propionate. Removal of the prpC gene in the chromosome of E. coli XL1-blue could increase the mole fraction of 2HB in copolymer, but the PHA content was decreased. The metabolic engineering strategy reported here suggests that propionyl-CoA can be successfully used as the precursor to provide PHA synthase with 2HB-CoA for the production of PHAs containing 2HB monomer.