• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.6
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• pp.371-374
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• 2002

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)［P(3HB/V)］, by fed-batch culture of recombinant Escherichia coli harboring a plasmid containing the Alcaligenes latus polyhy-droxyalkanoate (PHA) biosynthesis genes, was examined in two pilot-scale fermentors with air supply only, In a 30 L fermentor having a XLa value of 0.11 S­$^1$, the final P(3HB/V) concentration and the P(3HB/V) content obtained were 29.6 g/L and 70.1 wt%, respectively giving a productivity of 1.37 g P(3HB/V)/L-h. In a 300 L fermentor having a XLa of 0.03 S­$^1$, the P(3HB/V) concentration and the P(3HB/V) content were 20.4 g/L and 69 wt%, respectively giving a productivity of 1.06g P(3HB/V)/L-h. These results suggest that economical production of P(3HB/V) is possible by fed-batch culture of recombinant E. coli in a large-scale fermentor having low KLa value.

• Journal of Microbiology and Biotechnology
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• v.32 no.1
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• pp.110-116
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• 2022

Polyhydroxyalkanoates (PHAs) are emerging as alternatives to plastics by replacing fossil fuels with renewable raw substrates. Herein, we present the construction of engineered Escherichia coli strains to produce short-chain-length PHAs (scl-PHAs), including the monomers 4-hydroxyvalerate (4HV) and 3-hydroxyvalerate (3HV) produced from levulinic acid (LA). First, an E. coli strain expressing genes (lvaEDABC) from the LA metabolic pathway of Pseudomonas putida KT2440 was constructed to generate 4HV-CoA and 3HV-CoA. Second, both PhaAB enzymes from Cupriavidus necator H16 were expressed to supply 3-hydroxybutyrate (3HB)-CoA from acetyl-CoA. Finally, PHA synthase (PhaC_Cv) from Chromobacterium violaceum was introduced for the subsequent polymerization of these three monomers. The resulting E. coli strains produced four PHAs (w/w% of dry cell weight): 9.1 wt% P(4HV), 1.7 wt% P(3HV-co-4HV), 24.2 wt% P(3HB-co-4HV), and 35.6 wt% P(3HB-co-3HV-co-4HV).

• Journal of Microbiology and Biotechnology
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• v.3 no.2
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• pp.123-128
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• 1993

The polyesters (polyhydroxyalkanoates; PHAs) production capability in a two-step cultivation of Alcaligenes eutrophus H16(ATCC 17699) was investigated by using various organic carbon sources. The carbon sources used included linear $C_2~C_10$ monocarboxylic acids, $C_3~C_10$ dicarboxylic acids, crotonic acid, and several linear vicinal and $\omega$-diols. The polyesters synthesized were characterized by 500 MHz $^1 H-NMR$ spectroscopy, intrinsic viscosity$[\eta]$ measurement in chloroform and differential scanning calorimetry (DSC). The PHAs synthesis data showed that the use of C-odd ($C_3, C_5, and C_7$) monocarboxylic acids resulted in poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(P(3HB-co-3HV) (3HV content ranging 40 to 70 mol%) while the use of $C_9$ substrate gave the copolyester containing only 4 mol% of 3HV. All culture products obtained on $C_3$~C$_{10}$ dicarboxylic acids gave exclusively P(3HB). 500 MHz $^1 H-NMR$ analysis showed that all polyesters synthesized generally contained 1~2 mol% 3HV even for the unrelated substrates such as the carboxylic acids with even number of carbon. When $\alpha, \omega$-diols with even number of carbon were used as substrates, 4-hydroxybutyrate(4HB) was inserted into the polyester chain composed of P(3HB-co-4HB). Vicinal diols were generally not utilized by the bacterium for polyester production.n.

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.127-132
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• 2014

This study was carried out to investigate the effects of tissue inhibitor of matalloproteinase-1 (TIMP-1), Activin A and Heparin binding epidermal growth factor (HB-EGF) on in vitro production of bovine embryos. In experiment 1, presumptive zygotes were cultured in the medium supplemented with TIMP-1 ($0.5{\mu}g/ml$), Activin A (100 ng/ml), or HB-EGF (100 ng/ml) at $39^{\circ}C$ in a humidified atmosphere of 5% (v/v) $CO_2$, 5% (v/v) $O_2$ and 90% (v/v) $N_2$. In experiment 2, TIMP-1 + HB-EGF or Activin A + HB-EGF combinations were supplemented in the culture medium. The developmental rate to blastocysts, hatching rate and total cell numbers of the blastocysts were evaluated in both experiments. The embryos cultured in medium without growth factor supplementation was used as control group. In experiment 1, the embryos cultured in medium supplemented with TIMP-1 and Activin A showed significantly higher developmental rate to blastocysts than those cultured with HB-EGF and control (36.9%, 34.1%, 21.2% and 23.1%, respectively) (P<0.0001). However, the hatching rate of blastocyst was significantly higher in embryos with HB-EGF than those with TIMP-1, Actvin A and Control groups (84.4%, 58.8%, 51.4% and 49.3%, respectively) (P<0.001). Total cell number per blastocyst was also significantly higher in embryos with HB-EGF group ($174.3{\pm}2.5$) than those with TIMP-1, Activin A (149.7 and 150.0, respectively) (P<0.05) and Control (119.0) (P<0.001). In experiment 2, embryos cultured with combined treatment of Activin A and HB-EGF resulted in significantly higher rates of blastocysts formation (48.0%), hatching rate (89.7%) and total cell number in blastocyst ($182.3{\pm}2.1$) than those with TIMP-1 and HB-EGF combination group (32.0%, P<0.001; 76.6%, P<0.05; $165.7{\pm}4.2$, P<0.001, respectively). Our data demonstrate that in vitro production of bovine embryos could be improved by combined supplementation of Activin A and HB-EGF in culture medium.

• KSBB Journal
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• v.31 no.1
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• pp.27-32
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• 2016

Several CoA transferases from Clostridium beijerinckii, C. perfringens and Klebsiella pneumoniae were examined for biosynthesis of lactate-containing polyhydroxyalkanoates (PHAs) in recombinant Escherichia coli XL1-Blue strain. The CB3819 gene and the CB4543 gene from C. beijerinckii, the pct gene from C. perfringens and the pct gene from K. pneumoniae, which encodes putative CoA transferase gene, respectively, was co-expressed with the Pseudomonas sp. MBEL 6-19 phaC1437 gene encoding engineered Pseudomonas sp. MBEL 6-19 PHA synthase 1 ($PhaC1_{Ps6-19}$) to examine its activity for the construction of key metabolic pathway to produce poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)]. The recombinant E. coli XL1-Blue expressing the phaC1437 gene and CB3819 gene synthesized poly(3-hydroxybutyrate) [P(3HB)] homopolymer to the P(3HB) content of 60.5 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 3-hydroxybutyrate. Expression of the phaC1437 gene and CB4543 gene in recombinant E. coli XL1-Blue also produced P(3HB) homopolymer to the P(3HB) content of 51.2 wt% in the same culture condition. Expression of the phaC1437 gene and the K. pneumoniae pct gene in recombinant E. coli XL1-Blue could not result in the production of PHAs in the same culture condition. However, the recombinant E. coli XL1-Blue expressing the phaC1437 gene and the C. perfringens gene could produce poly(3-hydroxybutyrate-co-lactate [P(86.4mol%3HB-co-13.7 mol%LA) up to the PHA content of 10.6 wt% in the same culture condition. Newly examined CoA transfereases in this study may be useful for the construction of engineered E. coli strains to produce PHA containing novel monomer such lactate.

• Nutritional Sciences
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• v.2 no.2
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• pp.88-92
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• 1999

The purpose of this study was to study the intake of heavy metals such af arsenic, lead and cobalt and the relationship of dietary heavy metals with serum iron, topper, and zinc, which play important roles in hematopoiesis, in healthy college women living in Choongnam Korea, where we have detected heavy metals (As, Pb, Co) in some marine products in previous studies. The nutritional status of the subjects (35 women) was evaluated by anthropometric measurements, 24-hr dietary recall for 3 days. And 3-day diets (by weighing method) and blood were collected to analyze As, Pb, Co, Fe, Cu, Zn, Hb, Hct, and MCHC. The mean age, height, weight, and BMI were 20 years, 158 cm, 55 kg and 22.42 kg/$m^2$, respectively. The mean daily energy intake was 85.85% of RDA for Koreans. The ratio of energy from carbohydrate, protein, and fat was 60 : 24 : 16. The mean daily intake of heavy metals (As, Pb, Co) was 1.77 mg/day, 75.21 $\mu$g/day and 21.12 $\mu$g/day. And the mean daily intake of iron, copper, and Zinc concentrations were 97, 68, and 92% of normal values. The mean serum heavy metals (As, Pb, Co) were 16.14 $\mu$g/dl, 4.32 $\mu\textrm{g}$/dl and 0.02 $\mu$g/dr, respectively Mean blood levels of Fe, Cu, Zn, Hb, Hct, and MCHC were at normal levels. Dietary heavy metals except Co were not significantly different from serum Fe, Cu, Zn and Hb, Hct, and MCHC. However, there was a tendency toward lower serum concentration of Fe, Hb, Hct, and MCHC in the subjects with higher heavy metals (As) intake. Among heavy metals, only dietary Co showed a significant negative correlation with Hb (p< 0.001) and Hct (p < 0.001).

• KSBB Journal
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• v.29 no.6
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• pp.443-447
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• 2014

Biosynthesis of lactate-containing polyhydroxyalkanoates (PHAs) was examined in recombinant Escherichia coli W strain from sucrose. The Pseudomonas sp. MBEL 6-19 phaC1437 gene and the Clostridium propionicum pct540 gene, which encode engineered Pseudomonas sp. MBEL 6-19 PHA synthase 1 ($PhaC1_{Ps6-19}$) and engineered C. propionicum propionyl-CoA transferase ($Pct_{Cp}$), respectively, were expressed in E. coli W to construct key metabolic pathway to produce poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)]. The recombinant E. coli W expressing the phaC1437 gene and the pct540 gene could synthesize P(3HB-co-13mol%LA) up to the polymer content of 31.3 wt% when it was cultured in chemically defined MR medium containing 20 g/L of sucrose and 2 g/L of sodium 3-hydroxybutyrate. When Ralstonia eutropha phaAB genes were additionally expressed to provide 3-hydroxybutyrate-CoA (3HB-CoA) from sucrose, P(3HB-co-16mol%LA) could be synthesized from sucrose as a sole carbon source without supplement of sodium 3-hydroxybutyrate in culture medium, but the PHA content was decreased to 12.2 wt%. The molecular weight of P(3HB-co-16 mol%LA) synthesized in E. coli W using sucrose as carbon source were $1.53{\times}10^4$ ($M_n$) and $2.78{\times}10^4$ ($M_w$), respectively, which are not different from those that have previously been reported by other recombinant E. coli strains. Engineered E. coli strains developed in this study should be useful for the production of lactate-containing PHAs from sucrose, one of the most abundant and least expensive carbon sources.

• Macromolecular Research
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• v.14 no.4
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• pp.408-415
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• 2006

Temperature-dependent, wide-angle, x-ray diffraction (WAXD) patterns and infrared (IR) spectra were measured for biodegradable poly(3-hydroxybutyrate) (PHB) and its copolymers, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) P(HB-co-HHx) (HHx=2.5, 3.4, 10.5, and 12 mol%), in order to explore their crystal and lamellar structure and their pattern of C-H...O=C hydrogen bonding. The WAXD patterns showed that the P(HB-co-HHx) copolymers have the same orthorhombic system as PHB. It was found from the temperature-dependent WAXD measurements of PHB and P(HB-co-HHx) that the a lattice parameter is more enlarged than the b lattice parameter during heating and that only the a lattice parameter shows reversibility during both heating and cooling processes. These observations suggest that an interaction occurs along the a axis in PHB and P(HB-co-HHx). This interaction seems to be due to an intermolecular C-H...O=C hydrogen bonding between the C=O group in one helical structure and the $CH_3$ group in the other helical structure. The x-ray crystallographic data of PHB showed that the distance between the O atom of the C=O group in one helical structure and the H atom of one of the three C-H bonds of the $CH_3$ group in the other helix structure is $2.63{\AA}$, which is significantly shorter than the sum of the van der Waals separation ($2.72{\AA}$). This result and the appearance of the $CH_3$ asymmetric stretching band at $3009 cm^{-1}$ suggest that there is a C-H...O=C hydrogen bond between the C=O group and the $CH_3$ group in PHB and P(HB-co-HHx). The temperature-dependent WAXD and IR measurements revealed that the crystallinity of P(HB-co-HHx) (HHx =10.5 and 12 mol%) decreases gradually from a fairly low temperature, while that of PHB and P(HB-co-HHx) (HHx = 2.5 and 3.5 mol%) remains almost unchanged until just below their melting temperatures. It was also shown from our studies that the weakening of the C-H...O = C interaction starts from just above room temperature and proceeds gradually increasing temperature. It seems that the C-H...O=C hydrogen bonding stabilizes the chain holding in the lamellar structure and affects the thermal behaviour of PHB and its copolymers.

• Korean journal of applied entomology
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• v.40 no.1
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• pp.69-76
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• 2001

The entomopathogenic nematodes, Steinernema carpocapsae Pocheon strain (ScP) and Heterorhabditis bacteriophora Hamyang strain (HbH) were evaluated against chestnut insect pests, The farmers＇handling methods of chestnuts were taken into consideration to develop practical biological control with entomopathogenic nematodes . The major insect pests found with chestnuts were Curculio sikkimensis, Seichocrocis punctiferalis, and Cydia kurokoi. Although individual chestnut contained one species of insect was 58% representing 18% by C. sikkimensis, 27.7% by D. punctiferalis and 12.3% by C. kurokoi. The percentage of co-infection of C. sikkimensis with D. punctiferalis was 3.3%, C. sikkimensis with C. kurokoi 5.0%, D. punctiferalis with C. kurokoi 7.7%, and C. sikkimensis with D. punctiferalis and C. kurokoi 5.0%. The entomopathogenic nematodes, ScP and HbH were effective against all the species of chestnut insect pests. The $LC_{50}$ of ScP was 14.6 for C. sikkimensis, 4.6 for D. punctiferalis, and 5.6 for C. kurokoi and that of HbH was 49.2 for C. sikkimensis, 5.8 for D. punctiferalis, and 13.9 for C. kurokoi, respectively. When ScP was applied into pot including harvested chestnuts at the rate of 4,813 infective juveniles (Ijs)/pot $(=1\times10^9/ha)$, mortality of C. sikkimensis, D. punctiferalis, and C. kurokoi was 85.3%, 96.9%, and 68.1%, respectively. The mortality of C. sikkimensis, D. punctiferalis, and C. kurokoi was 60.73%, 96.5%, and 66.8%, respectively when HbH was applied at the same rate. Combination of two nematode species produced similar effects and insects were more infected by ScP than HbH. When chestnuts were soaked in the suspension of ScP at the rate of 300, 3,000, and 30,000 Ijs for 10 minutes or 30 minutes, mortalities of all chestnut insects were high irrespective of soaking time, concentration , and nematode species.

• Korean Journal of Microbiology
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• v.39 no.2
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• pp.76-82
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• 2003

To prepare evolved PHB depolymerase with increased activity for PHB or P(3HB-co-3HV) compared to the activity of the original PHB depolymerase from Alcaligenes faecalis T1, random mutation of the cloned PHB depolymerase gene was performed by using a DNA shuffling method. A library of mutated PHB depolymerase genes from A. faecalis T1 was fused to the ice nucleation protein (INP) gene from Pseudomonas syringae in pJHCl 1 and approximately 7,000 transformants were isolated. Using M9 minimal medium containing PHB or P(3HB-co-3HV) as the carbon source, mutants showing alteration in PHB depolymerase activity were selected from the transformants. The PHB depolymease activity of the transformants was confirmed by the formation of halo around colony and the turbidity decrease tests using culture supermatants. The catalytic activity of PHB depolymerase of the best mutant II-4 for PHB or P(3HB-co-13 mol% 3HV) was approximately 1.8-fold and 3.2-fold, respectively, higher than that of the original PHB depolymerase. DNA sequence analysis revealed that three amino acid residues (Ala209Val, Leu258Phe, and Asp263Thr) were substituted in II-4. From the mutational analysis, it was presumed that the substitution of amino acids near catalytic triad to more hydrophobic amino acids enhance the catalytic activity of PHB depolymerase from A. faecalis T1.