• 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.

• Korean Journal of Microbiology
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• v.48 no.3
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• pp.200-206
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• 2012

A bacterial strain capable of synthesizing polyhydroxyalkanoates (PHAs) with an unusual pattern of monomer units was isolated from activated sludge using the enrichment culture technique. The organism, identified as Pseudomonas aeruginosa P-5, produced polyesters consisting of 3-hydroxyvalerate and medium-chain-length (MCL) 3-hydroxyalkanoate monomer units when $C_{-odd}$ alkanoic acids such as nonanoic acid and heptanoic acid were fed as the sole carbon source. Solvent fractionation experiments using chloroform and hexane revealed that the 3-hydroxyalkanoate monomer units in these polyesters were copolymerized. The molar concentration of 3-hydroxyvalerate in the polyesters produced were significantly elevated up to 26 mol% by adding 1.0 g/L valeric acid as the cosubstrate. These copolyesters were sticky with low degrees of crystallinity. The PHA synthase genes were cloned, and the deduced amino acid sequences were determined. P. aeruginosa P-5 possessed genes encoding MCL-PHA synthases (PhaC1 and PhaC2) but lacked the short-chain-length PHA synthase gene, suggesting that the MCL-PHA synthases from P. aeruginosa P-5 are uniquely active for polymerizing (R)-3-hydroxyvaleryl-CoA as well as MCL (R)-3-hydroxyacyl-CoAs.

• 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.

• KSBB Journal
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• v.22 no.6
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• pp.371-377
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• 2007

Polyhydroxyalkanoates (PHAs) are a family of microbial polyesters that can be produced by fermentation from renewable resources. PHAs can be used as completely biodegradable plastics or elastomers. In this paper, novel applications of PHAs in biosensor are described. A general platform technology was developed by using the substrate binding domain (SBD) of PHA depolymerase as a fusion partner to immobilize proteins of interest on PHA surface. It could be shown that the proteins fused to the SBD of PHA depolymerase could be specifically immobilized onto PHA film, PHA microbead, and microcontact printed PHA surface. We review the results obtained for monitoring the specific interaction between the SBO and PHA by using enhanced green fluorescent protein, red fluorescent protein, single chain antibody against hepatitis B virus preS2 surface protein and severe acute respiratory syndrome coronavirus surface antigen as model proteins. Thus, this system can be efficiently used for studying protein-protein and possibly protein-biomolecule interactions for various biotechnological applications.

• Journal of Korean Society of Environmental Engineers
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• v.35 no.2
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• pp.109-114
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• 2013

Polyhydroxyalkanoates (PHAs) are synthesized by numerous bacteria as carbon and energy storage compounds and are raw materials for biocompatible plastics. In this paper, the effect of $CO_2$ concentrations on the growth of C. necator and the accumulation of Poly(3-hydroxybutyrate) (P(3HB)) are investigated by increasing the $CO_2$ concentration in the substrate gas mixture. During 6 d cultivation in a nitrogen-present mineral medium, the $CO_2$ concentration did not affect the growth of the cells, while the Poly(3- hydroxybutyrate) (P(3HB)) content decreased with increasing $CO_2$ concentrations from 1% to 20%. During 4 d cultivation in the nitrogen-limited medium, the P(3HB) accumulation was the greatest at 3% $CO_2$; however, the total amount of accumulated P(3HB) was the greatest at 1% $CO_2$, which decreased with increasing $CO_2$ concentrations. The results indicate that the gas mixture with 1% $CO_2$ is the most effective in both growing the cells and accumulating P(3HB) under our experimental conditions.

• KOREAN JOURNAL OF PACKAGING SCIENCE & TECHNOLOGY
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• v.30 no.1
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• pp.1-14
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• 2024

As awareness of environmental issues continues to grow, there is an escalating demand for recycling and repurposing byproducts of agricultural and food production processes and their conversion to high-value products. Coffee is the most widely consumed beverage globally; during coffee beverage processing and consumption, byproducts such as coffee silverskin (CS), spent coffee grounds (SCGs), and oil are generated. Despite containing beneficial materials such as cellulose, hemicellulose, lignin, lipids, and bioactive substances, these byproducts are typically discarded in landfills or incinerated. The utilization of CS, SCGs, and oil in the development of packaging materials holds significant potentials toward the realization of a sustainable society. To this end, considerable research efforts have been dedicated to the development of high-value materials derived from coffee byproducts, including functional fillers, polymer composites, and biodegradable polymers. Notably, CS and SCGs have been employed as functional fillers in polymer composites. Additionally, lipids extracted from SCGs have been used as plasticizers for polymers and cultured with microorganisms to produce biodegradable polymers. This review focuses on the research and development of polymer/CS and polymer/SCG composites as well as cellulose extraction and utilization from CS and SCGs and its applications, oil extraction from SCGs, and cultivation with microorganisms using extracted oil for polyhydroxyalkanoates(PHA) production.

• Journal of Microbiology and Biotechnology
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• v.3 no.2
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• pp.115-122
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• 1993

Poly(3-hydroxybutyrate)(P(3HB)) accumulation under nutrient-rich condition with various amounts of $(NH_4)_2 SO_4$ was systematically investigated. The results of the electron-microscopy and the solvent extraction showed that the P(3HB) accumulation is unavoidable even under nutrient-rich condition. This indicates that in a two-step culture of Alcaligenes eutrophus H16, the researches should be careful in interpreting the data of polyhydroxyalkanoates(PHAs) accumulation in terms of the carbon-source fed in the second step because the two-step culture product contains the P(3HB) produced under nutrient-rich condition. The polyester production capability in a two-step batch culture of A. eutrophus H16(ATCC 17699) was also investigated using various saccharides and their derivatives such as glucose, fructose, gluconic acid, glucaric acid, sorbitol, lactose, galactose, and mannose. The polyesters synthesized were characterized by 500 MHz$^{1}H-NMR$ spectroscopy, intrinsic viscosity$[\eta]$ measurement in chloroform and differential scanning calorimetry(DSC). 500 MHz $^{1}H-NMR$ analysis showed that all polyesters synthesized generally contained 1~2 mol% of 3HV. Another finding is that the glucose utilization can be increased by changing the autoclaving procedure of the substrate to enhance the P(3HB) production yield up to 46 wt% of P(3HB) in dry cells.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1120-1124
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• 2005

Pseudomonas putida KCTC 1639 was newly identified as a potential producer of biodegradable medium chain length polyhydroxyalkanoates. It exhibited a carbon assimilation pattern quite different from other known P. putida strains, but a more similar pattern with P. oleovorans, which assimilates the carbon sources mainly through ${\beta}$-oxidation rather than the fatty acid biosynthesis pathway. The PHA synthesis gene cluster from P. putida KCTC1639 was composed of two gene loci; the PHA synthase gene locus and granule-associated gene locus, which were cloned and deposited in the GenBank under accession numbers AY286491 and AY750858 as a new nucleotide sequence, respectively. The molecular structure and amino acid homology of the new gene cluster were compared with those from Pseudomonas species, including other P. putida strains and P. oleovorans, and a higher than $90\%$ homology was observed.

• Journal of Microbiology
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• v.35 no.2
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• pp.127-133
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• 1997

Biosynthesis of polyhydroxybutyrate and copolymer consisting of 3-hydroxybutyrate and 3-hydroxyvalerate [poly(3HB-co-3HV)] by Bacillus thuringiensis R-510 grown with glucose or with mixtures of glucose and propionate was investigated. n-Alkanoic acids other than propionate were not precursors of 3HV units. The fraction of 3HV unit in the copolymer increased from 0 to 84 mol% of 3HV. Polymer yield decreased as the fraction of propionate was increased but the molecular weight distribution was not affected by the composition of carbon substrate. The minimum melting temperature (around 65.deg.C) of poly (3HB-co-3HV) copolymers was observed for the polymer bearing approximately 35 mol% of 3HV. Polyhydroxyalkanoates production by this organism was not dependent on nutritional limitation, but remarkably influenced by dissolved oxygen concentration in the culture medium. Low level of dissolved oxygen concentration prevented spore formation in the cells and stimulated the synthesis of polyhydroxyalkanoate. The composition of poly (3HB-co-3HV) produced by B. thuringiensis R-510 lyhydroxyalkanoate. The composition of poly(3HB-co-3HV) propduced by B. thuringiensis R-510 varied according to the growth time. However, there was no evidence that polymers isolated from cells were mixtures of immiscible polymers.

• Journal of Microbiology and Biotechnology
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• v.18 no.12
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• pp.1958-1965
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• 2008

A denitrifying polyphosphate-accumulating bacterium (YKP-9) was isolated from activated sludge of a 5-stage biological nutrient removal process with step feed system. This organism was a Gram-negative, coccus-shaped, facultative aerobic chemoorganotroph. It had a respiratory type of metabolism with oxygen, nitrate, and nitrite as terminal electron acceptors. The 16S rRNA gene sequence of strain YKP-9 was most similar to the 16S rRNA gene sequence of Paracoccus sp. OL18 (AY312056) (similarity level, 97%). Denitrifying polyphosphate accumulation by strain YKP-9 was examined under anaerobic-anoxic and anaerobic-oxic batch conditions. It was able to use external carbon sources for polyhydroxyalkanoates(PHA) synthesis and to release phosphate under anaerobic condition. It accumulated polyphosphate and grew a little on energy provided by external carbon sources under anoxic condition, but did neither accumulate polyphosphate nor grow in the absence of external carbon sources under anoxic condition. Cells with intracellular PHA cannot accumulate polyphosphate in the absence of external carbon sources under anoxic condition. Under oxic condition, it grew but could not accumulate polyphosphate with external carbon sources. Based on the results from this study, strain YKP-9 is a new-type denitrifying polyphosphate-accumulating bacterium that accumulates polyphosphate only under anoxic condition, with nitrate and nitrite as the electron acceptors in the presence of external carbon sources.