Earticle

Polyhydroxyalkanoates (PHA)는 N, P, S, Mg, O2 등의 영양소가 제한된 성장 조건 하에서 여러 종류의 미생물들에 의해 세포 내 탄소와 에너지 저장 물질의 형태로 축적되는 생분해성 고분자이다. PHA는 가수분해나 효소에 의해 분해 되기 때문에 농업, 해양, 의약 등 다양한 분야의 적용에 있어 서 환경적으로 분해 가능한 열가소성 물질로써 많은 관심을 불러 일으키고 있으며 상업적 관심의 초점이 되고 있다 [1]. PHA 중 가장 단순한 형태인 poly(3-hydroxybutyrate) homopolymer는 부서지기 쉽고 가공성이 용이하지 않아 이를 보다 유연하게 만든 poly(3-hydroxybutyrate-co-3- hydroxyvalerate) [P(3HB-co-3HV)] 및 poly(3-hydroxybutyrateco- 4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer가 propionic acid, pentanoic acid, γ-butyrolactone 등의 기질을 함께 공급하여 합성되었다. 특히 P(3HB-co-4HB)는 4HB의 함량에 따라 딱딱한 결정성 플라스틱에서 탄성이 큰 고무에 이르기까지 다양한 물성을 나타내며 [2], 다른 PHA에 비해 생체 내 가수분해 속도도 크고 4HB 함량에 따라 조절 가능 하여 의약분야 소재로서의 활용이 기대되고 있다 [3]. 식물성 오일은 값싸고 풍부하며 대량생산 가능한 대표적 인 천연자원으로 이를 어떻게 유용한 고부가가치 제품으로 만드는지에 관한 연구가 활발히 진행되고 있다. 또한 미생물 배양시 탄소원으로 식물성 오일을 사용할 경우 식물성 오일 내의 높은 탄소 함량 때문에 높은 수율을 얻을 수 있음이 보고되고 있다 [4]. 식물성 오일은 다른 원료물질들에 비해 비교적 저가의 물질로 미생물 산업분야에서 매력적인 원 료로 부각되고 있다. 식물성 오일의 주성분인 oleic acid와 linoleic acid와 같은 불포화 지방산의 함량은 soybean oil의 경우 각각 22%와 55%, olive oil의 경우 78%와 7%에 달 한다 [5,6]. 또한 이론적으로 식물성 오일을 사용하였을 경우 약 1.0 g-PHA per g-vegetable oil의 수율 [4]을 얻을 수 있다고 보고되어 있으며, 이는 glucose를 사용하였을 경우 0.48 g-PHA per g-glucose [7]와 비교하였을 때 두 배 이 상에 달한다. Ralstonia eutropha (Cupriavidus necato)는 성장이 용이하 고 균체내 PHA 축적율이 높아 PHA 합성에 관해 가장 많이 연구되어 온 균주로서 본 연구진은 glucose 또는 fructose 를 주요 기질로 R. eutropha의 고농도 유가식 배양을 통해 최대 약 200 g/L의 균체농도 및 80 wt%의 PHA 함량 을 얻은 바 있다 [8-12]. 본 연구에서는 비교적 저가이고 높 은 수율을 얻을 수 있는 식물성 오일 또는 바이오디젤 제조 공정의 부산물인 glycerol을 탄소원으로 이용하여 R. eutropha 여섯 균주를 배양하여 최종 균체농도 및 PHA 함량 면에서 최적 균주를 결정하였다. 또한 식물성 오일에 γ-butyrolactone 또는 pentanoic acid를 첨가하여 이용하여 P(3HB) homopolymer 외에 P(3HB-co-4HB) 및 P(3HB-co- 3HV) copolymer의 합성 특성을 조사하였다.

Six strains of Ralstonia eutropha were grown to investigate characteristics of polyhydroxyalkanoates (PHA) synthesis from vegetable oils or glycerol. Poly(3-hydroxybutyrate) homopolymer was formed using soybean oil, olive oil, or glycerol as carbon source, while poly(3-hydroxybutyrate-co-4-hydroxybutyrate) or poly(3-hydroxybutyrateco- 3-hydroxyvalerate) copolymers were synthesized by co-feeding γ-butyrolactone or pentanoic acid, respectively. Optimum strain was determined as R. eutropha KCTC 2662 in terms of final cell concentration and PHA content. From 20 g/L of soybean oil (optimum substrate), cell concentration and PHA content at 72 h ranged 1.7~9.2 g/L and 70~92 wt%, respectively.

For mass production of polyhydroxyalkanoates (PHA), high cell density cultures of Alcaligenes eutrophus by fed-batch culture under phosphate-limitation condition has been investigated. PHA accumulation by the regulation by the regulation of initial phosphate concentration could be automatically induced, and high density cell culture above 200 g/L also could be successfully produced. The production of Poly-B-hydroxybutyrate (PHB) and dry cell weight increased with increasing the initial phosphate concentration. When the initial concentrations of phosphate were in the ranges of 1.5~4.5 g-PO4/L, PHB and dry cell weight obtained were 83~266 g/L and 61~216 g/L, respectively, and PHB productivity was in the ranges of 1.35~3.10 g/L.h. When a mixture of glucose and propionic acid is used as carbon sources, poly(3-hydroxybutyrate-co-poly-3-hydroxyvalerate), P(3HB-co-3HV), could be also successfully produced under phosphate limitation condition. When the mole ratio of propionic acid to glucose in the feeding solution is 0.22, a final dry cell weight of 150 g/L and a P(3-HB-co-3HV) of 90 g/L were produced. Morphological changes and size distribution of PHB granules synthesized in A. eutrophus under phosphate-limitation condition are determined by TEM during the course of fed-batch. Mean granule diameters of PHB produced are in the range of 0.36~0.39 m, and mean cell size was elongated from 0.54~0.59 um 1.3~1.5 m to 0.83~0.89 m 2.0~2.3 m. Phosphate concentration in media did not affect size distribution of PHB granule and cell.

Polyhydroxyalkanoates[PHAs] are the polyester of hydroxyalkanoates(HAs) synthesized by numerous bacteria as an intracellular carbon and energy storage compound and accumulated as granules in the cytoplasm of the cells under unbalanced growth condition in the presence of excess carbon source. Even though PHAs have been recognized as good candidates for biodegradable plastics, their high price compared with conventional plastics has limited their use in a wide mange of applications. To reduce the high production cost of PHAs, many group of scientists have devoted much efforts to improve productivity by employing various microorganisms and by developing efficient culture techniques. The strategies of producing PHAs to a high concentration with high productivity and their potential applications are reviewed.

재조합 E. coli를 이용한 MCL-PHA의 생산에서 fatty acid pathway로부터 PHA 생합성 전구체 물질들이 만들어진다는 사실과 함께 이에 관여하는 enzymes이 밝혀지고 있다. 본 논문에서는 protein homology search로부터 탐색된 paaG와 ydbU genes의 PHA 생합성에서의 역할을 확인하기 위하여 paaG와 ydbU gene이 각각 knock-out된 mutant E. coli strains를 제작하였다. 제작된 mutant E. coli들은 모균주들보다 낮은 PHA 농도와 함량을 가졌으며, 이러한 결과들로부터 paaG와 ydbU는 fatty acid pathway에서 PHA synthesis의 전구체 물질들을 공급한다는 사실을 확인하였다. 또한, 새로운 FadB homologous enzyme YgfG를 탐색하였으며, ygfG gene이 overexpression된 균주와 ygfG mutant를 제작하여 PHA 합성을 실험한 결과 ygfG도 paaG나 ydbU와 유사한 역할을 한다는 사실을 밝혔다. 이러한 연구결과들은 E. coli에서의 MCL-PHA 단량체들의 합성 경로를 확인하여 효과적인 PHA 생산 균주를 제작할 수 있게 할 것이다.

In vivo characterization of FadB homologous enzymes including PaaG, YdbU and YgfG for medium-chain-length (MCL) polyhydroxyalkanoate (PHA) biosynthesis was carried out in fadB mutant Escherichia coli. Previously, it was reported that amplification of FadB homologous enzymes such as PaaG and YdbU in fadB mutant E. coli resulted in enhanced biosynthesis of MCL-PHA by greater than two fold compared with control strain. In this study, we constructed paaG fadB double mutant E. coli WB114 and ydbU fadB double mutant E. coli WB115 to investigate the roles of PaaG and YdbU in biosynthesis of MCL-PHA. Inactivation of paaG and ydbU genes in fadB mutant E. coli harboring Pseudomonas sp. 61-3 phaC2 gene reduced the MCL-PHA production to 0.16 and 0.16 PHA g/L, respectively from 2 g/L of sodium decanoate, which are much lower than 0.43 PHA g/L obtained with fadB mutant E. coli WB101 harboring the phaC2 gene. Also, we identified new FadB homologous enzyme YgfG, and examined its roles by overexpression of ygfG and construction of ygfG fadB double mutant E. coli WB113.

As completely biodegradable polyesters, polyhydroxyalkanoates (PHAs) are synthesized by various bacterial strains. Usually PHAs are synthesized under stress conditions as organic carbon. Ralstonia eutropha H16 (KCTC 22469) is a famous lithoautotrophic bacterium. R. eutropha H16 can use both organic compounds and H2 as its energy sources and it has ability of storing PHAs. Since many studies for increasing products using electrochemical bioreactors were reported, increasing production of PHAs could be expected when R. eutropha H16 could use electron from electrode. Electricity is considered an energy source because R. eutropha H16 uses H2 and CO2 as its energy and carbon sources, respectively. Consuming of H2 and CO2 was observed in R. eutropha H16 when headspace was made up of H2, CO2, and O2. Instead of H2, electron from electrode could be energy source. R. eutropha H16 was cultured in H-type Bio-Electro-Reactor (H-BER) and serum bottle, respectively. As control experiments, R. eutropha H16 was grown under autotrophic condition at 30℃ in Basal Mineral Medium with Ar : CO2 : O2 = 8 : 1 : 1 and H2 : CO2 : O2 = 8 : 1 : 1 as the gas phase respectively. Dry cell weight of R. eutropha H16 was measured for the comparison between H-BER and serum bottle. As a result, R. eutropha H16 was grown under supplying CO2 and electron as a carbon and an energy source, respectively.

We have previously reported in vivo biosynthesis of polylactic acid (PLA) and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] employing recombinant Escherichia coli strains by the introduction of evolved Clostridium propionicum propionyl-CoA transferase (PctCp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps6-19). Using this in vivo PLA biosynthesis system, we engineered E. coli to synthesize PHAs containing 2-hydroxybutyrate (2HB) monomer from glucose. A heterologous metabolic pathway for the generation of 2HB from propionyl-CoA was constructed. Conversion of 2-ketobutyrate into 2HB was mediated by 2HB dehydrogenase encoded by the panE gene. Recombinant E. coli strain expressing the phaC1437, pct540, and prpE genes together with the panE gene successfully produced PHAs consisting of 2HB, 3HB, and a small fraction of lactate by varying the 3HB and propionic acid concentrations in the culture medium.

나노기술과 바이오기술의 융합연구에 의해 나노바이오기술이 발전되고 있다. 나노바이오기술의 중요한 응용연구 중의 하나로서, 진단이나 바이오센서 분야에서 단백질-단백질 및 단백질-바이오물질간의 상호작용을 연구하기 위한 단백질 센서 칩이 개발되어 왔다. 본 논문에서는 단백질의 선택적 고정화를 위한 새로운 생체고분자 기질로 PHA를 이용하는 첫 번째 예로서, 단백질-단백질 및 항원-항체 반응의 구현을 나타내고자 하였다. 본 시스템은 PHA 표면 위에서 PHA depolymerase의 SBD와의 선택적 결합에 기반한 것으로, PHA depolymerase의 SBD와 융합된 단백질이 PHA가 코팅된 표면 위에 spotting 될 수 있고 미세접촉인쇄방법에 의해 PHA 위에 미세패턴이 제조되어지는 것을 알 수 있었다(52, 53). 이러한 새로운 전략이 PHA depolymerase의 SBD와 다른 단백질을 융합함으로서 미세 spotting과 미세패터닝이 가능하게 되었고 항원-항체의 생물학적 반응을 통해 많은 바이오센서 칩 연구에 응용될 수 있음을 확인하였다. 또한, PHA 마이크로비드에도 PHA depolymerase의 SBD와 융합된 단백질을 고정시킴으로서 항원-항체 반응을 유도할 수 있음을 확인하였다(54). PHA의 구조를 변경하여 PHA 기판, PHA 필름, PHA 미세패턴, PHA 마이크로 비드 등을 이용할 수 있으며 multiplex assay를 동시에 진행할 수 있는 다양한 융합 단백질을 사용할 수 있을 것이다. 생분해성 플라스틱으로서 성공적으로 개발된 PHA를 이용한 새로운 플랫폼 기술이 PHA depolymerase의 SBD를 이용함으로서 특이적이고 선택적인 단백질의 고정화에 이용될 수 있음을 확인하였다. 본 전략이 다양한 단백질-단백질 및 단백질-바이오물질반응을 이용한 바이오칩 및 바이오센서의 응용연구에 유용하게 사용될 것이다.

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 SBD 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.

인공폐수로 연속회분식 하수처리시스템에서 바이오플라스틱인 PHAs의 생산에 대하여 연구하였다. 실험은 하수처리장에서 식종슬러지를 채취하고, 실험실 내에서 제작된 인공하수를 이용하여 4 L 규모의 2조의 연속회분식반응기로 수행하였다. 인공하수의 영양조건(C/N/P)은 42:10:1로 운전되었으며, 연속회분식반응기는 호기상태에서 Feast/Famine 조건을 부여하는 ADF (aerobic dynamic feeding)의 SBR 1과 혐기/호기조건에서 Feast/Famine 조건을 부여하는 AODF (anaerobic/oxic dynamic feeding)의 SBR 2로 운전하였다. PHAs의 생산은 AODF가 ADF 보다 우수하였으며, ADOF 에서 바이오매스 대비 최대 40.0%(w/w)로 높게 생산되었으며, 구조적 및 열적 물성도 우수한 것으로 나타났다.

The production of polyhydroxyalkanoates (PHAs) using Sequencing Batch Reactor (SBR) was investigated. The experiments were performed in two fabricated SBRs (4 L) of different oxidation state. Synthetic wastewater was used as substrate, using C/N/P ratio of 42:10:1. SBR 1 and SBR 2 were operated in aerobic dynamic feeding (ADF) and anaerobic/oxic dynamic feeding (AODF) condition, respectively. ADF provide feast and famine in aerobic condition, while AODF in anaerobic/oxic condition. PHAs production was found high in AODF than AOF. Maximum PHAs content of 40.0% (w/w)of biomass were produced in AODF mode. Produced PHAs structural and thermal property were good.

Polyhydroxyalkanoates (PHA) are a family of microbial polyesters that are used as biodegradable plastics in replacement of conventional plastics for various applications. However, the high production cost is the barrier for PHA market expansion. This study aimed to utilize food waste as low-cost feedstock to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Haloferax mediterranei. The effects of acetate (Ac), propionate (Pr), butyrate (Bu), and the short-chain carboxylates derived from food waste were examined on the microbial growth and PHBV production. Results showed that a mixture of carboxylates provided a 55% higher PHBV yield than glucose. The food-waste-derived nutrients achieved the yields of 0.41 to 0.54 g PHBV/g Ac from initial loadings of 450 mg/l to 1,800 mg/l Ac of total carboxylates. And the consumption of individual carboxylate varied between different compositions of the carbon source. The present study demonstrates the potential of using food waste as feedstock to produce PHBV by Haloferax mediterranei, which can provide economic benefits to the current PHA industry. Meanwhile, it will also help promote organic waste reduction in landfills and waste management in general.

Polyhydroxyalkanoates (PHAs) are biodegradable plastics produced by bacteria, but their use in diverse applications is prohibited by high production costs. To reduce these costs, the conversion by Pseudomonas strains of PHAs from crude sludge palm oil (SPO) as an inexpensive renewable raw material was tested. Pseudomonas putida S12 was found to produce the highest yield (~41%) of elastomeric medium-chain-length (MCL)-PHAs from SPO. The MCL-PHA characteristics were analyzed by gas-chromatography/mass spectrometry, gel permeation chromatography, and differential scanning calorimetry. These findings may contribute to more widespread use of PHAs by reducing PHA production costs.

Polyhydroxyalkanoates (PHAs) are homo or hetero polyesters of (R)-hydroxyalkanoates accumulated in various microorganisms under growth-limiting condition in the presence of excess carbon source. They have been suggested as biodegradable substitutes for chemically synthesized polymers. Recombinant Escherichia coli is one of the promising host strains for the economical production of PHAs, and has been extensively investigated for the process development. The heterologous PHA biosynthetic pathways have been established through the metabolic engineering and inherent metabolic pathways of E. coli have been redirected to supply PHA precursors. Fermentation strategies for cultivating these recombinant E. coli strains have also been developed for the efficient production of PHAs. Nowadays, short-chain-length (SCL) PHAs are being re-invited due to its improved mechanical properties and possible applications in the biomedical area. In this article, recent advances in the development of metabolically engineered E. coli strains for the enhanced production of SCL-PHAs are reviewed. Also, medical applications of SCL-PHAs are discussed.

Rhodopseudomonas sp. KCTC1437균주를 이용하여 Polyhydroxyalkanoate (PHA)와 5-aminolevulinic acid (ALA)를 생산하기 위한 배양조건과 이들 생합성 조건의 상호관련성에 대하여 조사하였다. PHA생합성을 위한 탄소원으로는 acetic acid가 가장 효과적이었으나, succinic acid를 보조 탄소원으로 사용하였을 때의 세포건체량은 2.5g/ι, PHA함량은 건체량의 73%로서 주 탄소원만을 사용할 때에 비하여 크게 증가하였다. 조사된 탄소원으로부터 생합성된 PHA는 모두 polyhydroxybutyrate 단일중합체 이었으나, valeric acid로부터는 3-hydroxybutyrate와 3-hydroxyvalerate로 구성된 공중합체가 생산되었다. ALA의 생합성을 위하여서는 환원제 인 sodium thioglycolate를 첨가하여 혐기적 조건을 만들고, 탄소원인 acetic acid와 propionic acid 이외에 전구물질인 levulinic acid, succinic acid와 glycine을 반복적으로 공급해주었을 때 가장 좋았으며, 약 400 mg/ι의 ALA를 생산할 수 있었다. 그러나 ALA 생합성의 필수물질인 glycine, levulinic acid와 환원제는 세포생장과 PHA의 생합성을 저해하는 것으로 나타났다. 이러한 실험 결과, Rhodopseudomonas sp. KCTC1437균주로부터 PHA와 ALA를 동시에 생산할 수 있으나, 두 가지 유용산물을 효율적으로 생합성하기 위한 각각의 배양조건이 상호 배타적임을 확인하였다.

For elucidating the relationship between the biosynthetic pathways for polyhydroxyslkanoates (PHAs) and 5-aminolevulinic acid (ALA), culture conditions for the production of these two biomaterials by Rhodopseudomonas sp. KCTC 1437 were investigated. Of the carbon substrates tested, acetic acid was the best carbon source for cell growth and PHA biosynthesis. When succinic acid was added as a co-substrate into culture medium, cell growth and PHA production were greatly increased up to 2.5 g/ι and 73% of dry cell weight, respectively. The PHA obtained from the carbon substrates tested was homopolyester of 3-hydroxybutyrate, while valeric acid was only effective for the production of copolyester consisting of 3-hydroxybutyrate and 3-hydroxyvalerate. Anaerobic light culture condition was better for PHA production and cell growth than anaerobic dark or aerobic dark culture condition. The organism was capable of synthesizing ALA when glycine and succinic acid were added to the culture medium. ALA was produced to ca.400 mg/ι when levulinic acid, soccinic acid, and glycine were repeatedly added with a reductant (sodim thioglycolate). However, the presence of glycine, levulinic acid and sodium glycolate inhibited the cell growth and the conversion of carbon substrates to PHA. From these results it is apparent that the production yields of PHA and ALA could not be increased simultaneously because the optimal conditions for the production of PHA and ALA are opposed to each other.

Five microorganisms capable of degrading an aromatic medium-chain-length polyhydroxyalkanoate ($PHA_{MCL}$), poly(3-hydroxy-5-phenylvalerate) (PHPV), were isolated from wastewater-treatment sludge. Among the isolates, JS02 showed degrading activity consistantly during several transfers. The isolate JS02 could hydrolyze another aromatic MCL copolyester, poly(3-hydroxy-5-phenoxyvalerate-co-3-hydroxy-7-phenoxyheptanoate), ［P(5POHV-co-7POHH)］, and other short-chain-length PHAs ($PHA_{SCL}$) such as poly(3-hydroxybutyrate) ［P3(HB)］, poly(3-hydroxybutyrate-co-4-hydroxybutyrate) ［P(3 HB-co-4 HB)］, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) ［P(3HB-co-3HV)］ with relatively low activity. The culture supernatant of JS02 showed hydrolyzing activity for the p-nitrophenyl esters of fatty acids.

One of the major challenges in metabolic engineering for enhanced synthesis of value-added chemicals is to design and develop new strains that can be translated into well-controlled fermentation processes using bioreactors. The aim of this study was to assess the influence of various fed-batch strategies in the performance of metabolically engineered Pseudomonas putida strains, ${\Delta}gcd$ and ${\Delta}gcd-pgl$, for improving production of medium-chain-length polyhydroxyalkanoates (mcl-PHAs) using glucose as the only carbon source. First we developed a fed-batch process that comprised an initial phase of biomass accumulation based on an exponential feeding carbon-limited strategy. For the mcl-PHA accumulation stage, three induction techniques were tested under nitrogen limitation. The substrate-pulse feeding was more efficient than the constant-feeding approach to promote the accumulation of the desirable product. Nonetheless, the most efficient approach for maximum PHA synthesis was the application of a dissolved-oxygen-stat feeding strategy (DO-stat), where P. putida ${\Delta}gcd$ mutant strain showed a final PHA content and specific PHA productivity of 67% and $0.83g{\cdot}l^{-1}{\cdot}h^{-1}$, respectively. To our knowledge, this mcl-PHA titer is the highest value that has been ever reported using glucose as the sole carbon and energy source. Our results also highlighted the effect of different fed-batch strategies upon the extent of realization of the intended metabolic modification of the mutant strains.

토양으로부터 oleic acid 를 탄소원으로 이용하여 MCL-PHA의 생산능이 우수한 균주를 분리하여 Pseudomonas sp. MBEL21이라 명명하였다. Pseudomonas sp. MBEL21의 PHA함량을 증가시키기 위하여 여러 가지 필수 영양분의 제한 조건에서 배양한 결과, 인산이 제한된 조건에서 가장 높은 함량의 PHA를 축적하였다. 고농도의 MCL-PHA생산을 위한 Pseudomonas sp. MBEL21의 유가식 배양 전략을 개발하여 28.1 wt％의 함량으로 23 g/L. MCL-PHA를 생산하였다. 또한, 분리된 Pseudomonas sp. MBEL21을 olive oil을 탄소원으로 이용하여 배양한 결과 9.3 wt％의 PHA가 축적되어졌으며, MCL-hydroxyalkanoate와 함께 3-hyoxy-butyrate 의 단량체들로 이루어진 PHA가 확인되어졌다. 이러한 결과들은 분리된 신규 Pseudomonas sp. MBEL21이 저가의 olive oil로부터 생분해성 탄성체의 응용분야를 갖는 MCL-PHA를 효율적으로 생산할 수 있다는 것을 보여준다.

Pseudomonas sp. MBEL21 was newly isolated from soil samples and found to accumulate medium-chain-length Polyhydroxyalkanoates(MCL-PHAs) using oleic acid as a sole carbon source. Among the various nutrient limiting conditions examined, including nitrogen, sulfur and phosphorus, only phosphorus limitation supported the accumulation of MCL-PHAs up to 15 wt％ of dry cell weight in flask cultures. MCL-PHAs produced by Pseudomonas sp. MBEL21 was mainly composed of 3-hydroxy-5-cis-tetradecenoate. Fed-batch culture of Pseudomonas sp. MBEL21 by novel feeding strategies based on cell growth charcteristics was carried out under phosphorus limitation using oleic acid as a sole carbon source. The final cell concentration and PHA content of 82 g/L and 28 wt％, respectively, were obtained. Furthermore, PHA consisted of MCL-hydroxyalkanoates and 3-hydroxybutyrate could be produced using olive oil as a sole carbon source.