Polyhydroxyalkanoates (PHAs) are biopolymers accumulated in a number of microorganisms as carbon and energy storage materials under the nutrient-limiting condition in the presence of an excess carbon source. PHAs are considered to be one of the potential alternatives to petrochemically-derived plastics due to their versatile material properties. Unlike short-chain-length (SCL) PHAs, medium-chain-length (MCL) PHAs possess a much lower crystallinity and higher elasticity. With the effort of improving production of MCL PHA, a number of studies have been carried out for the past few decades. However, MCL PHA production is still economically poor in order that it is able to be brought into a practical application. In this study, by introducing yeast β-oxidation pathway into different E. coli mutants, we obtained an improved amount of MCL PHA produced in fadAB mutant E.coli strain (WAB101) and fadR atoC (Con) mutant E.coli strain (LS5218), which produced MCL PHA 1.5 and 5 times higher than the control strains, respectively. In addition, this is the first time eukaryotic system has been introduced into prokaryotic system for MCL PHA production.
Recently, environmental issues, which have a direct influence on human health, have been paid special attention. Petroleum-based materials, which have long lives in the environment, contributing to waste in landfills and the oceans and posing a direct threat to wildlife, are necessary to be substituted. PHAs belong to a growing family of biopolyesters which are accumulated in a large number of bacteria as storage materials under nutrient-limited conditions in the presence of excess carbon source (Anderson and Dawes 1990). PHAs consist of various monomer units in D(-) configuration ranging from 3 to 16 carbon monomers. This wide range of monomers makes PHAs to have diverse material properties classified by thermoplastics as SCL PHAs consisting of 3- to 5-carbon monomers, elastomers as MCL PHAs consisting of 6- to 16-carbon monomers and their copolymers showing intermediate properties. Since poly(3-hydroxybutyrate) (P(3HB)) homopolymer was discovered in Bacillus megaterium by Maurice Lemoigne in 1926, more than 150 kinds of (R)-hydroxyalkanoic acid monomers have been found to be incorporated into bacterial PHAs (Steinb??chel and Valentin 1995). The monomer composition of PHAs highly depends on the metabolic capability of host microorganism and on the substrate specificity of PHA synthase, and subsequently determines the physicochemical properties of PHAs (Fig. 1.1). This implies that tailor-made PHA production using microorganisms can be optimized by metabolic engineering and key enzyme engineering, and finally be maximized by fine-controlled fermentation. Furthermore, recently, the genome sequences of the most widely used PHA producers, Cupriavidus necator H16 (formerly, Ralstonia eutropha H16) and Pseudomonas putida KT2440, as the starting point of engineering, have been released by Pohlmann et al (2007) and Nelson et al. (2002), respectively, which has encouraged strain development for the novel PHA production through systems metabolic engineering started from numerous invaluable genetic sources of strong PHA producers.
Poly(3-hydroxybutyrate) [P(3HB) has been studied most extensively and recognized as the first well-known PHA. P(3HB) is a homopolymer whose monomer is 3-hydroxybutyrate. P(3HB) is a compact right-handed helix with a twofold screw axis and a fiber repeat of 0.596 nm. The structure of PHA which composed of 3-hydroxy fatty acid monomers is linear, head-to-tail. In these polymers, there is formation of an ester bond between the carboxyl group of one monomer and hydroxyl group of the neighboring monomer. An alkyl group which can vary from methyl to tridecyl located at the C-3 or β position. This alkyl side chain is likely to be one of the following types: aromatic, unsaturated, halogenated, expoxidized and branched monomers. The monomer units in PHAs are actively optical and all in the D-(-) configuration owing to the stereo specificity of the biosynthetic enzyme (Anderson and Dawes 1990).
MCL PHAs have been structurally characterized and compared between two different recovery methods which are solvent extraction and enzyme treatment methods (Kathiraser et al. 2007). By using FTIR, it was seen that the straight-chain alkyl group (C??H) with a frequency range (cm??1) of 2962??2853 with a strong intensity presented for the major part in MCL PHA. In addition, it demonstrated the presence of the intense ester group shown by the carbonyl (C=O) stretch at 1750??1735cm??1 as well as the characteristic absorption in the fingerprint region at 1300??1000cm??1 from the couplings of C??O and C??C stretches. There are also a number of medium to strong signals at wavenumber values between 1500 and 1000cm??1 due to methyl (CH3) and methylene deformations (CH2). At around 721cm??1, the transmission signal could be associated with extended contribution of CH2 at the side chain. It was observed that using two different recovery methods resulted in the similar of major functional groups of the biopolymers. The only difference between them was a slightly broad peak with a wavenumber of 3453.49 with a weak intensity shown by PHA recovered from enzyme digestion. It was attributed to the O??H group bonded with H, suggesting the presence of the H2O group due to incomplete drying in enzymatic digestion procedure. By using H-NMR analysis, molecular structure of PHA has been confirmed. A set of double doublets at 2.58 and 2.51 ppm, a signal at 5.1ppm, and other weak signals at 5.30ppm and 5.52ppm represented the presence of the CH2, CH group and two ethylene carbon sequences, respectively. The first side chain of the monomers showed a peak appearing at 1.50 ppm. The remaining methyl (CH2) was observed through the signal at 1.30 ppm. A small signal at 2.01 ppm is attributed to double bonds (C5 ?? C6). The terminal methyl (CH3) group was determined at the peak of 0.89 ppm.
Polyhydroxyalkanoates(PHAs)는 과도한 탄소원의 존재 및 제한적인 영양소 조건에서 탄소원과 에너지원을 비축하는 역할을 하는 생고분자 물질(biopolymer)이다. PHA는 다양한 물성으로 인해 석유화학을 기반으로 만들어지는 기존 플라스틱의 가능성 있는 대체물질로 여겨지고 있다. 짧은 길이의 PHA와 다르게 중간 길이(MCL) PHA는 낮은 결정성(crystallinity)와 높은 탄력성(elasticity)을 가지고 있다. 지난 수 년간, MCL PHA의 생산성을 높이려는 노력이 진행되었으나, MCL PHA를 실질적으로 응용하기는 경제적으로 어렵다. 이 연구에서는 돌연변이 대장균에 효소의 β-oxidation 경로를 도입하여 fadAB 유전자가 결실된 대장균 돌연변이균과(WAB101)과 이에 추가적으로 atoC 유전자가 변이된 대장균(LS5218) 돌연변이 균을 제작하여 MCL PHA의 양을 향상시켰다. 이는 control과 비교해보았을 때, 1.5배와 5배가 각각 증가하였다. 이는 MCL PHA 생성을 위해 진핵생물의 system을 원핵생물에 도입한 첫 연구로서 더욱 의미가 있다.
Polyhydroxyalkanoates(PHAs)는 과도한 탄소원의 존재 및 제한적인 영양소 조건에서 탄소원과 에너지원을 비축하는 역할을 하는 생고분자 물질(biopolymer)이다. PHA는 다양한 물성으로 인해 석유화학을 기반으로 만들어지는 기존 플라스틱의 가능성 있는 대체물질로 여겨지고 있다. 짧은 길이의 PHA와 다르게 중간 길이(MCL) PHA는 낮은 결정성(crystallinity)와 높은 탄력성(elasticity)을 가지고 있다. 지난 수 년간, MCL PHA의 생산성을 높이려는 노력이 진행되었으나, MCL PHA를 실질적으로 응용하기는 경제적으로 어렵다. 이 연구에서는 돌연변이 대장균에 효소의 β-oxidation 경로를 도입하여 fadAB 유전자가 결실된 대장균 돌연변이균과(WAB101)과 이에 추가적으로 atoC 유전자가 변이된 대장균(LS5218) 돌연변이 균을 제작하여 MCL PHA의 양을 향상시켰다. 이는 control과 비교해보았을 때, 1.5배와 5배가 각각 증가하였다. 이는 MCL PHA 생성을 위해 진핵생물의 system을 원핵생물에 도입한 첫 연구로서 더욱 의미가 있다.
Polyhydroxyalkanoates(PHAs)는 과도한 탄소원의 존재 및 제한적인 영양소 조건에서 탄소원과 에너지원을 비축하는 역할을 하는 생고분자 물질(biopolymer)이다. PHA는 다양한 물성으로 인해 석유화학을 기반으로 만들어지는 기존 플라스틱의 가능성 있는 대체물질로 여겨지고 있다. 짧은 길이의 PHA와 다르게 중간 길이(MCL) PHA는 낮은 결정성(crystallinity)와 높은 탄력성(elasticity)을 가지고 있다. 지난 수 년간, MCL PHA의 생산성을 높이려는 노력이 진행되었으나, MCL PHA를 실질적으로 응용하기는 경제적으로 어렵다. 이 연구에서는 돌연변이 대장균에 효소의 β-oxidation 경로를 도입하여 fadAB 유전자가 결실된 대장균 돌연변이균과(WAB101)과 이에 추가적으로 atoC 유전자가 변이된 대장균(LS5218) 돌연변이 균을 제작하여 MCL PHA의 양을 향상시켰다. 이는 control과 비교해보았을 때, 1.5배와 5배가 각각 증가하였다. 이는 MCL PHA 생성을 위해 진핵생물의 system을 원핵생물에 도입한 첫 연구로서 더욱 의미가 있다.
