Hokamura Ayaka, Wakida Izumi, Miyahara Yuki, Tsuge Takeharu, Shiratsuchi Hideki, Tanaka Kenji, Matsusaki Hiromi
Department of Food and Health Sciences, Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, 3-1-100 Tsukide, Higashi-ku, Kumamoto 862-8502, Japan.
Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan.
J Biosci Bioeng. 2015 Sep;120(3):305-10. doi: 10.1016/j.jbiosc.2015.01.022. Epub 2015 Feb 24.
The polyhydroxyalkanoate (PHA) copolymers consisting of short-chain-length (scl) and medium-chain-length (mcl) monomers have various properties ranging from stiff to flexible depending on the molar fraction of the monomer compositions. It has been reported that PhaG, which is first known as a (R)-3-hydroxyacyl-acyl carrier protein (ACP)-CoA transferase, actually functions as a 3-hydroxyacyl-ACP thioesterase, and the product of PP0763 gene from Pseudomonas putida KT2440 has a (R)-3-hydroxyacyl (3HA)-CoA ligase activity (Wang et al., Appl. Environ. Microbiol., 78, 519-527, 2012). In this study, we found a new (R)-3HA-CoA ligase (the product of PA3924 gene) from Pseudomonas aeruginosa PAO. The PA3924 gene was coexpressed with PHA synthase 1 gene (phaC1Ps) and phaGPs gene from Pseudomonas sp. 61-3, and β-ketothiolase gene (phbARe) and acetoacetyl-CoA reductase gene (phbBRe) from Ralstonia eutropha in Escherichia coli LS5218 at 25°C. As a result, the copolymer containing 94.6 mol% 3-hydroxybutyrate (3HB) and 5.4 mol% mcl-3-hydroxyalkanoates (3HA) consisting of C8, C10, C12 and C14 was synthesized by recombinant E. coli LS5218 from glucose as the sole carbon source. The concentration of P(3HB-co-3HA) (scl-co-mcl-PHA) synthesized by the recombinant E. coli LS5218 harboring phaC1Ps, phaGPs, phbABRe and the PA3924 genes was approximately 7-fold higher than that of the recombinant LS5218 harboring phaC1Ps, phaGPs, phbABRe and the PP0763 genes. The number-average molecular weight of the P(3HB-co-5.4% 3HA) copolymer was 233 × 10(3), which was relatively high molecular weight. In addition, the physical and the mechanical properties of the copolymer were demonstrated to improve the brittleness of P(3HB) homopolymer.
由短链长度（scl）和中链长度（mcl）单体组成的聚羟基脂肪酸酯（PHA）共聚物具有从硬到软的各种性质，这取决于单体组成的摩尔分数。据报道，最初被认为是（R）-3-羟基酰基-酰基载体蛋白（ACP）-CoA转移酶的PhaG，实际上起着3-羟基酰基-ACP硫酯酶的作用，恶臭假单胞菌KT2440的PP0763基因产物具有（R）-3-羟基酰基（3HA）-CoA连接酶活性（Wang等人，《应用与环境微生物学》，78，519 - 527，2012）。在本研究中，我们从铜绿假单胞菌PAO中发现了一种新的（R）-3HA-CoA连接酶（PA3924基因产物）。PA3924基因与来自假单胞菌属61 - 3的PHA合酶1基因（phaC1Ps）和phaGPs基因，以及来自真养产碱菌的β-酮硫解酶基因（phbARe）和乙酰乙酰-CoA还原酶基因（phbBRe）在大肠杆菌LS5218中于25°C共表达。结果，重组大肠杆菌LS5218以葡萄糖作为唯一碳源合成了含有94.6摩尔％3-羟基丁酸酯（3HB）和5.4摩尔％由C8、C10、C12和C14组成的mcl-3-羟基脂肪酸酯（3HA）的共聚物。含有phaC1Ps、phaGPs、phbABRe和PA3924基因的重组大肠杆菌LS5218合成的P（(3HB-co-3HA）（scl-co-mcl-PHA）的浓度比含有phaC1Ps、phaGPs、phbABRe和PP0763基因的重组LS5218高出约7倍。P（3HB-co-5.4% 3HA）共聚物数均分子量为233×10³，属于相对较高的分子量。此外，共聚物的物理和机械性能被证明可改善P（3HB）均聚物的脆性。
