Biosynthesis of Polyhydroxyalkanoate from Steamed Soybean Wastewater by a Recombinant Strain of Pseudomonas sp. 61-3

Pseudomonas sp. 61-3 accumulates a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer, poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA)], consisting of 3HA units of 4–12 carbon atoms. Pseudomonas sp. 61-3 possesses two types of PHA synthases, PHB synthase (PhbC) and PHA synthases (PhaC1 and PhaC2), encoded by the phb and pha loci, respectively. The P(94 mol% 3HB-co-6 mol% 3HA) copolymer synthesized by the recombinant strain of Pseudomonas sp. 61-3 (phbC::tet) harboring additional copies of phaC1 gene is known to have desirable physical properties and to be a flexible material with moderate toughness, similar to low-density polyethylene. In this study, we focused on the production of the P(3HB-co-3HA) copolymer using steamed soybean wastewater, a by-product in brewing miso, which is a traditional Japanese seasoning. The steamed soybean wastewater was spray-dried to produce a powder (SWP) and used as the sole nitrogen source for the synthesis of P(3HB-co-3HA) by the Pseudomonas sp. 61-3 recombinant strain. Hydrolyzed SWP (HSWP) was also used as a carbon and nitrogen source. P(3HB-co-3HA)s with relatively high 3HB fractions could be synthesized by a recombinant strain of Pseudomonas sp. 61-3 (phbC::tet) harboring additional copies of the phaC1 gene in the presence of 2% glucose and 10–20 g/L SWP as the sole nitrogen source, producing a PHA concentration of 1.0–1.4 g/L. When HSWP was added to a nitrogen- and carbon-free medium, the recombinant strain could synthesize PHA without glucose as a carbon source. The recombinant strain accumulated 32 wt% P(3HB-co-3HA) containing 80 mol% 3HB and 20 mol% medium-chain-length 3HA with a PHA concentration of 1.0 g/L when 50 g/L of HSWP was used. The PHA production yield was estimated as 20 mg-PHA/g-HSWP, which equates to approximately 1.0 g-PHA per liter of soybean wastewater

バイオマスからのグリーンプラスチック生産を目指した遺伝子組換え微生物の作製

Polyhydroxyancanoate (PHA) is a bacterial polyester which is thermoplastic, flexible and biodegradable. PHA is expected as “green plastic” material therefore many researchers have been studying the development of new type PHAs, their physical property, biodegradability and the production method. In commercial production of PHA, use of substances obtained from biomass as economic and ecological carbon sources is necessary. Hemicellulose, which is mainly composed of glucose, xylose and other monosaccharaides is an important biomass, however xylose is not very feasible sugar to be used as substrate in fermentation. Our isolated bacterium, Enterobacter sp.TF grow vigorously on xylose, arabinose and other many kind of sugars but it does not accumulate PHA. Hence, we made genetic engineering to the strain TF by inserting the genes for PHA biosynthesis from Ralstonia eutropha which is well known as PHB producer, and from Pseudomonas sp.61-3 which is one of the few bacteria to produce PHA copolymer from glucose. As, result, our recombinant of Enterobacter sp.TF did not produce PHA copolymer but produced about 20wt% PHB homopolymer per dried cells.キシロースは、農産廃棄物や廃材など植物性バイオマスに含まれるヘミセルロースの主要構成単糖であり、発酵原料等への有効利用法の開発が急がれている。しかしながら、キシロースは微生物にとって利用しやすい糖であるとは言い難く、また工業的に利用価値のある発酵生産物も少ないのが現状である。ある種の微生物が産生するポリヒドロキシ酪酸（PHB）やポリヒドロキシアルカン酸（PHA）などのポリエステルは、熱可塑性と生分解性を併せ持つため“グリーンプラスチック”として期待されており、植物由来の有機物を原料基質として使えばCO2の排出量を増やさない“カーボンニュートラル”なプラスチックにもなりえる。本研究では、独自に分離したキシロース高資化性のEnterobacter属細菌にPHAやPHBの生合成遺伝子を導入した組換え株を作製し、実際にキシロースからこれらグリーンプラスチックを合成するか検証した