Goto Saki, Miyahara Yuki, Taguchi Seiichi, Tsuge Takeharu, Hiroe Ayaka
Department of Chemistry for Life Sciences and Agriculture, Faculty of Life Sciences, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan.
MIRAI, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
Microorganisms. 2022 Feb 16;10(2):458. doi: 10.3390/microorganisms10020458.
The biodegradable polyester poly-()-3-hydroxybutyrate [P(3HB)] is synthesized by a polymerizing enzyme called polyhydroxyalkanoate (PHA) synthase and accumulates in a wide variety of bacterial cells. Recently, we demonstrated the secretory production of a ()-3HB oligomer (3HBO), a low-molecular-weight P(3HB), by using recombinant expressing PHA synthases. The 3HBO has potential value as an antibacterial substance and as a building block for various polymers. In this study, to construct an efficient 3HBO production system, the coexpression of molecular chaperones and a PHA synthase derived from YB-4 (PhaRC) was examined. First, genes encoding enzymes related to 3HBO biosynthesis (, and derived from H16) and two types of molecular chaperones (, , and ) were introduced into the strains BW25113 and BW25113Δ. As a result, coexpression of the chaperones promoted the enzyme activity of PHA synthase (approximately 2-3-fold) and 3HBO production (approximately 2-fold). The expression assay of each chaperone and PHA synthase subunit (PhaR and PhaC) indicated that the combination of the two chaperone systems (GroEL-GroES and TF) supported the folding of PhaR and PhaC. These results suggest that the utilization of chaperone proteins is a valuable approach to enhance the formation of active PHA synthase and the productivity of 3HBO.
可生物降解的聚酯聚（-）-3-羟基丁酸酯[P（3HB）]由一种名为聚羟基链烷酸酯（PHA）合酶的聚合酶合成，并在多种细菌细胞中积累。最近，我们通过使用表达PHA合酶的重组体证明了低分子量P（3HB）即（-）-3-羟基丁酸酯寡聚物（3HBO）的分泌生产。3HBO作为一种抗菌物质和各种聚合物的构建单元具有潜在价值。在本研究中，为构建一个高效的3HBO生产系统，研究了分子伴侣与源自YB-4的PHA合酶（PhaRC）的共表达。首先，将编码与3HBO生物合成相关的酶（源自H16的、和）以及两种分子伴侣（、和）的基因导入大肠杆菌菌株BW25113和BW25113Δ。结果，分子伴侣的共表达促进了PHA合酶的酶活性（约2-3倍）和3HBO的产量（约2倍）。对每个分子伴侣和PHA合酶亚基（PhaR和PhaC）的表达分析表明，两种分子伴侣系统（GroEL-GroES和TF）的组合支持PhaR和PhaC的折叠。这些结果表明利用伴侣蛋白是增强活性PHA合酶形成和3HBO生产力的一种有价值的方法。
