以重組枯草桿菌生產海藻糖合成酶

Production of Trehalose Synthase in Recombinant Bacillus subtilis

枯草桿菌目前常被運用來將重組蛋白質外泌到胞外，這種優勢特性可以簡化蛋白質下游純化步驟。然枯草桿菌中使用的質體往往產生不穩定問題，且會造成細胞生理的壓力，以致難以穩定和大量分泌生產重組蛋白質。海藻糖 (Trehalose)屬於非還原性雙糖，在自然界中廣泛的存在於生物體內，以保護生物體生存於環境壓力，如乾旱、冷凍、高溫與高鹽等。近年來，有許多研究對於海藻糖在各種生物體的功能進行探討，進而大幅提昇了海藻糖的應用性。嗜酸性古細菌 Picrophilus torridus DSM 9790擁有海藻糖合成酶trehalose synthase (PTTS)，該酵素具有高海藻糖轉化效率，極具有工業化的發展潛力。在本計畫中，我們以染色體工程技術，發展一株穩定且高效率生產重組海藻糖合成的枯草桿菌生產菌株，並使用Box-Behnken回應曲面法來將培養基進行最佳化以量產海藻糖合成酶。以5公升醱酵槽進行生產，海藻糖合成PTTS最大產量可達175.89 ± 6.83 mg/L。

Bacillus subtilis is most commonly employed for secretion of recombinant proteins, which could facilitate the downstream processing of proteins. However, plasmids used in B. subtilis are usually unstable and increase the metabolic burden in the cells. This makes stable and mass production of secreted recombinant proteins very difficult. Trehalose is a non-reducing disaccharide and is widespread in microorganisms in nature. This sugar confers on living cells the ability to survive in stressed environments such as desiccation, freezing, high temperature and high salt condition. Recently, many studies on biological properties of trehalose have substantially extended its application range. Picrophilus torridus DSM 9790, a thermoacidophilic archaea, carries trehalose synthase (PTTS) and exhibits a high activity for trehalose synthesis. Based on genomic engineering technique, a potent recombinant trehalose synthase producer strain will be constructed. Meanwhile, the response surface methodology of Box-Behnken method will be applied to optimize medium for mass-producing trehalose synthase PTTS. The highest production of trehalose synthase PTTS could reach 175.89 ± 6.83 mg/L for fermentation of 5 L fermentor.