Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization.
Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization.
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解脂耶氏酵母中吸水链霉菌过量生产转谷氨酰胺酶原及其生化特性
DOI：
10.1186/s12896-015-0193-1
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发表时间：
2015-08-14
影响因子：
3.5
通讯作者：
Chen J
中科院分区：
文献类型：
--
作者：
Liu S;Wan D;Wang M;Madzak C;Du G;Chen J
转谷氨酰胺酶（TGase）是链霉菌中作为酶原（前TGase）合成的，是食品工业中的重要酶。由于TGase在食品工业中的重要应用，获得强健且食品安全的TGase生产菌株在过去十年中引起了广泛关注。在这项研究中，吸水链霉菌前 TGase 由食品级宿主解脂耶氏酵母有效表达和分泌，无需抗生素标记。将pro-TGase基因克隆到整合载体pINA1296（单拷贝）和pINA1297（多拷贝）中，并分别用于转化解脂耶氏酵母Po1g或Po1h菌株。表达由重组 hp4d 启动子驱动，并使用 XPR2 前序列作为信号肽获得分泌。重组Po1h菌株产生的胞外前TGase的最高产量相当于5.3 U/mL TGase，比使用重组Po1g菌株获得的水平高8.8倍。来自 pro-TGase 的两个潜在 Asn 连接糖基化位点（Asn160 和 Asn355）中的天冬酰胺单独或同时突变为谷氨酰胺，产生去糖基化变体 N160Q、N355Q 和 N160Q/N355Q。 N160Q、N355Q 和 N160Q/N355Q 构建体的活性分别为 5.3 U/mL、7.8 U/mL 和 3.0 U/mL，相当于野生型 pro-TGase 的 100%、147% 和 57%。通过在 3 L 发酵罐中使用甘油补料策略，N355Q 变体的 TGase 产量提高至 35.3 U/mL。活化的前 TGase 及其去糖基化变体的最佳 pH 值和温度分别在 5.0-6.0 pH 和 40-45 °C 范围内。重组野生型pro-TGase在37℃下的半衰期达到34.0min，变异体的半衰期为24.2min至11.5min。与野生型pro-TGase相比，所有变体的比活性均降低，并且变体的Km和kcat值均相应降低。这项研究首次报道了解脂耶氏酵母中前 TGase 的异源表达，并为高效生产用于食品加工的 TGase 提供了新的可能性。
Transglutaminases (TGase), synthesized as a zymogen (pro-TGase) in Streptomyces sp., are important enzymes in food industry. Due to the important applications of TGase in food industry, obtaining robust and food-safe TGase-producing strains has attracted much attention during the past decade. In this study, Streptomyces hygroscopicus pro-TGase was efficiently expressed and secreted by a food-grade host, Yarrowia lipolytica, without antibiotic markers. The pro-TGase gene was cloned into integrative vectors pINA1296 (monocopy) and pINA1297 (multicopy), and was used to transform the Y. lipolytica Po1g or Po1h strain, respectively. Expression was driven by a recombinant hp4d promoter and secretion obtained using a XPR2 pre-sequence as a signal peptide. The highest yield of extracellular pro-TGase produced by the recombinant Po1h strain corresponded to 5.3 U/mL of TGase, a level 8.8 fold higher than that obtained using the recombinant Po1g strain. Asparagines in two potential Asn-linked glycosylation sites (Asn160 and Asn355) from pro-TGase were mutated to glutamine individually or simultaneously, yielding the deglycosylated variants N160Q, N355Q, and N160Q/N355Q. The activities of N160Q, N355Q and N160Q/N355Q constructs were respectively 5.3 U/mL, 7.8 U/mL, and 3.0 U/mL, equivalent to 100 %, 147 %, and 57 % of that from wild-type pro-TGase. The TGase yield of N355Q variant was raised to 35.3 U/mL of by using a glycerol feeding strategy in a 3 L fermenter. The optimal pH and temperature of the activated pro-TGase, and of its deglycosylated variants, were in the range of 5.0-6.0 pH and 40-45 °C, respectively. The half-life of the recombinant wild-type pro-TGase at 37 °C reached 34.0 min, and those of the variants were from 24.2 min to 11.5 min. In contrast to the wild-type pro-TGase, all of the variants had decreased specific activities, and both the Km and kcat values of the variants decreased accordingly. This study constitutes the first report of the heterologous expression of a pro-TGase in Y. lipolytica, and provides new possibilities for the efficient production of TGases used in food processing.