Improving the active expression of transglutaminase in Streptomyces lividans by promoter engineering and codon optimization.
Improving the active expression of transglutaminase in Streptomyces lividans by promoter engineering and codon optimization.
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通过启动子工程和密码子优化提高浅青紫链霉菌中转谷氨酰胺酶的活性表达
DOI：
10.1186/s12896-016-0304-7
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发表时间：
2016-10-28
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;Wang M;Du G;Chen J
背景：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。由于这种前肽对于链霉菌 TGase 的正确折叠至关重要，因此 TGase 通常以无活性的前 TGase 形式表达，然后通过在体外添加激活的蛋白酶将其转化为活性 TGase。本研究通过启动子工程和密码子优化，由变青链霉菌主动产生吸水链霉菌TGase。结果：从吸水链霉菌WSH03-13中扩增出编码pro-TGase及其内源启动子区、信号肽和终止子的基因片段（tg1，2.6 kb），并克隆到质粒pIJ86中，得到pIJ86/tg1。发酵2天后，携带pIJ86/tg1的S.lividans TK24产生了1.8 U/mL的TGase，并且在培养物上清液中检测到清晰的TGase条带（38 kDa）。这些结果表明，通过使用TGase启动子，pro-TGase在变青链球菌TK24中成功表达并正确加工成活性TGase。根据缺失分析，TGase启动子的完整序列仅限于-693至-48区域。我们还在 TGase 启动子中鉴定了一个负元件（-198 至 -148），与 S. lividans 表达 pIJ86/tg1 的方法相反，该元件的缺失使 TGase 产量增加了 81.3%。结合启动子负元件的缺失和基因密码子的优化，重组变青链霉菌中TGase的产量和产率分别达到5.73 U/mL和0.14 U/mL/h。结论：我们构建了高产、高产的活性TGase菌株，优化后的TGase启动子可以作为链霉菌中其他蛋白表达的良好候选启动子。
Background:Transglutaminases (TGase), which are synthesized as a zymogen (pro-TGase) in Streptomyces sp., are important enzymes in the food industry. Because this pro-peptide is essential for the correct folding of Streptomyces TGase, TGase is usually expressed in an inactive pro-TGase form, which is then converted to active TGase by the addition of activating proteases in vitro. In this study, Streptomyces hygroscopicus TGase was actively produced by Streptomyces lividans through promoter engineering and codon optimization.Results:A gene fragment (tg1, 2.6 kb) that encoded the pro-TGase and its endogenous promoter region, signal peptide and terminator was amplified from S. hygroscopicus WSH03-13 and cloned into plasmid pIJ86, which resulted in pIJ86/tg1. After fermentation for 2 days, S. lividans TK24 that harbored pIJ86/tg1 produced 1.8 U/mL of TGase, and a clear TGase band (38 kDa) was detected in the culture supernatant. These results indicated that the pro-TGase was successfully expressed and correctly processed into active TGase in S. lividans TK24 by using the TGase promoter. Based on deletion analysis, the complete sequence of the TGase promoter is restricted to the region from -693 to -48. We also identified a negative element (-198 to -148) in the TGase promoter, and the deletion of this element increased the TGase production by 81.3 %, in contrast to the method by which S. lividans expresses pIJ86/tg1. Combining the deletion of the negative element of the promoter and optimization of the gene codons, the yield and productivity of TGase reached 5.73 U/mL and 0.14 U/mL/h in the recombinant S. lividans, respectively.Conclusions:We constructed an active TGase-producing strain that had a high yield and productivity, and the optimized TGase promoter could be a good candidate promoter for the expression of other proteins in Streptomyces.
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DOI：
10.1007/s10295-008-0373-2
发表时间：
2008-09-01
影响因子：
3.4
作者：
Lin, Shie-Jea;Hsieh, Yi-Fang;Chu, Wen-Shen
通讯作者：
Chu, Wen-Shen
影响因子：
4.4
作者：
Kikuchi, Y;Date, M;Matsui, H
通讯作者：
Matsui, H
影响因子：
2.8
作者：
Ali, N;Herron, PR;Dyson, PJ
通讯作者：
Dyson, PJ
影响因子：
8.8
作者：
Cui, Li;Du, Guocheng;Chen, Jian
通讯作者：
Chen, Jian
影响因子：
3.9
作者：
Noda, Shuhei;Miyazaki, Takaya;Kondo, Akihiko
通讯作者：
Kondo, Akihiko