Enhanced secretory production of hemolysin-mediated cyclodextrin glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system
Enhanced secretory production of hemolysin-mediated cyclodextrin glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system
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DOI：
10.1016/j.jbiotec.2010.10.001
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发表时间：
2010-12-01
影响因子：
4.1
通讯作者：
Illias, Rosli Md.
中科院分区：
文献类型：
--
作者：
Low, Kheng Oon;Mahadi, Nor Muhammad;Illias, Rosli Md.
当溶血素转运系统与 HlyA (HlyAs(61)) C 端 61 个氨基酸融合时，它会介导环糊精葡聚糖转移酶 (CGTase) 释放到细胞外介质中。为了产生改进的分泌变体，百合成分（hlyAs、hlyB 和 hlyD）通过使用易错 PCR 的定向进化进行改造。在固体LB-淀粉平板上筛选Hly突变体的晕区大于亲本菌株。通过筛选约1 x 10(4)个大肠杆菌BL21 (DE3)转化体，我们成功分离出5个突变体，其CGTase-HlyAs(61)的分泌水平相对于野生型菌株增加了35-217%。每个突变体的突变位点位于HlyB，主要沿着跨膜结构域，这意味着相应的区域对于改善靶蛋白的分泌非常重要。在这项研究中，我们描述了 HlyB 中负责增强大肠杆菌 CGTase 分泌的新位点的发现。 (C) 2010 Elsevier B.V. 保留所有权利。
The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferase (CGTase) into the extracellular medium when it was fused to the C-terminal 61 amino acids of HlyA (HlyAs(61)). To produce an improved-secretion variant, the lily components (hlyAs, hlyB and hlyD) were engineered by directed evolution using error-prone PCR. Hly mutants were screened on solid LB-starch plate for halo zone larger than the parent strain. Through screening of about 1 x 10(4) Escherichia coli BL21 (DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs(61), relative to the wild-type strain. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain, implying that the corresponding region was important for the improved secretion of the target protein. In this study we describe the finding of novel site(s) of HlyB responsible for enhancing secretion of CGTase in E. coli. (C) 2010 Elsevier B.V. All rights reserved.