Combinatorial Sec pathway analysis for improved heterologous protein secretion in Bacillus subtilis: identification of bottlenecks by systematic gene overexpression.
Combinatorial Sec pathway analysis for improved heterologous protein secretion in Bacillus subtilis: identification of bottlenecks by systematic gene overexpression.
复制标题
用于改善枯草芽孢杆菌异源蛋白分泌的组合 Sec 途径分析：通过系统基因过表达识别瓶颈。
DOI：
10.1186/s12934-015-0282-9
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发表时间：
2015-06-26
影响因子：
6.4
通讯作者：
Wen J
中科院分区：
文献类型：
--
作者：
Chen J;Fu G;Gai Y;Zheng P;Zhang D;Wen J
通过B分泌表达有价值的蛋白质。枯草芽孢杆菌及其相关物种在过去的三十年里吸引了大量的工作。虽然同源蛋白质可以获得非常高的产量，但是通过B.不幸的是，subtilis不是直接的。Sec途径是B中蛋白质分泌的主要途径。枯草杆菌。因此，本研究的目的是通过分子遗传学技术来确定Sec途径的瓶颈，并提高异源蛋白的分泌。两种α-淀粉酶（AmyL和AmyS）均在PHpaII启动子的控制下，并配备有其天然信号肽SPamyl和SPamyS，成功分泌，但表达水平显着不同。为了提高分泌效率，通过在染色体上增加额外的拷贝来单独过表达参与Sec途径或与Sec途径密切相关的23个主要基因或基因操纵子，并且prsA的过表达使α-淀粉酶（AmyL和AmyS）的产量分别提高了3.2倍和5.5倍。通过不同浓度木糖的诱导，优化了prsA的过表达水平，进一步提高了α-淀粉酶的分泌效率。将prsA与9个筛选出的基因或基因操纵子组合过表达，与单独过表达prsA相比，prsA与部分dnaK操纵子组合过表达使AmyL和AmyS的α-淀粉酶活性分别提高了160%和173%。最后，对重组B.在7.5L发酵罐中对菌株1A 237进行分批补料发酵，AmyL和AmyS的分泌量分别达到1,352和2, 300 U/mL，生产能力分别为16.1U/mL·h和27.4U/mL·h。我们设计了系统的基因过表达方法来研究B中Sec途径的瓶颈。枯草杆菌。PrsA脂蛋白和DnaK系列分子伴侣的缺乏是外源蛋白分泌的主要限速因素。深入系统地了解Sec途径各组分之间的相互作用可能是彻底提高异源蛋白产量的关键。本文的在线版本（doi：10.1186/s12934-015-0282-9）包含补充材料，可供授权用户使用。
Secretory expression of valuable proteins by B. subtilis and its related species has attracted intensive work over the past three decades. Although very high yields can be achieved with homologous proteins, production of heterologous proteins by B. subtilis is unfortunately not the straight forward. The Sec pathway is the major route for protein secretion in B. subtilis. Therefore, the aim of this work was to identify the bottlenecks of the Sec pathway and improve the secretion of heterologous proteins by molecular genetic techniques. Two α-amylases (AmyL and AmyS) both under the control of the PHpaII promoter and equipped with their native signal peptides SPamyl and SPamyS were successfully secreted with significantly different expression levels. To improve the secretion efficiency, 23 main genes or gene operons involved in or closely related to the Sec pathway were overexpressed singly by increasing an additional copy on the chromosome, and the overexpression of prsA enhanced the production of α-amylases (AmyL and AmyS) by 3.2- and 5.5-fold, respectively. With the induction by xylose of different concentrations, prsA overexpression level was optimized and the secretion efficiency of α-amylase was further improved. Moreover, combinatorial overexpression of prsA and nine screened genes or gene operons, respectively, was performed, and the overexpression of prsA combined with partial dnaK operon improved the α-amylase activity of AmyL and AmyS by 160 and 173%, respectively, compared with the overexpression of prsA singly. Finally, the performance of the recombinant B. subtilis 1A237 was evaluated with the fed-batch fermentation in 7.5 L fermentor, and the level of secreted AmyL and AmyS reached 1,352 and 2,300 U/mL with the productivity of 16.1 U/mL h and 27.4 U/mL h, respectively. Our systematic gene overexpression approach was designed to investigate the bottleneck of Sec pathway in B. subtilis. The deficiency of PrsA lipoprotein and chaperones of DnaK series was main rate-limiting factors for heterologous proteins secretion. Systematic and deep insight into how components of Sec pathway interact with each other may be the key to improving the yield of heterologous proteins thoroughly. The online version of this article (doi:10.1186/s12934-015-0282-9) contains supplementary material, which is available to authorized users.