Functional heterologous expression of an engineered full length CipA from Clostridium thermocellum in Thermoanaerobacterium saccharolyticum.
Functional heterologous expression of an engineered full length CipA from Clostridium thermocellum in Thermoanaerobacterium saccharolyticum.
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DOI：
10.1186/1754-6834-6-32
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发表时间：
2013-03-01
影响因子：
6.3
通讯作者：
Lynd LR
中科院分区：
文献类型：
--
作者：
Currie DH;Herring CD;Guss AM;Olson DG;Hogsett DA;Lynd LR
纤维素是高度顽固的，因此需要一套特殊的酶将其溶解成可发酵的糖。在C. thermocellum中，这些细胞外酶以一种称为纤维素体的高活性多组分系统存在。本研究探讨了一种关键的C. thermocellum cellulosomal component在T. saccharolyticum中的表达，作为利用异源表达的纤维素体创造一种能够巩固生物处理的嗜热细菌的一步。本研究以天然糖溶菌xynA启动子为基础，建立了木聚糖和木糖诱导的诱导启动子体系。该启动子用于表达C. thermocellum野生型cipA的工程形式的胞体成分cipA*。验证了CipA*的表达和在上清液上的定位。将ΔcipA突变型C. thermocellum菌株与表达CipA*的T. saccharolyticum菌株一起培养，观察到每升10克高结晶纤维素SigmaCell 101的水解和发酵效果。这种cipA缺失的跨物种互补证明了cipA *组装功能性纤维素的能力。本研究是工程嗜热菌异源表达纤维素结构成分的第一个例子。为了实现这一目标，我们开发并测试了一种可诱导的启动子，用于糖溶菌的控制表达，以及一种合成的cipA。此外，我们证明了微晶纤维素的高度水解（高达93%）。
Cellulose is highly recalcitrant and thus requires a specialized suite of enzymes to solubilize it into fermentable sugars. In C. thermocellum, these extracellular enzymes are present as a highly active multi-component system known as the cellulosome. This study explores the expression of a critical C. thermocellum cellulosomal component in T. saccharolyticum as a step toward creating a thermophilic bacterium capable of consolidated bioprocessing by employing heterologously expressed cellulosomes. We developed an inducible promoter system based on the native T. saccharolyticum xynA promoter, which was shown to be induced by xylan and xylose. The promoter was used to express the cellulosomal component cipA*, an engineered form of the wild-type cipA from C. thermocellum. Expression and localization to the supernatant were both verified for CipA*. When a ΔcipA mutant C. thermocellum strain was cultured with a CipA*-expressing T. saccharolyticum strain, hydrolysis and fermentation of 10 grams per liter SigmaCell 101, a highly crystalline cellulose, were observed. This trans-species complementation of a cipA deletion demonstrated the ability for CipA* to assemble a functional cellulosome. This study is the first example of an engineered thermophile heterologously expressing a structural component of a cellulosome. To achieve this goal we developed and tested an inducible promoter for controlled expression in T. saccharolyticum as well as a synthetic cipA. In addition, we demonstrate a high degree of hydrolysis (up to 93%) on microcrystalline cellulose.
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影响因子：
4.1
作者：
Caspi, Jonathan;Irwin, Diana;Bayer, Edward A.
通讯作者：
Bayer, Edward A.
影响因子：
6
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影响因子：
3
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影响因子：
4.4
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通讯作者：
Schwarz, Wolfgang H.
影响因子：
4.4
作者：
Caspi, Jonathan;Barak, Yoav;Bayer, Edward A.
通讯作者：
Bayer, Edward A.