Production of multifunctional chimaeric enzymes in plants: a promising approach for degrading plant cell wall from within
Production of multifunctional chimaeric enzymes in plants: a promising approach for degrading plant cell wall from within
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DOI：
10.1111/j.1467-7652.2009.00484.x
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发表时间：
2010-04-01
影响因子：
13.8
通讯作者：
Yuan, Ling
中科院分区：
文献类型：
--
作者：
Fan, Zhanmin;Yuan, Ling
P>；多功能嵌合水解酶可以通过共价连接单一多肽中的异源催化结构域和功能结构域来产生。以前，我们已经产生了一些嵌合的木质纤维素水解酶，包含两到五个模块[Biotechnol Bioeng(2009)102：1045；Appl Environ Microbiol(2009)75：1754]。这些嵌合体在动力学和其他酶性质上与亲本酶非常相似，并且与亲本酶的混合物相比，一些嵌合体在降解天然底物方面表现出更好的协同作用。除了在发酵酶生产中的应用外，嵌合基因还可用于构建携带多个基因的单一植物转化双元载体，这些基因编码一套完整的木质纤维素水解酶活性。这种方法的优点包括易于构建和转化载体，以及下游植物分析和育种。固定在生物质原料中的水解酶可能有助于酶的预处理和糖的转化。在这里，我们报道了一种嵌合型半纤维素酶在转基因烟草中的基因表达和功能特性。T1转基因植株每克总可溶性叶蛋白产生高达19毫克的活性酶。结果表明，在植物中生产多功能木质纤维素水解酶是可行的。讨论了嵌合基因的设计、构建和植物表达中的关键问题。
P>Multifunctional chimaeric hydrolases can be created by covalently linking heterologous catalytic and functional domains in a single polypeptide. Previously, we have generated a number of chimaeric lignocellulosic hydrolases that contain two to five modules [Biotechnol Bioeng (2009) 102: 1045; Appl Environ Microbiol (2009) 75: 1754]. These chimaeras closely resemble the parental enzymes in kinetics and other enzymatic properties, and some exhibit improved synergy in degrading natural substrates when compared to mixtures of parental enzymes. In addition to the applications in fermentative enzyme production, the chimaeric genes can be used in the construction of a single plant transformation binary vector carrying several genes that encode a complete set of lignocellulosic hydrolase activities. The advantages of this approach include ease in vector construction and transformation, as well as downstream plant analysis and breeding. The hydrolases sequestered in biomass feedstock can potentially assist enzymatic pretreatment and sugar conversion. Here, we report the gene expression and functional characterization of a chimaeric hemicellulase in transgenic tobacco plants. T1 transgenic plants produced up to 19-mg active enzymes per gram of total-soluble leaf proteins. The results demonstrate the feasibility of producing multifunctional lignocellulosic hydrolases in plants. Key considerations in the design, construction and plant expression of the chimaeric genes are discussed.