Department of Genetics, University of Georgia, Athens, Georgia, United States of America.
PLoS One. 2012;7(8):e43844. doi: 10.1371/journal.pone.0043844. Epub 2012 Aug 22.
Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to transformation (in this case apparently absolute) and that methylation with an endogenous unique α-class N4-Cytosine methyltransferase is required for transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA transformation and the combined frequencies of transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation.
能够利用复杂基质的嗜热微生物在将木质纤维素生物质转化为生物燃料和生物制品方面具有特殊优势。属于革兰氏阳性菌属的 Caldicellulosiruptor 成员是厌氧嗜热菌，最适生长温度在 65°C 到 78°C 之间，是已知最耐热的纤维素分解菌。事实上，它们可以有效地利用未经预处理的生物质作为唯一的碳源，并且在连续几轮的应用中消化了 70%的总柳枝稷基质。对这些生物体进行基因操作的能力是将它们用于转化这些复杂基质以获得感兴趣的产物以及鉴定对其利用未经预处理的生物质的能力至关重要的基因产物的前提。在这里，我们报告了该属的一个成员 C. bescii 的 DNA 转化的第一个例子。我们表明，DNA 的限制是转化的主要障碍（在这种情况下显然是绝对的），并且来自大肠杆菌的 DNA 的转化需要内源性独特的α类 N4-胞嘧啶甲基转移酶的甲基化。使用修饰的 DNA 导致了一种高效且可重复的 DNA 转化方法的发展，并且转化和重组的组合频率允许在非复制质粒和染色体基因之间进行标记替换，为快速有效的遗传操作方法提供了基础。
