Population level analysis of evolved mutations underlying improvements in plant hemicellulose and cellulose fermentation by Clostridium phytofermentans.
Population level analysis of evolved mutations underlying improvements in plant hemicellulose and cellulose fermentation by Clostridium phytofermentans.
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DOI：
10.1371/journal.pone.0086731
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Blanchard JL
中科院分区：
文献类型：
--
作者：
Mukherjee S;Thompson LK;Godin S;Schackwitz W;Lipzen A;Martin J;Blanchard JL
植物细胞壁的复杂性给微生物分解带来了许多挑战。Clostridiumphytofermentans是一种从森林土壤中分离出来的厌氧细菌，它直接分解和利用许多植物细胞壁碳水化合物。本研究的目的是了解植物分解率的限制条件，并确定可能克服这些限制的分子机制。通过在指数生长期间重复的连续转移的实验进化被用于选择C。phytofermentans基因型在纤维二糖、纤维素和木聚糖上生长更快。为了鉴定潜在的突变，每个群体产生平均13，600，000个配对末端读段，导致基因组中每个位点的300倍覆盖。等位基因频率为5%或更高的突变可以用统计学置信度鉴定。许多突变发生在碳水化合物相关基因中，包括糖苷水解酶的启动子区域和参与碳水化合物摄取的ABC转运蛋白中的氨基酸取代、检测特定碳水化合物的信号转导传感器、影响胞外酶输出的蛋白质以及未知特异性的调节剂。ABC转运蛋白复合物蛋白的结构模型表明，这些基因中的突变可能会改变底物结合蛋白对碳水化合物的识别，以及跨膜和ATP酶结合蛋白的细胞间面之间的通信。实验进化在识别半纤维素和纤维素发酵速率的分子限制方面是有效的，并且被选择用于通常不出现在传统分子遗传筛选中的功能突变的推定增益。这些结果揭示了进化和工程微生物的新策略，以便在植物碳水化合物上更快地生长。
The complexity of plant cell walls creates many challenges for microbial decomposition. Clostridium phytofermentans, an anaerobic bacterium isolated from forest soil, directly breaks down and utilizes many plant cell wall carbohydrates. The objective of this research is to understand constraints on rates of plant decomposition by Clostridium phytofermentans and identify molecular mechanisms that may overcome these limitations. Experimental evolution via repeated serial transfers during exponential growth was used to select for C. phytofermentans genotypes that grow more rapidly on cellobiose, cellulose and xylan. To identify the underlying mutations an average of 13,600,000 paired-end reads were generated per population resulting in ∼300 fold coverage of each site in the genome. Mutations with allele frequencies of 5% or greater could be identified with statistical confidence. Many mutations are in carbohydrate-related genes including the promoter regions of glycoside hydrolases and amino acid substitutions in ABC transport proteins involved in carbohydrate uptake, signal transduction sensors that detect specific carbohydrates, proteins that affect the export of extracellular enzymes, and regulators of unknown specificity. Structural modeling of the ABC transporter complex proteins suggests that mutations in these genes may alter the recognition of carbohydrates by substrate-binding proteins and communication between the intercellular face of the transmembrane and the ATPase binding proteins. Experimental evolution was effective in identifying molecular constraints on the rate of hemicellulose and cellulose fermentation and selected for putative gain of function mutations that do not typically appear in traditional molecular genetic screens. The results reveal new strategies for evolving and engineering microorganisms for faster growth on plant carbohydrates.
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影响因子：
4.4
作者：
Guimaraes, Pedro M. R.;Francois, Jean;Domingues, Lucilia
通讯作者：
Domingues, Lucilia
影响因子：
12.3
作者：
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通讯作者：
Zhang J
DOI：
10.1016/j.bbrc.2009.12.013
发表时间：
2010-01-01
影响因子：
3.1
作者：
Hu, Hongbo;Wood, Thomas K.
通讯作者：
Wood, Thomas K.
影响因子：
12.3
作者：
Chu VT;Gottardo R;Raftery AE;Bumgarner RE;Yeung KY
通讯作者：
Yeung KY
影响因子：
5.8
作者：
Arnold, K;Bordoli, L;Schwede, T
通讯作者：
Schwede, T