Low-mutation-rate, reduced-genome Escherichia coli: an improved host for faithful maintenance of engineered genetic constructs.
Low-mutation-rate, reduced-genome Escherichia coli: an improved host for faithful maintenance of engineered genetic constructs.
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DOI：
10.1186/1475-2859-11-11
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发表时间：
2012-01-20
影响因子：
6.4
通讯作者：
Pósfai G
中科院分区：
文献类型：
--
作者：
Csörgo B;Fehér T;Tímár E;Blattner FR;Pósfai G
产生遗传变异的分子机制为种群在不断变化的环境中进化和长期生存提供了基础。在稳定的实验室条件下，变异产生机制是可有可无的，因为细胞适应不利条件的需求有限。事实上，当研究高度精细、精确的分子和合成生物学任务时，新出现的、进化的特征可能是不可取的。通过构建低突变率变异，我们降低了MDS42的进化能力，MDS42是一种减少基因组的大肠杆菌菌株，缺乏大多数与实验室/工业应用无关的基因。消除多样性产生，容易出错的DNA聚合酶参与诱变实现了基因组的显著稳定。所得到的菌株在保持正常生长的同时，总体突变率显著下降，在各种胁迫条件下最为明显。此外，易出错的无聚合酶宿主允许表达甲基转移酶的毒性克隆相对稳定地维持。相比之下，亲本菌株产生的突变克隆无法产生功能性甲基转移酶，这些突变克隆在培养物中迅速过度生长（在24小时的诱导期内，50%的克隆缺乏功能性甲基转移酶活性）。两种菌株之间令人惊讶的巨大稳定性差异是由于亲本菌株的高压诱变，有毒蛋白表达的生长抑制以及不再产生活性有毒酶的突变体的选择/生长的综合作用。通过消除应激诱导的易出错dna聚合酶，可移动的无遗传元件的大肠杆菌菌株MDS42的基因组进一步稳定。由此产生的菌株在各种合成和分子生物学应用中代表了一种改进的宿主，允许更稳定地生产生长抑制生物分子。
Molecular mechanisms generating genetic variation provide the basis for evolution and long-term survival of a population in a changing environment. In stable, laboratory conditions, the variation-generating mechanisms are dispensable, as there is limited need for the cell to adapt to adverse conditions. In fact, newly emerging, evolved features might be undesirable when working on highly refined, precise molecular and synthetic biological tasks. By constructing low-mutation-rate variants, we reduced the evolutionary capacity of MDS42, a reduced-genome E. coli strain engineered to lack most genes irrelevant for laboratory/industrial applications. Elimination of diversity-generating, error-prone DNA polymerase enzymes involved in induced mutagenesis achieved a significant stabilization of the genome. The resulting strain, while retaining normal growth, showed a significant decrease in overall mutation rates, most notably under various stress conditions. Moreover, the error-prone polymerase-free host allowed relatively stable maintenance of a toxic methyltransferase-expressing clone. In contrast, the parental strain produced mutant clones, unable to produce functional methyltransferase, which quickly overgrew the culture to a high ratio (50% of clones in a 24-h induction period lacked functional methyltransferase activity). The surprisingly large stability-difference observed between the strains was due to the combined effects of high stress-induced mutagenesis in the parental strain, growth inhibition by expression of the toxic protein, and selection/outgrowth of mutants no longer producing an active, toxic enzyme. By eliminating stress-inducible error-prone DNA-polymerases, the genome of the mobile genetic element-free E. coli strain MDS42 was further stabilized. The resulting strain represents an improved host in various synthetic and molecular biological applications, allowing more stable production of growth-inhibiting biomolecules.
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影响因子：
16
作者：
Berdichevsky, A;Izhar, L;Livneh, Z
通讯作者：
Livneh, Z
影响因子：
3.2
作者：
Frisch, Ryan L.;Su, Yang;Hastings, P. J.
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Hastings, P. J.
影响因子：
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作者：
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Romesberg, FE
影响因子：
14.9
作者：
AMSTER, O;SALOMON, D;ZAMIR, A
通讯作者：
ZAMIR, A
影响因子：
6.4
作者：
Ferrer-Miralles N;Domingo-Espín J;Corchero JL;Vázquez E;Villaverde A
通讯作者：
Villaverde A