Comparative genomics and experimental evolution of Escherichia coli BL21(DE3) strains reveal the landscape of toxicity escape from membrane protein overproduction.
Comparative genomics and experimental evolution of Escherichia coli BL21(DE3) strains reveal the landscape of toxicity escape from membrane protein overproduction.
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DOI：
10.1038/srep16076
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发表时间：
2015-11-04
影响因子：
4.6
通讯作者：
Kim JF
中科院分区：
文献类型：
--
作者：
Kwon SK;Kim SK;Lee DH;Kim JF
获得足够的蛋白质产量是当代生物科学和生物技术的第一个瓶颈。为了在一个重要的生物体如大肠杆菌中成功地过表达膜蛋白。大肠杆菌，定义和合理的优化策略的基础上的理解的遗传背景的毒性逃逸机制是可取的。为此，我们对E. coli C41（DE3）及其衍生物C43（DE3），并对它们进行了初步筛选。并与其祖先菌株E. coli BL21（DE3）中，两株菌均发生了不同程度的遗传变异。一系列的E.通过体外进化产生能够耐受膜蛋白转化或膜蛋白过表达的大肠杆菌变体。进化菌株的靶向测序揭示了获得性遗传变化中的突变热点。通过这些组合的方法，我们发现在乳糖操纵子的乳糖阻遏基因的非同义变化，以及在DE 3区的lacUV5启动子的核苷酸取代，通过它的毒性作用，对主机所造成的过度表达的膜蛋白可以减轻。lacI的突变被证明是至关重要的赋予耐受膜蛋白过表达。
Achieving sufficient yields of proteins in their functional form represents the first bottleneck in contemporary bioscience and biotechnology. To accomplish successful overexpression of membrane proteins in a workhorse organism such as E. coli, defined and rational optimization strategies based on an understanding of the genetic background of the toxicity-escape mechanism are desirable. To this end, we sequenced the genomes of E. coli C41(DE3) and its derivative C43(DE3), which were developed for membrane protein production. Comparative analysis of their genomes with those of their ancestral strain E. coli BL21(DE3) revealed various genetic changes in both strains. A series of E. coli variants that are able to tolerate transformation with or overexpression of membrane proteins were generated by in vitro evolution. Targeted sequencing of the evolved strains revealed the mutational hotspots among the acquired genetic changes. By these combinatorial approaches, we found non-synonymous changes in the lac repressor gene of the lac operon as well as nucleotide substitutions in the lacUV5 promoter of the DE3 region, by which the toxic effect to the host caused by overexpression of membrane proteins could be relieved. A mutation in lacI was demonstrated to be crucial for conferring tolerance to membrane protein overexpression.