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首页> 外文期刊>Proceeding of the Singapore National Academy of Science >Design and Construction of a Synthetic Riboregulator-Based Platform for Metabolic Shunting of Pathways in Lactococcus lactis
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Design and Construction of a Synthetic Riboregulator-Based Platform for Metabolic Shunting of Pathways in Lactococcus lactis

机译：合成的设计和施工Riboregulator-Based平台代谢分流途径在Lactococcus lactis

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Microbial cell factories are subjected to rewiring of basic metabolism to enhance the carbon flux towards the desired product pathway. Conventionally, this metabolic engineering approach often involves over-expression of pathway genes and knocking out genes in the competing pathways. However, these approaches result in severe metabolic burden and eventual poor performance of the cells. Particularly, where biomass formation and product synthesis depend on common precursors, permanent changes like knocking out genes will only result in poor titer. Hence, temporary decoupling of biomass formation and product synthesis is considered to be a potential alternative. In this study, we designed synthetic riboregulators, which are RNA-based genetic switches, to shunt metabolic pathways in Lactococcus lactis bacteria, a GRAS organism employed as a cell factory for many biocompounds. The riboregulators are then subjected to evaluation by tagging the cis-repressive sequence (crRNA) to mCherry, a fluorescent reporter and regulated by the constitutive promoter, PpepN. The trans-activating RNA (taRNA) that interacts with the crRNA is placed under the control of another inducible promoter, Pnis. First, we observed that when there is no induction of taRNA, there is negligible fluorescence of mCherry indicating the successful repression of translation by the cis-sequence as expected. This has been further verified by comparing this expression level with the expression level of PpepN-mCherry without the cis-sequence, using fluorometer. Results from this analysis suggest that there is ~97% repression by the designed crRNA sequence. Next, we induced the cells with 2 ng/mL of nisin in the mid-log phase. Upon induction, there is a maximum of three fold increase in the fluorescence levels when compared to the uninduced cells, suggesting that the trans-activation takes place inside the live cells. However, further studies are necessary to optimize the cis-trans ratio to achieve better dynami
机译：微生物细胞工厂进行重新布线基本的代谢增强的碳通量对所需的产品通路。一般来说,这种代谢工程方法往往需要表达的通路基因和基因敲出竞争的途径。并最终导致严重的代谢负担表现不佳的细胞。生物量的形成和产品合成在哪里依靠共同的前体,永久的改变像敲基因只会导致贫穷效价。合成是形成和产品是一个潜在的替代。设计合成riboregulators,RNA-based基因开关、分流代谢通路Lactococcus lactis细菌,肝微生物细胞工厂很多biocompounds。评估通过标记mCherry cis-repressive序列(crRNA),荧光记者和监管组成型启动子,PpepN。trans-activating RNA (taRNA)交互crRNA放置的控制下诱导启动子句。当没有taRNA感应,有可以忽略不计的荧光mCherry指示成功的翻译的镇压cis-sequence如预期。通过比较这个表达水平与验证PpepN-mCherry没有的表达水平cis-sequence,使用荧光计。这一分析表明,有~ 97%设计crRNA镇压的序列。我们用2 ng / mL诱导细胞的乳酸链球菌肽mid-log阶段。三个褶皱增加荧光水平uninduced细胞相比,暗示内部的trans-activation发生活细胞。需要优化顺反比率取得更好的dynami

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