Functional Genetic Elements for Controlling Gene Expression in Cupriavidus necator H16.
Functional Genetic Elements for Controlling Gene Expression in Cupriavidus necator H16.
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DOI：
10.1128/aem.00878-18
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发表时间：
2018-10-01
影响因子：
4.4
通讯作者：
Minton NP
中科院分区：
文献类型：
--
作者：
Alagesan S;Hanko EKR;Malys N;Ehsaan M;Winzer K;Minton NP
这份报告提供了强大的和可预测的控制基因表达的工具，在模型石质自生菌H16。为了满足当前的需求，我们设计、构建和测试了启动子和限制性内切酶来控制链球菌H16中的基因表达。为了回答现有和新开发的诱导体系的比较问题，对两个正调控的诱导体系(AraC/ParaBAD-L-阿拉伯糖和RhaRS/PrhaBAD-L-鼠李糖)和两个负调控的诱导体系(ACUR/PacuRI-丙烯酸酯和Cymr/PCMT-Cumate)进行了定量评价和诱导动力学分析。为了确定是否可以进一步提高基因表达，评估了遗传因素，如mRNA茎环结构和富含A/U序列对基因表达的影响。以异戊二烯生产为例，研究化合物产量是否以及在多大程度上与产物合成酶的基因表达水平相关。稳健和可预测的基因表达控制在合成生物学和生物技术应用中发挥着重要作用。需要开发和定量评估功能性遗传元件，如构成启动子和诱导启动子以及核糖体结合位点(RBSS)。在这项研究中，我们设计、构建并测试了用于控制结石自养细胞Cupriavidus Necator H16中基因表达的启动子和RBSS。一系列可变强度、绝缘的结构性启动子在700倍的动态范围内表现出可预测的活性，与本地PphaC进行比较，大多数启动子的活性最高可达9倍。对正向(AraC/ParaBAD-L-阿拉伯糖和RhaRS/PrhaBAD-L-鼠李糖)和负向(ACUR/PACURI-丙烯酸酯和CymR/PCMT-Cumate)调节的诱导体系进行了评估。通过提供不同浓度的诱导剂，基因表达水平达到了1000倍的范围。应用可诱导系统控制异戊二烯合成酶基因ISPS的表达导致异戊二烯产量与报告蛋白合成水平显着相关。此外，还评估了设计的RBSS和其他遗传因素，如mRNA茎环结构和富含A/U序列对基因表达的影响。RBS活性与异戊二烯产率之间呈二次多项式关系。这份报告提供了调控遗传元件的定量数据，并扩展了C.necator的遗传工具箱。重要性这份报告提供了健壮的和可预测的控制基因表达的工具，在模型石质自生菌H16中。为了满足当前的需求，我们设计、构建和测试了启动子和限制性内切酶来控制链球菌H16中的基因表达。为了回答现有和新开发的诱导体系的比较问题，对两个正调控的诱导体系(AraC/ParaBAD-L-阿拉伯糖和RhaRS/PrhaBAD-L-鼠李糖)和两个负调控的诱导体系(ACUR/PacuRI-丙烯酸酯和Cymr/PCMT-Cumate)进行了定量评价和诱导动力学分析。为了确定是否可以进一步提高基因表达，评估了遗传因素，如mRNA茎环结构和富含A/U序列对基因表达的影响。以异戊二烯生产为例，研究化合物产量是否以及在多大程度上与产物合成酶的基因表达水平相关。
This report provides tools for robust and predictable control of gene expression in the model lithoautotroph C. necator H16. To address a current need, we designed, built, and tested promoters and RBSs for controlling gene expression in C. necator H16. To answer a question on how existing and newly developed inducible systems compare, two positively (AraC/ParaBAD-l-arabinose and RhaRS/PrhaBAD-l-rhamnose) and two negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems were quantitatively evaluated and their induction kinetics analyzed. To establish if gene expression can be further improved, the effect of genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was evaluated. Using isoprene production as an example, the study investigated if and to what extent chemical compound yield correlates to the level of gene expression of product-synthesizing enzyme. A robust and predictable control of gene expression plays an important role in synthetic biology and biotechnology applications. Development and quantitative evaluation of functional genetic elements, such as constitutive and inducible promoters as well as ribosome binding sites (RBSs), are required. In this study, we designed, built, and tested promoters and RBSs for controlling gene expression in the model lithoautotroph Cupriavidus necator H16. A series of variable-strength, insulated, constitutive promoters exhibiting predictable activity within a >700-fold dynamic range was compared to the native PphaC, with the majority of promoters displaying up to a 9-fold higher activity. Positively (AraC/ParaBAD-l-arabinose and RhaRS/PrhaBAD-l-rhamnose) and negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems were evaluated. By supplying different concentrations of inducers, a >1,000-fold range of gene expression levels was achieved. Application of inducible systems for controlling expression of the isoprene synthase gene ispS led to isoprene yields that exhibited a significant correlation to the reporter protein synthesis levels. The impact of designed RBSs and other genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was also evaluated. A second-order polynomial relationship was observed between the RBS activities and isoprene yields. This report presents quantitative data on regulatory genetic elements and expands the genetic toolbox of C. necator. IMPORTANCE This report provides tools for robust and predictable control of gene expression in the model lithoautotroph C. necator H16. To address a current need, we designed, built, and tested promoters and RBSs for controlling gene expression in C. necator H16. To answer a question on how existing and newly developed inducible systems compare, two positively (AraC/ParaBAD-l-arabinose and RhaRS/PrhaBAD-l-rhamnose) and two negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems were quantitatively evaluated and their induction kinetics analyzed. To establish if gene expression can be further improved, the effect of genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was evaluated. Using isoprene production as an example, the study investigated if and to what extent chemical compound yield correlates to the level of gene expression of product-synthesizing enzyme.