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Obtaining a series of native gradient promoter-5'-UTR sequences in Corynebacterium glutamicum ATCC 13032.
Microbial Cell Factories ( IF 4.3 ) Pub Date : 2020-06-03 , DOI: 10.1186/s12934-020-01376-3
Ning Li 1, 2, 3 , Weizhu Zeng 1, 2, 3 , Sha Xu 1, 2, 3 , Jingwen Zhou 1, 2, 3
Affiliation  

Corynebacterium glutamicum is an important industrial microorganism used for the production of many valuable compounds, especially amino acids and their derivatives. For fine-tuning of metabolic pathways, synthetic biological tools are largely based on the rational application of promoters. However, the limited number of promoters make it difficult. In this study, according to the analysis of RNA-Seq data, 90 DNA fragments with lengths of 200-500 bp that may contain promoter-5′-UTR (PUTR) sequences were amplified and linked to a fluorescent protein gene. When compared with the common strong PUTR PsodUTR, 17 strong PUTRs were obtained, which maintained stable expression strengths from the early to post stationary phase. Among them, PNCgl1676UTR was the strongest and its fluorescent protein expression level was more than five times higher than that of PsodUTR. Furthermore, nine typical chemicals related to the biosynthesis of sulfur-containing amino acids (such as l-methionine, l-cysteine) were selected as stress substances to preliminarily explore the stress on these PUTRs. The results showed that the expression of PbrnFUTR was activated by l-methionine, while that of PNCgl1202UTR was severely inhibited by l-lysine. These findings demonstrated that the selected PUTRs can stably express different genes, such as the red fluorescence protein gene, and can be useful for fine-tuning regulation of metabolic networks in C. glutamicum or for establishing high-throughput screening strategies through biosensor for the production of useful compounds.

中文翻译:

在谷氨酸棒杆菌ATCC 13032中获得一系列天然梯度启动子5'-UTR序列。

谷氨酸棒杆菌是一种重要的工业微生物,用于生产许多有价值的化合物,尤其是氨基酸及其衍生物。对于代谢途径的微调,合成生物学工具很大程度上基于启动子的合理应用。然而,启动子的数量有限使之困难。在这项研究中,根据对RNA-Seq数据的分析,扩增了90个长度为200-500 bp的DNA片段,其中可能包含启动子5'-UTR(PUTR)序列,并将其与荧光蛋白基因连接。与常见的强PUTR PsodUTR相比,获得了17个强PUTR,它们从早期到固定期都保持了稳定的表达强度。其中,PNCgl1676UTR最强,其荧光蛋白表达水平是PsodUTR的五倍以上。此外,选择了九种与生物合成含硫氨基酸有关的典型化学物质(如L-蛋氨酸,L-半胱氨酸)作为胁迫物质,初步探索了这些PUTR的胁迫。结果表明,L-蛋氨酸激活了PbrnFUTR的表达,而L-赖氨酸则严重抑制了PNCgl1202UTR的表达。这些发现表明,所选择的PUTR可以稳定表达不同的基因,例如红色荧光蛋白基因,并且可以用于谷氨酸棒状杆菌的代谢网络的微调调节或通过生物传感器建立高通量筛选策略以用于生产。有用的化合物。选择了九种与含硫氨基酸生物合成有关的典型化学物质(如L-蛋氨酸,L-半胱氨酸)作为胁迫物质,初步探讨了这些PUTR的胁迫。结果表明,L-蛋氨酸激活了PbrnFUTR的表达,而L-赖氨酸严重抑制了PNCgl1202UTR的表达。这些发现表明,所选择的PUTR可以稳定表达不同的基因,例如红色荧光蛋白基因,并且可以用于谷氨酸棒状杆菌的代谢网络的微调调节或通过生物传感器建立高通量筛选策略以用于生产。有用的化合物。选择了9种与含硫氨基酸生物合成有关的典型化学物质(如L-蛋氨酸,L-半胱氨酸)作为胁迫物质,初步探讨了这些PUTR的胁迫。结果表明,L-蛋氨酸激活了PbrnFUTR的表达,而L-赖氨酸严重抑制了PNCgl1202UTR的表达。这些发现表明,所选择的PUTR可以稳定地表达不同的基因,例如红色荧光蛋白基因,并且可以用于微调谷氨酸棒状杆菌的代谢网络的调节或通过生物传感器建立高通量筛选策略以用于生产。有用的化合物。选择L-半胱氨酸作为应激物质,以初步探索这些PUTR上的应激。结果表明,L-蛋氨酸激活了PbrnFUTR的表达,而L-赖氨酸则严重抑制了PNCgl1202UTR的表达。这些发现表明,所选择的PUTR可以稳定地表达不同的基因,例如红色荧光蛋白基因,并且可以用于微调谷氨酸棒状杆菌的代谢网络的调节或通过生物传感器建立高通量筛选策略以用于生产。有用的化合物。选择L-半胱氨酸作为应激物质,以初步探索这些PUTR上的应激。结果表明,L-蛋氨酸激活了PbrnFUTR的表达,而L-赖氨酸则严重抑制了PNCgl1202UTR的表达。这些发现表明,所选择的PUTR可以稳定表达不同的基因,例如红色荧光蛋白基因,并且可以用于谷氨酸棒状杆菌的代谢网络的微调调节或通过生物传感器建立高通量筛选策略以用于生产。有用的化合物。
更新日期:2020-06-03
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