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首页> 外文期刊>Applied Microbiology >Discovery and Characterization of Native Deinococcus radiodurans Promoters for Tunable Gene Expression
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Discovery and Characterization of Native Deinococcus radiodurans Promoters for Tunable Gene Expression

机译：发现和表征天然Deinococcus radiodurans启动子的可调基因表达。

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The potential utilization of extremophiles as a robust chassis for metabolic engineering applications has prompted interest in the use of Deinococcus radiodurans for bioremediation efforts, but current applications are limited by the lack of availability of genetic tools, such as promoters. In this study, we used a combined computational and experimental approach to identify and screen 30 predicted promoters for expression in D. radiodurans using a fluorescent reporter assay. The top eight candidates were further characterized, compared to currently available promoters, and optimized for engineering through minimization for use in D. radiodurans. Of these top eight, two promoter regions, PDR_1261 and PrpmB, were stronger and more consistent than the most widely used promoter sequence in D. radiodurans, PgroES. Furthermore, half of the top eight promoters could be minimized by at least 20% (to obtain final sequences that are approximately 24 to 177?bp), and several of the putative promoters either showed activity in Escherichia coli or were D. radiodurans specific, broadening the use of the promoters for various applications. Overall, this work introduces a suite of novel, well-characterized promoters for protein production and metabolic engineering in D. radiodurans.IMPORTANCE The tolerance of the extremophile, Deinococcus radiodurans, to numerous oxidative stresses makes it ideal for bioremediation applications, but many of the tools necessary for metabolic engineering are lacking in this organism compared to model bacteria. Although native and engineered promoters have been used to drive gene expression for protein production in D. radiodurans, very few have been well characterized. Informed by bioinformatics, this study expands the repertoire of well-characterized promoters for D. radiodurans via thorough characterization of eight putative promoters with various strengths. These results will help facilitate tunable gene expression, since these promoters demonstrate strong and consistent performance compared to the current standard, PgroES. This study also provides a methodology for high-throughput promoter identification and characterization using fluorescence in D. radiodurans. The promoters identified in this study will facilitate metabolic engineering of D. radiodurans and enable its use in biotechnological applications ranging from bioremediation to synthesis of commodity chemicals.
机译：极端嗜热菌作为代谢工程应用的强大底盘的潜在用途已引起人们对放射性杜氏球菌用于生物修复工作的兴趣，但当前的应用受到缺乏遗传工具（如启动子）的限制。在这项研究中，我们使用了一种计算和实验相结合的方法，使用荧光报告基因检测法鉴定和筛选了30种预测的启动子，以在D. radiodurans中表达。与目前可用的启动子相比，对前八名候选基因进行了进一步表征，并通过最小化进行工程优化，以用于D. radiodurans。在这前八位中，两个启动子区域PDR_1261和PrpmB比D. radiodurans中最广泛使用的启动子序列PgroES更强，更一致。此外，可以将前八个启动子中的一半最小化至少20％（以获得大约24至177？bp的最终序列），并且推定的启动子中有几个在大肠杆菌中显示出活性，或者是D. radiodurans特异的，扩大了启动子在各种应用中的用途。总的来说，这项工作引入了一套新颖的，特征明确的启动子，用于放射性杜氏螺菌的蛋白质生产和代谢工程。重要提示极端微生物嗜热菌Deinococcus radiodurans对多种氧化胁迫的耐受性使其非常适合生物修复应用，但许多与模型细菌相比，该生物缺乏代谢工程所需的工具。尽管天然的和工程化的启动子已被用来驱动D. radiodurans中蛋白质生产的基因表达，但是很少有人被很好地表征。受生物信息学的影响，这项研究通过对八种推定的具有不同强度的启动子进行了全面表征，扩展了D. radiodurans的特征明确的启动子。这些结果将有助于促进可调基因的表达，因为与目前的标准PgroES相比，这些启动子表现出强大而一致的性能。这项研究还提供了一种利用放射线虫中的荧光进行高通量启动子鉴定和表征的方法。在这项研究中确定的启动子将促进放射性杜鹃的代谢工程，使其能够用于从生物修复到商品化学合成的生物技术应用中。

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