Recent efforts to sequence, survey, and functionally characterize the diverse biosynthetic capabilities of bacteria have identified numerous Biosynthetic Gene Clusters (BGCs). Genes found within BGCs are typically transcriptionally silent, suggesting their expression is tightly regulated. To better elucidate the underlying mechanisms and principles that govern BGC regulation on a DNA sequence level, we employed high-throughput DNA synthesis and multiplexed reporter assays to build and to characterize a library of BGC-derived regulatory sequences. Regulatory sequence transcription levels were measured in the Actinobacteria Streptomyces albidoflavus J1074, a popular model strain from a genus rich in BGC diversity. Transcriptional activities varied over 1000-fold in range and were used to identify key features associated with expression, including GC content, transcription start sites, and sequence motifs. Furthermore, we demonstrated that transcription levels could be modulated through coexpression of global regulatory proteins. Lastly, we developed and optimized a S. albidoflavus cell-free expression system for rapid characterization of regulatory sequences. This work helps to elucidate the regulatory landscape of BGCs and provides a diverse library of characterized regulatory sequences for rational engineering and activation of cryptic BGCs.

中文翻译：

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细菌生物合成基因簇调控序列的高通量转录表征

最近对细菌的多种生物合成能力进行测序、调查和功能表征的努力已经确定了许多生物合成基因簇 (BGC)。在 BGC 中发现的基因通常是转录沉默的，表明它们的表达受到严格调控。为了更好地阐明在 DNA 序列水平上控制 BGC 调控的潜在机制和原则，我们采用高通量 DNA 合成和多路报告基因分析来构建和表征 BGC 衍生调控序列库。在 Actinobacteria Streptomyces albidoflavus中测量调节序列转录水平J1074，一种流行的模型菌株，来自富含 BGC 多样性的属。转录活动范围变化超过 1000 倍，用于识别与表达相关的关键特征，包括 GC 含量、转录起始位点和序列基序。此外，我们证明了转录水平可以通过全局调节蛋白的共表达来调节。最后，我们开发并优化了S. albidoflavus无细胞表达系统，用于快速表征调控序列。这项工作有助于阐明 BGC 的调控格局，并为合理工程化和激活神秘的 BGC 提供一个多样化的特征调控序列库。