Department of Systems Biology, Columbia University Irving Medical Center, New York, New York 10032, United States.
Integrated Program in Cellular, Molecular and Biomedical Studies, Columbia University Irving Medical Center, New York, New York 10032, United States.
ACS Synth Biol. 2021 Aug 20;10(8):1859-1873. doi: 10.1021/acssynbio.0c00639. Epub 2021 Jul 21.
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 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 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.
最近的努力对细菌的多样化生物合成能力进行了测序、调查和功能表征，确定了许多生物合成基因簇（BGCs）。BGC 内发现的基因通常转录沉默，表明它们的表达受到严格调控。为了更好地阐明 DNA 序列水平上调控 BGC 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检测来构建和表征 BGC 衍生调控序列文库。在放线菌 J1074 中测量了调控序列的转录水平，放线菌 J1074 是一种来自富含 BGC 多样性的属的流行模型菌株。转录活性在 1000 倍范围内变化，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，我们证明通过共表达全局调控蛋白可以调节转录水平。最后，我们开发并优化了一种无细胞表达系统，用于快速表征调控序列。这项工作有助于阐明 BGC 的调控景观，并为理性工程和激活隐匿 BGC 提供了多样化的特征调控序列文库。
