High-Throughput Transcriptional Characterization of Regulatory Sequences from Bacterial Biosynthetic Gene Clusters

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.