Harnessing systems biology to elucidate multi-level regulation of gene expression in $\textit{Streptomyces}$ genomes

Abstract

Streptomyces, the largest genus of actinobacteria, has received great attention as an industrial producer of secondary metabolites, exhibiting a broad range of pharmaceutical bioactivities, such as antimicrobial, antifungal, anticancer and immunosuppressive activities. Streptomyces is also promising producers of many bio-sustainable secondary metabolites that can be used as bioherbicides, biosurfactants, vitamins, pigments, nematocides, insecticides, and agrochemicals to combat pests and parasites. Such secondary metabolites are typically synthesized via a multi-step conversion of precursor molecules, such as CoA pool and amino acids, by multi-enzyme complexes encoded in secondary metabolite biosynthetic gene clusters (BGCs). On an average, one Streptomyces strain possesses >30 different BGCs in its genome. However, their genetic potential has been poorly studied because many BGCs are not activated under standard culture conditions. Moreover, the BGCs are tightly controlled by complex regulatory systems at transcriptional and translational levels to effectively utilize precursors that are supplied by primary metabolism. Determining transcriptional and translational regulatory elements in GC-rich Streptomyces genomes is essential to elucidating the complex regulatory networks that govern secondary metabolite biosynthetic gene cluster (BGC) expression. However, information about such regulatory elements has been limited for Streptomyces genomes. To overcome this limitation, I harnessed systems biology to elucidate multi-level regulation of gene expression in Streptomyces genomes by integration of multi-omics data including Genome-Seq, RNA-Seq, Ribosome profiling, dRNA-Seq, and TermSeq, providing the comprehensive understanding of the primary to secondary metabolisms in Streptomyces for the rational design of Streptomyces engineering. First, the genome-wide regulatory elements on transcription initiation in Streptomyces were systematically analyzed by integration of Genome-Seq, RNA-Seq, and dRNA-Seq. I obtained a high-quality genome sequence of β-lactam antibiotic producer Streptomyces clavuligerus ATCC 27064 by integration of Illumina short reads and PacBio long reads genome sequencing, and 58 potential BGCs were predicted based on the genome information. Then, the functional enrichment of differential expressed genes during growth in S. clavuligerus and model strain Streptomyces griseus NBRC 13350 was demonstrated from transcriptomic data. Using dRNA-Seq, the genome-scale TSSs of S. clavuligerus and S. griseus was precisely determined. Based on the TSS information, the potential regulons of sigma(σ) factors and transcription factors of S. griseus and S. clavuligerus affecting the differential transcription pattern during growth were elucidated, including β-lactam biosynthesis. This information will improve the understanding of complex transcription regulatory networks in Streptomyces BGCs. Second, the genome-wide regulatory elements on transcription termination and posttranscriptional processing in Streptomyces were systematically analyzed by integration of RNA-Seq, dRNA-Seq, and Term-Seq. I determined the genome-wide transcription 3’-end positions (TEPs), transcription units (TUs), and transcription unit clusters (TUCs) in S. clavuligerus and S. griseus. Then, unique structural and regulatory features of TEPs in Streptomyces were elucidated. TU architecture showed that the transcript abundance in TU isoforms of a TUC was potentially affected by the sequence context of their TEPs, suggesting that the regulatory elements of TEPs could control the transcription level in additional layers. In particular, potential regulatory elements located in the middle of the polycistronic operon of hopanoid BGC of S. griseus and the potential regulatory transcription unit XRE-DUF397 of S. clavuligerus were investigated. These findings highlight the role of transcriptional regulatory elements in transcription termination and post-transcriptional processing in Streptomyces. Third, the genome-wide regulatory elements on translation in Streptomyces were systematically analyzed by integration of ribosome profiling and other multi-omics data. Ribosome profiling data corrects mis-annotation of open reading frames. Then, I investigated the dynamic translational landscape during growth, revealing the translational buffering at the later growth phases. The regulatory elements on translational initiation level encoded in the 5-UTR was elucidated including the abundant leaderless mRNA and RNA structures. The regulatory elements on translation elongation level encoded in the CDS was also elucidated related to the ribosome pausing by different codon usage. Understanding of the translational features of Streptomyces will provide the potential engineering targets for the secondary metabolite production. Fourth, the primary to secondary metabolisms of the seven Streptomyces strains including model strain Streptomyces coelicolor A3(2), avermectin producer Streptomyces avermitilis MA-4680, S. griseus, S. clavuligerus, model strain Streptomyces lividans TK24, pikromycin producer S. venezuelae ATCC15439, and FK506 producer S. tsukubaensis NRRL 18488 were comparatively analyzed for the comprehensive understanding of the dynamic regulations. I constructed pan-reactome of these strains, and integrate the multi-omics (dRNA-Seq, RNA-Seq, Term-Seq, Ribosome profiling) information to elucidate the potential regulatory elements governing the transcriptome change of the hopene biosynthesis pathway. Systematic analysis of this study will provide not only the pipeline of comprehensive analysis of the pan-reactome of the secondary metabolisms, but also the foundation of the synthetic design of the precursor biosynthetic modules for the specialized chassis libraries of BGC heterologous expressions. Overall, systems biology to elucidate multi-level regulation of gene expression in Streptomyces genomes provides the regulatory elements, potential genetic expression parts, engineering targets, and the design principle of precursor biosynthetic modules for the further rational engineering to improve the secondary metabolite productions and to discover novel secondary metabolites.