Transcriptome and translatome profiles of Streptomyces species in different growth phases.

Streptomyces are efficient producers of various bioactive compounds, which are mostly synthesized by their secondary metabolite biosynthetic gene clusters (smBGCs). The smBGCs are tightly controlled by complex regulatory systems at transcriptional and translational levels to effectively utilize precursors that are supplied by primary metabolism. Thus, dynamic changes in gene expression in response to cellular status at both the transcriptional and translational levels should be elucidated to directly reflect protein levels, rapid downstream responses, and cellular energy costs. In this study, RNA-Seq and ribosome profiling were performed for five industrially important Streptomyces species at different growth phases, for the deep sequencing of total mRNA, and only those mRNA fragments that are protected by translating ribosomes, respectively. Herein, 12.0 to 763.8 million raw reads were sufficiently obtained with high quality of more than 80% for the Phred score Q30 and high reproducibility. These data provide a comprehensive understanding of the transcriptional and translational landscape across the Streptomyces species and contribute to facilitating the rational engineering of secondary metabolite production

Adaptive laboratory evolution of a genome-reduced Escherichia coli.

Synthetic biology aims to design and construct bacterial genomes harboring the minimum number of genes required for self-replicable life. However, the genome-reduced bacteria often show impaired growth under laboratory conditions that cannot be understood based on the removed genes. The unexpected phenotypes highlight our limited understanding of bacterial genomes. Here, we deploy adaptive laboratory evolution (ALE) to re-optimize growth performance of a genome-reduced strain. The basis for suboptimal growth is the imbalanced metabolism that is rewired during ALE. The metabolic rewiring is globally orchestrated by mutations in rpoD altering promoter binding of RNA polymerase. Lastly, the evolved strain has no translational buffering capacity, enabling effective translation of abundant mRNAs. Multi-omic analysis of the evolved strain reveals transcriptome- and translatome-wide remodeling that orchestrate metabolism and growth. These results reveal that failure of prediction may not be associated with understanding individual genes, but rather from insufficient understanding of the strain's systems biology

Genome-scale reconstruction of the sigma factor network in Escherichia coli: topology and functional states

Background: At the beginning of the transcription process, the RNA polymerase (RNAP) core enzyme requires a sigma-factor to recognize the genomic location at which the process initiates. Although the crucial role of sigma-factors has long been appreciated and characterized for many individual promoters, we do not yet have a genome-scale assessment of their function. Results: Using multiple genome-scale measurements, we elucidated the network of s-factor and promoter interactions in Escherichia coli. The reconstructed network includes 4,724 sigma-factor-specific promoters corresponding to transcription units (TUs), representing an increase of more than 300% over what has been previously reported. The reconstructed network was used to investigate competition between alternative sigma-factors (the sigma(70) and sigma(38) regulons), confirming the competition model of sigma substitution and negative regulation by alternative s-factors. Comparison with sigma-factor binding in Klebsiella pneumoniae showed that transcriptional regulation of conserved genes in closely related species is unexpectedly divergent. Conclusions: The reconstructed network reveals the regulatory complexity of the promoter architecture in prokaryotic genomes, and opens a path to the direct determination of the systems biology of their transcriptional regulatory networks

The dynamic transcriptional and translational landscape of the model antibiotic producer Streptomyces coelicolor A3(2)

Individual Streptomyces species have the genetic potential to produce a diverse array of natural products of commercial, medical and veterinary interest. However, these products are often not detectable under laboratory culture conditions. To harness their full biosynthetic potential, it is important to develop a detailed understanding of the regulatory networks that orchestrate their metabolism. Here we integrate nucleotide resolution genome-scale measurements of the transcriptome and translatome of Streptomyces coelicolor, the model antibiotic-producing actinomycete. Our systematic study determines 3,570 transcription start sites and identifies 230 small RNAs and a considerable proportion (∼21%) of leaderless mRNAs; this enables deduction of genome-wide promoter architecture. Ribosome profiling reveals that the translation efficiency of secondary metabolic genes is negatively correlated with transcription and that several key antibiotic regulatory genes are translationally induced at transition growth phase. These findings might facilitate the design of new approaches to antibiotic discovery and development

The architecture of ArgR-DNA complexes at the genome-scale in<i> Escherichia coli</i>

DNA-binding motifs that are recognized by transcription factors (TFs) have been well studied; however, challenges remain in determining the in vivo architecture of TF-DNA complexes on a genome-scale. Here, we determined the in vivo architecture of Escherichia coli arginine repressor (ArgR)-DNA complexes using high-throughput sequencing of exonuclease-treated chromatin-immunoprecipitated DNA (ChIP-exo). The ChIP-exo has a unique peak-pair pattern indicating 5′ and 3′ ends of ArgR-binding region. We identified 62 ArgR-binding loci, which were classified into three groups, comprising single, double and triple peak-pairs. Each peak-pair has a unique 93 base pair (bp)-long (±2 bp) ArgR-binding sequence containing two ARG boxes (39 bp) and residual sequences. Moreover, the three ArgR-binding modes defined by the position of the two ARG boxes indicate that DNA bends centered between the pair of ARG boxes facilitate the non-specific contacts between ArgR subunits and the residual sequences. Additionally, our approach may also reveal other fundamental structural features of TF-DNA interactions that have implications for studying genome-scale transcriptional regulatory networks

Synthetic biology on acetogenic bacteria for highly efficient conversion of c1 gases to biochemicals

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Synthesis gas, which is mainly produced from fossil fuels or biomass gasification, consists of C1 gases such as carbon monoxide, carbon dioxide, and methane as well as hydrogen. Acetogenic bacteria (acetogens) have emerged as an alternative solution to recycle C1 gases by converting them into value-added biochemicals using the Wood-Ljungdahl pathway. Despite the advantage of utilizing acetogens as biocatalysts, it is difficult to develop industrial-scale bioprocesses because of their slow growth rates and low productivities. To solve these problems, conventional approaches to metabolic engineering have been applied; however, there are several limitations owing to the lack of required genetic bioparts for regulating their metabolic pathways. Recently, synthetic biology based on genetic parts, modules, and circuit design has been actively exploited to overcome the limitations in acetogen engineering. This review covers synthetic biology applications to design and build industrial platform acetogens

Primary transcriptome and translatome analysis determines transcriptional and translational regulatory elements encoded in the Streptomyces clavuligerus genome

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 address this limitation, a high-quality genome sequence of β-lactam antibiotic-producing Streptomyces clavuligerus ATCC 27 064 is completed, which contains 7163 newly annotated genes. This provides a fundamental reference genome sequence to integrate multiple genome-scale data types, including dRNA-Seq, RNA-Seq and ribosome profiling. Data integration results in the precise determination of 2659 transcription start sites which reveal transcriptional and translational regulatory elements, including -10 and -35 promoter components specific to sigma (σ) factors, and 5'-untranslated region as a determinant for translation efficiency regulation. Particularly, sequence analysis of a wide diversity of the -35 components enables us to predict potential σ-factor regulons, along with various spacer lengths between the -10 and -35 elements. At last, the primary transcriptome landscape of the β-lactam biosynthetic pathway is analyzed, suggesting temporal changes in metabolism for the synthesis of secondary metabolites driven by transcriptional regulation. This comprehensive genetic information provides a versatile genetic resource for rational engineering of secondary metabolite BGCs in Streptomyces