Synthetic RNA Silencing of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

Abstract

We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces.

Rapid and high-throughput construction of microbial cell-factories with regulatory noncoding RNAs

“…This shows that cnc2198.1 indeed affects glutamine synthetase I by interacting with glnA mRNA, leading to a stall of translation and/or its degradation (D'Alia et al, 2010). Like E. coli, the addition of paired-termini to asRNA (Nakashima et al, 2006) also enhanced the efficiency of translation repression in S. coelicolor MT1110 (Uguru et al, 2013). Briefly, Uguru et al computationally generated DNA sequences ranging between 120 and 160-bp complementary to the translation initiation region of polyketide beta-ketoacyl synthase subunit gene (actI-ORF1) involved in the actinorhodin biosynthetic pathway.…”

“…Like other bacteria, Streptomyces also utilizes trans-acting sRNAs to regulate gene expression at post-transcription level. For example, the 86 nucleotidelong MicX sRNA, which possess a paired-end structure for protection from RNase III-mediated cleavage, positively activates the translation of genes involved in the actinorhodin biosynthesis in Streptomyces lividans (Hjalt and Wagner, 1995;Romero and Mellado, 1995;Uguru et al, 2013). In addition, the scr5239 sRNA represses the translation of the agarose gene, dagA, in S. coelicolor.…”

“…Briefly, Uguru et al computationally generated DNA sequences ranging between 120 and 160-bp complementary to the translation initiation region of polyketide beta-ketoacyl synthase subunit gene (actI-ORF1) involved in the actinorhodin biosynthetic pathway. Of the generated sequences, those with weak secondary structure and high positional entropy in the mRNA binding portion of asRNA were identified using RNAfold (Hofacker, 2003;Uguru et al, 2013). Consequently, a PTasRNA with a 21-bp-long inverted repeat at both termini and 155-bp-long antisense region (−92 to +63) was experimentally constructed (Nakashima et al, 2006) reducing actI-ORF1 mRNA translation, and thereby a decrease in 4-fold actinorhodin production was observed (Fig.…”

“…In most studies on RNA interference in bacteria, asRNAs are produced mostly from a plasmid or genome (Uguru et al, 2013). Whereas, a synthetic nucleotide PNA that can hybridize to DNA or RNA can be directly delivered into bacterial cells in aid of a cell-penetrating peptide (Good et al, 2001).…”

Synthetic Biology of Antibiotic Production

“…For the major antibiotics producers, the regulatory effects of noncoding RNA were first described by D'Alia et al [103] for a cis-encoded antisense RNA in the glutamine synthetase I gene in S. coelicolor, followed by characterization of the resulting synthetic metabolic switch by detailed metabolomic profiling using high-resolution mass spectrometry [104]. This was complemented by the demonstration of a functional synthetic RNA silencer in the same species by Uguru et al [105], targeting the biosynthesis of the pigmented antibiotic actinorhodin.…”