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Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA in Escherichia coli.

ABSTRACT: RNA interference (RNAi) is a natural mechanism for protecting against harmful genetic elements and regulating gene expression, which can be artificially triggered by the delivery of homologous double-stranded RNA (dsRNA). This mechanism can be exploited as a highly specific and environmentally friendly pest control strategy. To this aim, systems for producing large amounts of recombinant dsRNA are necessary. We describe a system to efficiently produce large amounts of circular dsRNA in Escherichia coli and demonstrate the efficient insecticidal activity of these molecules against Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte), a highly damaging pest of corn crops. In our system, the two strands of the dsRNA are expressed in E. coli embedded within the very stable scaffold of Eggplant latent viroid (ELVd), a small circular non-coding RNA. Stability in E. coli of the corresponding plasmids with long inverted repeats was achieved by using a cDNA coding for a group-I autocatalytic intron from Tetrahymena thermophila as a spacer. RNA circularization and large-scale accumulation in E. coli cells was facilitated by co-expression of eggplant tRNA ligase, the enzyme that ligates ELVd during replication in the host plant. The inserted intron efficiently self-spliced from the RNA product during transcription. Circular RNAs containing a dsRNA moiety homologous to smooth septate junction 1 (DvSSJ1) gene exhibited excellent insecticide activity against WCR larvae. Finally, we show that the viroid scaffold can be separated from the final circular dsRNA product using a second T. thermophila self-splicing intron in a permuted form.

SUBMITTER: Ortola B

PROVIDER: S-EPMC8582998 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The actin-like FtsA protein is essential for function of the cell division machinery, or divisome, in many bacteria including Escherichia coli. Previous in vitro studies demonstrated that purified wild-type FtsA assembles into closed mini-rings on lipid membranes, but oligomeric variants of FtsA such as FtsAR286W and FtsAG50E can bypass certain divisome defects and form arc and double-stranded (DS) oligomeric states, respectively, which may reflect conversion of an inactive to an active form of FtsA. However, it remains unproven which oligomeric forms of FtsA are responsible for assembling and activating the divisome. Here, we used an in vivo crosslinking assay for FtsA DS filaments to show that they largely depend on proper divisome assembly and are prevalent at later stages of cell division. We also used a previously reported variant that fails to assemble DS filaments, FtsAM96E R153D, to investigate the roles of FtsA oligomeric states in divisome assembly and activation. We show that FtsAM96E R153D cannot form DS filaments in vivo, fails to replace native FtsA, and confers a dominant negative phenotype, underscoring the importance of the DS filament stage for FtsA function. Surprisingly, however, activation of the divisome through the ftsL* or ftsW* superfission alleles suppressed the dominant negative phenotype and rescued the functionality of FtsAM96E R153D. Our results suggest that FtsA DS filaments are needed for divisome activation once it is assembled, but they are not essential for divisome assembly or guiding septum synthesis.IMPORTANCECell division is fundamental for cellular duplication. In simple cells like Escherichia coli bacteria, the actin homolog FtsA is essential for cell division and assembles into a variety of protein filaments at the cytoplasmic membrane. These filaments not only help tether polymers of the tubulin-like FtsZ to the membrane at early stages of cell division but also play crucial roles in recruiting other cell division proteins to a complex called the divisome. Once assembled, the E. coli divisome subsequently activates synthesis of the division septum that splits the cell in two. One recently discovered oligomeric conformation of FtsA is an antiparallel double-stranded filament. Using a combination of in vivo crosslinking and genetics, we provide evidence suggesting that these FtsA double filaments have a crucial role in activating the septum synthesis enzymes.

Project description:BackgroundLate-stage fermentation broth contains high concentrations of target chemicals. Additionally, it contains various cellular metabolites which have leaked from lysed cells, which would exert multifactorial stress to industrial hyperproducers and perturb both cellular metabolism and product formation. Although adaptive laboratory evolution (ALE) has been wildly used to improve stress tolerance of microbial cell factories, single-factor stress condition (i.e. target product or sodium chloride at a high concentration) is currently provided. In order to enhance bacterial stress tolerance to actual industrial production conditions, ALE in late-stage fermentation broth is desired. Genome replication engineering assisted continuous evolution (GREACE) employs mutants of the proofreading element of DNA polymerase complex (DnaQ) to facilitate mutagenesis. Application of GREACE coupled-with selection under stress conditions is expected to accelerate the ALE process.ResultsIn this study, GREACE was first modified by expressing a DnaQ mutant KR5-2 using an arabinose inducible promoter on a temperature-sensitive plasmid, which resulted in timed mutagenesis introduction. Using this method, tolerance of a lysine hyperproducer E. coli MU-1 was improved by enriching mutants in a lysine endpoint fermentation broth. Afterwards, the KR5-2 expressing plasmid was cured to stabilize acquired genotypes. By subsequent fermentation evaluation, a mutant RS3 with significantly improved lysine production capacity was selected. The final titer, yield and total amount of lysine produced by RS3 in a 5-L batch fermentation reached 155.0 ± 1.4 g/L, 0.59 ± 0.02 g lysine/g glucose, and 605.6 ± 23.5 g, with improvements of 14.8%, 9.3%, and 16.7%, respectively. Further metabolomics and genomics analyses, coupled with molecular biology studies revealed that mutations SpeBA302V, AtpBS165N and SecYM145V mainly contributed both to improved cell integrity under stress conditions and enhanced metabolic flux into lysine synthesis.ConclusionsOur present study indicates that improving a lysine hyperproducer by GREACE-assisted ALE in its stressful living environment is efficient and effective. Accordingly, this is a promising method for improving other valuable chemical hyperproducers.

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Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA in Escherichia coli.

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