Efficient production of long double-stranded RNAs applicable to agricultural pest control by Corynebacterium glutamicum equipped with coliphage T7-expression system

Abstract

RNA-based pesticides exert their function by suppressing the expression of an essential gene in the target pest through RNA interference caused by double-stranded RNA (dsRNA). Here, we selected target genes for growth suppression of the solanaceous crop pests ladybird beetle (Henosepilachna vigintioctopunctata) and Colorado potato beetle (Leptinotarsa decemlineata)-the death-associated inhibitor of apoptosis protein 1 gene (diap1), and an orthologous gene of the COPI coatomer protein complex (copI), respectively. We constructed a cost-competitive overproduction system for dsRNA using Corynebacterium glutamicum as a host bacterium. The dsRNA expression unit was equipped with two sets of promoters and terminators derived from coliphage T7, and the convergent expression system was designed to be selectively transcribed by T7 RNA polymerase. This expression system efficiently overproduced both target dsRNAs. On culture in a jar fermentor, the yield of diap1-targeting dsRNA (approximately 360 bp) was > 1 g per liter of culture. Long-chain diap1-targeting dsRNAs (up to around 1 kbp) could be produced without a substantial loss of efficiency. dsRNA accumulated in C. glutamicum significantly suppressed larval growth of H. vigintioctopunctata. The dsRNA expression technology developed here is expected to substantially reduce dsRNA production costs. Our method can be applied for a wide range of industrial uses, including agricultural pest control. Key points • Overexpression of dsRNA was achieved in C. glutamicum using a coliphage T7 system. • The best strain produced > 1 g/L of the target dsRNA species, for use as an insecticide. • The developed system efficiently produced long dsRNA species, up to ~ 1 kbp.

Advances in the Development of Microbial Double-Stranded RNA Production Systems for Application of RNA Interference in Agricultural Pest Control

“…As most of these bacteria are probiotics and/or model species, they might be the most suitable microbial hosts for diverse dsRNA production. Corynebacterium glutamicum has also been shown to efficiently synthesize dsRNA longer than 1 kbp in a yield >1 g/L of culture ( Hashero et al, 2021 ). Besides, microalgae can also be engineered as dsRNA expression vectors, and shrimps and crabs can be protected from bacterial or viral infection by feeding on microalgae expressing dsRNA ( Saksmerprome et al, 2009 ; Somchai et al, 2016 ; Charoonnart et al, 2019 ).…”

RNA Interference-Based Pesticides and Antiviral Agents: Microbial Overproduction Systems for Double-Stranded RNA for Applications in Agriculture and Aquaculture

“…To further increase the productivity of dsRNA, the employment of T7 RNA polymerase within C. glutamicum was considered, since T7 RNA polymerase has the advantage of avoiding collisions between the RNA polymerases in the transcriptional process from T7 promoters in such a convergent transcription system (Figure 5c). Convergent transcription in C. glutamicum using the combination of T7 RNA polymerase and the T7 promoter allowed the production of longer dsRNA strands than was possible using the endogenous multisubunit RNA polymerase and the F1 promoter; it was possible to achieve the production of dsRNAs as long as ~1 kbp without a significant decrease in productivity [72]. In addition, by installing T7 terminators in the transcription system and further improving the high-copy-number plasmid vector [73] carrying the expression system, diap1*-dsRNA was produced at a yield of approximately 1 g per liter of culture medium in jar fermentation of C. glutamicum C. glutamicum-driven production of dsRNAs that can be applied as RNAi-based pesticides was subsequently investigated.…”

“…Convergent transcription in C. glutamicum using the combination of T7 RNA polymerase and the T7 promoter allowed the production of longer dsRNA strands than was possible using the endogenous multisubunit RNA polymerase and the F1 promoter; it was possible to achieve the production of dsRNAs as long as ~1 kbp without a significant decrease in productivity [72]. In addition, by installing T7 terminators in the transcription system and further improving the high-copy-number plasmid vector [73] carrying the expression system, diap1*-dsRNA was produced at a yield of approximately 1 g per liter of culture medium in jar fermentation of C. glutamicum strain 2256L∆rnc [72]. As far as we know, this is the highest reported microbial production level of dsRNA so far.…”

“…Sterilization of the C. glutamicum strain was achieved by simply suspending the bacterial cells in each alcohol solution at a concentration of >50%; no noticeable degradation of dsRNA in the cells was observed. When larvae of the target pest H. vigintioctopunctata ingested the dsRNA encapsulated in killed C. glutamicum cells prepared in this way, the feeding activity of the pest on potato leaves was reduced and weight gain was significantly suppressed, indicating that diap1*-dsRNA-containing sterilized C. glutamicum cells were effective in inhibiting the growth of the pest [71,72]. A similar method of ethanol sterilization of a dsRNA-producing microorganism for use as an RNAi-based pesticide has recently been reported in E. coli producing dsRNA for protection against potato virus infecting N. benthamiana [34].…”

Strategies for the production of dsRNA biocontrols as alternatives to chemical pesticides

“…Recently, higher yields of over 1 g/L of dsRNA targeting the H. vigintioctopunctata gene diap1 were achieved in C. glutamicum using a similar convergent promoter arrangement, though utilising T7 RNA polymerase and the associated promoters, supplied by co-transfection of the pVC7T7pol1 plasmid ( Hashiro et al, 2021 ). Ethanol-sterilised C. glutamicum containing recombinantly produced diap1 -targeting dsRNA were again used in H. vigintioctopunctata feeding assays, and once more ingestion of the dsRNA-containing bacteria significantly reduced larval weight gain in a 48-h period.…”