## 2 Production of Recombinant dsRNA in Microorganisms
### Corynebacterium glutamic
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 \(\sim\)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\(\Delta\)_rnc_[72]. As far as we know, this is the highest reported microbial production level of dsRNA so far. The capacity to produce long dsRNAs means that many dsRNA segments for various regions of the targeted essential gene of a desired pest can be carried on a single dsRNA molecule. This would improve the effectiveness of the pesticide, because even if the long dsRNA is somewhat degraded in the process of being taken into cells of the target pest, the probability that some of the remaining dsRNA segments are able to exert RNAi action will increase. In addition, since dsRNA segmented for RNAi induction against several different target pests can be carried on a single long dsRNA, in principle, one RNAi-based pesticide can be designed that works on several target species at once. Figure 5: Overproduction system of recombinant RNA species in _Corynebacterium glutamicum_: (**a**) Schematic structure of U1A*-RNA as a target RNA species. The SL-II domain of U1A*-RNA allows the RNA molecule to bind specifically to the U1A-RNA binding domain of U1 small nuclear ribonucleoprotein A. Schematic structures of _diap1_*-dsRNA expression system in pVH2-Hvlap-1 (**b**) and in pPH1-Hvlap1 (T7 pT7t) (**c**). A delivery method of dsRNA accumulated in _C. glutamicum_ cells to pests was also devised to facilitate the use of RNAi-pesticides at low cost. Generally, _E. coli_ (and other bacteria) that have accumulated dsRNA are sterilized by heat treatment and then the killed bacteria containing the dsRNA are administered to the target pest [74; 75]. To promptly sterilize dsRNA-producing _C. glutamicum_ and to eliminate the risk of latent degradation of the target dsRNA within the cells during heat treatment, a dsRNA preparation and delivery method using common alcohols (such as methyl and ethyl alcohols) was proposed [71]. 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. viginticotopuncata_ 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]. The antiviral protective effect of using killed bacteria containing the target dsRNA was comparable to that of using dsRNA extracted from the producing strain. These findings suggest that the sterilization of dsRNA-producing microbes by alcohol treatment and the resultant encapsulation of dsRNA may be effective for the formulation of RNAi-based pesticides in a variety of dsRNA-producing microbes.