## 2 Production of Recombinant dsRNA in Microorganisms
### Escherichia coli
In directed transcriptional mode (**right panel**), RNA polymerase makes single-stranded RNA species by first transcribing DNA in which the target RNA coding region is arranged as an inverted repeat sequence, and then the transcript forms hpRNA with an intramolecular dsRNA structure by self-annealing in vivo. dsRNA expressed using the L4440 vector with _E. coli_ HT115(DE3) as the host was employed. The use of RNAi-based products against tree pests is also being explored. Oral administration of a suspension of dsRNA-expressing _E. coli_ to larvae of the invasive forest pest emerald ash borer (EAB) was shown to inhibit growth and reduce survival of the pest [27]. EAB larvae feed on the cambial tissue of ash trees, thus killing them and causing significant damage to forest resources in North America. As the target genes, the _slibire_ gene (_shi_), which is involved in neuronal chemotransduction, and the _lsp_ gene, which encodes a 70 kDa-heat shock protein, were selected, and dsRNA corresponding to each gene was produced by convergent transcription using the T7 promoter of L4440 in _E. coli_ HT115(DE3), although the productivity was not specified. [PERSON] et al. [33] showed in 2011 that heat-sterilized _E. coli_ cells expressing dsRNA could be orally administered to Colorado potato beetle (CPB), a threatening crop pest, to induce RNAi and cause significant mortality in the target pest. In this case, the combination of _E. coli_ HT115(DE3) and vector L4440 was employed to produce dsRNAs targeting five genes, including genes encoding actin and subunits of V-ATPase. Recently, [PERSON] et al. [34] showed that topical application of sterilized _E. coli_ cells containing a target dsRNA produced by a recombinant RNA expression system could induce resistance of _N. benthamiana_ to potato viruses. The effectiveness of the _E. coli_-encapsulated dsRNA in providing RNAi-mediated protection against dual infection by two potato viruses was shown to be comparable to that of naked dsRNA crudely extracted from the producing bacteria. In addition, the oriental fruit fly _Bactrocera dorsalis_, which is a damaging pest that infects important fruits and vegetables, was fed with _E. coli_ cells equipped with the same dsRNA production system as above as part of an artificial diet [35]. The treated maggots, emerged pupae, and adults exhibited severe mortality and deformities. The application of dsRNA as an antiviral agent to protect shrimp from damage caused by white spot syndrome virus (WSSV) has been studied. For RNAi action in the shrimp, [PERSON] et al. [36] produced two kinds of dsRNA in _E. coli_ to target expression of VP28, the major structural protein of the virus, and WSSV015, a hub-protein that interacts with many proteins. They constructed _E. coli_ strains producing each dsRNA using strain HT115(DE3) as the host with a T7 promoter expression system, and co-cultured them in Terrific Broth by fed-batch culture with the addition of glycerol. The accumulation of total dsRNA reached approximately 95 mg per liter of culture broth. The use of such multiple-target dsRNAs will be of considerable interest as a method to effectively control the complex WSSV that has marked genetic variation. In an effort to further improve dsRNA productivity in the L4440-based recombinant RNA expression system in _E. coli_, [PERSON] et al. [37] analyzed the kinetics of target dsRNA (480 bp-segment) production in fed-batch fermentation and found that after 12 h of fermentation, the dsRNA/biomass yield was 0.06 g/g, the maximum productivity of dsRNA was 15.2 mg/L/h, and the final dsRNA concentration achieved was 182 mg/L. Thus, it was indicated that the dsRNA production by the standard expression system is growth-associated with the producing bacteria. _E. coli_ strains other than HT115(DE3) have also been generated as host strains for dsRNA production. One such strain is _E. coli_ M-JM109 lacY, which was constructed by deleting the _rnc_ and _lacY_ (encoding lactose permease) genes from strain JM109(DE3), a derivative of strain JM109 [38].