## 2 Production of Recombinant dsRNA in Microorganisms
### Escherichia coli
The authors examined the productivity of a target hpRNA (approximately 480 bp long) for suppression of gene expression of tobacco mosaic virus (TMV) coat protein, with several _E. coli_ strains as hosts including M-JM109 lacY, using a transcription system based on IPTG-induced expression of T7 RNA polymerase. It was shown that the productivity of the target hpRNA using strain M-JM109 lacY was superior to that using strains such as HT115(DE3) as the host. It was speculated that mutations in _endA_ (encoding endonuclease I, which is a periplasmic enzyme that cleaves within duplex DNA) and _recA_ (encoding a DNA strand exchange protein that is central to the process of homologous recombination) in M-JM109 lacY might have contributed to the stability of the expression plasmid and the dsRNA products in the host cells, but details of the under lying factors remain unclear. In addition, although it was suggested that _lacY_ mutation allows uniform entry of IPTG into all cells in the population of the culture and produces a homologous induction level of gene expression by the inducer, the involvement of the _lacY_ mutation in improving dsRNA productivity in the strain is also not certain. However, there have been some reports on successful dsRNA production using M-JM109 lacY as the host strain [38; 39; 40; 41; 42]. Recently, [PERSON] et al. [43] attempted to construct a production system that is more efficient than the conventional system for dsRNA production, L4440-HT115(DE3). When an _rnc_-deficient strain of _E. coli_ BL21(DE3) was used as the host and a hairpin-loop-type dsRNA expression system using pET28 (equipped with a single T7 promoter) was adopted as the dsRNA transcription system, it was reported that the productivity of the target dsRNA (4.23 mg/L) was approximately three times that in L4440-HT115(DE3). A unique system that allows microorganisms to produce and partially purify short interference RNA (siRNA) molecules of approximately 21 nucleotides for RNAi has been developed. [PERSON] et al. [44; 45] used an RNA-binding protein named p19 to stabilize target siRNAs produced within _E. coli_ and to purify the target siRNAs. p19 is a 19-kDa protein encoded by the plant RNA virus _Tombusvirus_, which selectively binds to siRNA molecules and is originally a suppressor of RNA silencing function in plants. Specifically, siRNA-embedded short hairpin RNA (shRNA) was co-expressed fused with glutathione 5-transferase (GST) and His-tagged p19 (GST-p19-His) (Figure 3). The target siRNA segment from the produced shRNA was separated by the action of endogenous RNase III within _E. coli_, and the siRNAs bound to GST-p19-His to form a stable complex. The siRNA-GST-p19-His complex was easily purified from the total protein extracted from the producing _E. coli_ by nickel affinity chromatography. Subsequently, the siRNA was isolated from the purified complex by SDS treatment, and then finally purified by anion exchange chromatography. Using this preparation method, the authors succeeded in producing a few kinds of functional siRNA, but the overall yield and productivity were low (several tens of micrograms per liter of _E. coli_ culture). This indicates that while this approach is very elegant for siRNA production, challenges remain for its use in agriculture and aquaculture where extremely low-cost dsRNA is required. When expressing recombinant RNA in a microorganism, it is critical to avoid attack on the RNA molecule by endogenous RNA-degrading enzymes in the cell. Besides reducing the activity of endogenous RNA-degrading enzymes, some measures have been developed to make the produced RNA molecules less susceptible to the action of ribonucleases. One  
Figure 3: Recombinant siRNA production in _Escherichia coli_. hpRNA is co-expressed with His-tagged glutathione S-transferase (GST)-fused p19 (GST-p19-His). The target siRNA segment is generated by fragmentation of hpRNA by endogenous RNase III in _E. coli_, and the siRNA species bind to GST-p19-His to form a stable GST-p19-His protein/siRNA complex, which is protected by p19.