## 2 Production of Recombinant dsRNA in Microorganisms
### Corynebacterium glutamic  
_C. glutamicum_ is a non-sportulating Gram-positive soil bacterium with a high G + C mol% content in its genomic DNA. This microorganism is nonpathogenic and does not produce endotoxins, making it a safe strain for humans. In 1965, this strain was reported as a microorganism capable of overproducing L-glutamic acid in the culture medium [60]. Since then, _C. glutamicum_, metabolically engineered, has been used as a workhorse for the production of various L-amino acids, proteins, and, recently, a variety of other useful biochemicals [61; 62; 63]. In particular, _C. glutamicum_ has been employed as an industrial L-amino acid-producing microorganism in large-scale cultivation for several decades, and is a GRAS host for the industrial production of biochemicals including L-glutamate and L-lysine [64; 65]. The advantages of this robust strain are also being exploited to make it a host for overproduction of recombinant RNA molecules. [PERSON] et al. first examined the possibility of overexpression of RNA molecules with a desired nucleotide sequence in a mutant _C. glutamicum_ strain (named 2256L\(\Delta\)_rnc_, in which the _rnc_ gene encoding RNase III was deleted from wild-type strain ATCC13869) [66; 67]. To enhance transcription of recombinant RNA molecules, the F1 promoter derived from bacteriophage BFK20 [67; 68], which infects _Corynebacterium_, was chosen as a strong promoter that functions in _C. glutamicum_. A newly acquired high-copy-number mutant plasmid carrying the transcription system was also employed [69]. Overproduction of a recombinant RNA named U1A*-RNA (approximately 160 nucleotides long) was examined as a model RNA for expression studies in strain 2256L\(\Delta\)_rnc_ (Figure 5a). U1A*-RNA consists of a stem/loop II (SL II) structure from U1 small nuclear RNA (snRNA) and is an RNA  
Figure 4: dsRNA production system in _Pseudomonas syringae_ exploiting the genetic functional components of bacteriophage phfi. \(\mathrm{M_{TMV}}\) and \(\mathrm{S_{TMV}}\) contain sequences derived from tobacco mosaic virus (TMV). \(\mathrm{L_{kan}}\)-ssRNA, \(\mathrm{M_{TMV}}\)-ssRNA, and \(\mathrm{S_{TMV}}\)-ssRNA are transcripts from the plasmids introduced; \(\mathrm{L_{kan}}\)-ssRNA is translated to produce phi6 polymerase complex (PC), in which \(\mathrm{L_{kan}}\)-ssRNA, \(\mathrm{M_{TMV}}\)-ssRNA, and \(\mathrm{S_{TMV}}\)-ssRNA are packaged. During the process, the complementary strand of each ssRNA species containing the phi6 replication signal is synthesized to generate the corresponding dsRNA species. As a result, a capsid structure filled with \(\mathrm{L_{kan}}\), \(\mathrm{M_{TMV}}\), and \(\mathrm{S_{TMV}}\) dsRNA (dsRNA\({}^{\mathrm{rep}}\)-MP) is formed, and the capsid is amplified in the host cell. molecule designed to contain a domain that binds to U1A protein to form a U1 small nuclear ribonucleoprotein (snRNP) involved in pre-mRNA splicing. The producer microbe showed prominent accumulation of U1A*-RNA (approximately 300 mg/L of culture medium), which opened the door to the production of recombinant RNAs using _C. glutamicum_ as the host microorganism [67]. _C. glutamicum_-driven production of dsRNAs that can be applied as RNAi-based pesticides was subsequently investigated. The _diap1_ gene (encoding death-associated inhibitor of apoptosis protein 1) [70] was selected as a target for RNAi action to combat the crop pest _H. viginiotopuncata_, and production of target dsRNA (named _diap1_*-dsRNA) corresponding to a region of approximately 360 nucleotides long was undertaken [71]. This production system in _C. glutamicum_ was designed to produce the target _diap1_*-dsRNA by convergent transcription using two strong F1 promoters (Figure 5b). This expression system led to the accumulation of _diap1_*-dsRNA to approximately 75 mg per liter of culture medium in jar fermentation of the producing microorganism [71]. To further increase the productivity of dsRNA, the employment of T7 RNA polymerase within _C.