Cell‐free translation systems have developed significantly over the last two decades and improvements in yield have resulted in their use for protein production in the laboratory. These systems have protein engineering applications, such as the production of proteins containing unnatural amino acids and development of proteins exhibiting novel functions. Recently, it has been suggested that cell‐free translation systems might be used as the fundamental basis for cell‐like systems. We review recent progress in the field of cell‐free translation systems and describe their use as tools for protein production and engineering.

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The marine photosynthetic bacterium Rhodovulum sulfidophilum produces extracellular nucleic acids involved in its flocculation. Previously, we showed that the RNA fraction of these extracellular nucleic acids released into the culture medium contains mainly non-aminoacylated fully mature-sized tRNAs and fragments of 16S and 23S rRNAs. Here, we report the characterization of extracellular DNA itself and its production during cultivation. No differences were detected in nucleotide sequence between the intracellular DNA and extracellular soluble DNA on Southern blotting. Whole intracellular DNA seemed to be released from the cell. The bacterial floc was degraded by deoxyribonuclease or ribonuclease treatment, indicating that at least the extracellular DNA and RNAs in the floc are involved in the maintenance of the floc. When cultivated in nutritionally rich medium, the bacteria formed small flocs and produced large amounts of extracellular DNA, which were solubilized in the medium. In nutritionally poor medium, however, huge flocs of cells appeared and almost no extracellular soluble DNA was observed in the medium. As the floc was degraded by deoxyribonuclease treatment, it seems likely that the extracellular soluble DNA observed in the rich medium may be incorporated into the large floc and play a role in floc maintenance in poor medium. Addition of an inhibitor of quorum sensing, alpha-cyclodextrin, inhibited huge floc maintenance in the nutritionally poor medium. In the presence of alpha-cyclodextrin, the floc was rapidly degraded and extracellular soluble DNA production increased.

Gene transfer agents (GTAs) are shaped like bacteriophage particles but have many properties that distinguish them from bacteriophages. GTAs play a role in horizontal gene transfer in nature and thus affect the evolution of prokaryotic genomes. In the course of studies on the extracellular production of designed RNAs using the marine bacterium Rhodovulum sulfidophilum, we found that this bacterium produces a GTA-like particle. The particle contains DNA fragments of 4.5 kb, which consist of randomly fragmented genomic DNA from the bacterium. This 4.5-kb DNA production was prevented while quorum sensing was inhibited. Direct observation of the particle by transmission electron microscopy revealed that the particle resembles a tailed phage and has a head diameter of about 40 nm and a tail length of about 60 nm. We also identified the structural genes for the GTA in the genome. Translated amino acid sequences and gene positions are closely related to those of the genes that encode the Rhodobacter capsulatus GTA. This is the first report of a GTA-like particle from the genus Rhodovulum. However, gene transfer activity of this particle has not yet been confirmed. The differences between this particle and other GTAs are discussed.

Natural noncoding small RNAs have been shown to be involved in a number of cellular processes as regulators. Using the mechanisms thus elucidated, artificial small interfering RNAs (siRNAs), ribozymes, and RNA aptamers are also expected to be potential candidates for RNA therapeutic agents. However, current techniques are too costly for industrial production of these RNAs for use as drugs. Here, we propose a new method for in vivo production of artificial RNAs using the marine phototrophic bacterium Rhodovulum sulfidophilum. Using engineered plasmids and this bacterium, which produces extracellular nucleic acids in nature, we developed a method for extracellular production of a streptavidin RNA aptamer. As the bacterium does not produce any RNases in the culture medium, at least within the cultivation period tested, the designed RNA itself is produced and retained in the culture medium of the bacterium without any specific mechanism for protection against degradation by nucleases. Here, we report that the streptavidin RNA aptamer is produced in the culture medium and retains its specific function. This is the first demonstration of extracellular production of a functional artificial RNA in vivo, which will pave the way for inexpensive production of RNA drugs.Recent studies have indicated that many small RNAs play key roles in the regulation of gene expression and that higherorder structures in RNA sequences, such as riboswitches or ribozymes, act as regulators of mRNA expression (3,4,17). In addition to these natural RNA functions, artificial small interfering RNAs (siRNAs), ribozymes, and RNA aptamers are also expected to be potential candidates for RNA therapeutics (10,20). An RNA aptamer has already been developed as an RNA drug for the inhibition of macular degradation by specifically targeting the vascular endothelial growth factor (18). In both basic studies of RNA and RNA drug production, efficient methods for preparation of homogeneous RNA molecules are very important. In the case of RNA drug production, economically efficient methods for large-scale production are required.At present, the most reliable methods for preparation of homogeneous RNAs are in vitro transcription using T7 RNA polymerase (15) and chemical synthesis (12). These methods, however, are not suitable for preparation in large quantities because they are both costly and labor intensive. For industrial production of RNAs, in vivo production using microorganisms is thought to be the most suitable method. Recently, Ponchon and Dardel reported in vivo production of recombinant RNAs using Escherichia coli (19). They proposed a system called a "tRNA scaffold" in which the product RNA is designed to be included in a tRNA structure to obtain a homogeneous RNA product. Their product RNA is produced efficiently in homogeneous form, because the in vivo transcript containing the product sequence is processed as a tRNA by the cellular processing system, although the product contains the flanking sequences of tRNA on both sides (19). We have reported...