The transglutaminase secreted by Streptoverticillium mobaraense is a useful enzyme in the food industry. A fragment of transglutaminase was secreted by Corynebacterium glutamicum when it was coupled on a plasmid to the promoter and signal peptide of a cell surface protein from C. glutamicum. We analyzed the signal peptide and the pro-domain of the transglutaminase gene and found that the signal peptide consists of 31 amino acid residues and the pro-domain consists of 45 residues. When the pro-domain of the transglutaminase was used, the pro-transglutaminase was secreted efficiently by C. glutamicum but had no enzymatic activity. However, when the plasmid carrying the S. mobaraense transglutaminase also encoded SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus, the peptide bond to the C side of 41-Ser of the pro-transglutaminase was hydrolyzed, and the pro-transglutaminase was converted to an active form. Our findings suggest that C. glutamicum has potential as a host for industrial-scale protein production.Transglutaminases (protein-glutamine ␥-glutamyltransferase, EC 2.3.2.13) are a family of enzymes that catalyze an acyl transfer reaction between a ␥-carboxyamide group of a glutamine residue in a peptide chain and a ␥-amino group of a lysine residue, resulting in the formation of an ε-(␥-glutamyl) lysine cross-link (6). Transglutaminases are widely distributed, and the physiological properties of several of them have been studied. Transglutaminases derived from animals, for example, human blood coagulation factor XIII, human epidermis keratinocyte transglutaminase, guinea pig liver transglutaminase, and fish liver transglutaminase, are calcium-dependent enzymes (6, 24, 38). Calcium-independent transglutaminases have been discovered in bacteria belonging to the actinomycetes, which include, for example, Streptoverticillium cinnamoneum (4) and Streptoverticillium mobaraense. The enzyme from S. mobaraense has been especially well characterized (1, 36).S. mobaraense transglutaminase (MTG [mature-form transglutaminase]) has been used in the food industry for the modification of proteins (9,13,22). It is used in binding meat or fish and gelled food products such as jelly, yogurt, and cheese. Moreover, it has great potential for use in manufacturing materials found in cosmetics, thermostable microcapsules, and carriers for immobilized enzymes. To date, it is produced by conventional fermentation, but it would be desirable to develop a more efficient system, and a number of reports have described the expression and production of MTG in hostvector systems such as Streptomyces lividans (36) and Escherichia coli (33,39). MTG was secreted in microorganisms such as S. lividans (no more than 0.1 mg/liter) (36) and E. coli (about 5 mg/liter) (33); moreover, it was produced by an inclusion body within E. coli (39). The levels of expression in these studies were low, and it would be very difficult to produce MTG on an industrial scale via an inclusion body.

BackgroundAmong other advantages, recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Although production of recombinant Fab using microbial expression systems has been reported, yields of active Fab have not been satisfactory. We recently developed the Corynebacterium glutamicum protein expression system (CORYNEX®) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production.ResultsThe Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX® system using the conventional C. glutamicum strain YDK010, but the productivity was very low. To improve the secretion efficiency, we investigated the effects of deleting cell wall-related genes. Fab secretion was increased 5.2 times by deletion of pbp1a, encoding one of the penicillin-binding proteins (PBP1a), mediating cell wall peptidoglycan (PG) synthesis. However, this Δpbp1a mutation did not improve Fab secretion in the wild-type ATCC13869 strain. Because YDK010 carries a mutation in the cspB gene encoding a surface (S)-layer protein, we evaluated the effect of ΔcspB mutation on Fab secretion from ATCC13869. The Δpbp1a mutation showed a positive effect on Fab secretion only in combination with the ΔcspB mutation. The ΔcspBΔpbp1a double mutant showed much greater sensitivity to lysozyme than either single mutant or the wild-type strain, suggesting that these mutations reduced cell wall resistance to protein secretion.ConclusionThere are at least two crucial permeability barriers to Fab secretion in the cell surface structure of C. glutamicum, the PG layer, and the S-layer. The ΔcspBΔpbp1a double mutant allows efficient Fab production using the CORYNEX® system.

We previously observed secretion of active-form transglutaminase in Corynebacterium glutamicum by coexpressing the subtilisin-like protease SAM-P45 from Streptomyces albogriseolus to process the prodomain. However, the N-terminal amino acid sequence of the transglutaminase differed from that of the native Streptoverticillium mobaraense enzyme. In the present work we have used site-directed mutagenesis to generate an optimal SAM-P45 cleavage site in the C-terminal region of the prodomain. As a result, native-type transglutaminase was secreted.Transglutaminases (protein-glutamine ␥-glutamyltransferase [EC 2.3.2.13]) are a family of enzymes that catalyze an acyl transfer reaction between a ␥-carboxyamide group of a glutamine residue in a peptide chain and a ␥-amino group of a lysine residue, resulting in the formation of an ⑀-(␥-glutamyl) lysine cross-linkage (2). Transglutaminases are widely distributed, and their physiological properties have been studied. Animal transglutaminases are calcium-dependent enzymes (2, 12, 17), while calcium-independent transglutaminases have been discovered in bacteria belonging to the actinomycetes (1, 16). Streptoverticillium mobaraense transglutaminase (matureform transglutaminase [MTG]) has been used in the food industry to modify protein (3,7,11). Presently the enzyme is produced by conventional fermentation, but it would be desirable to develop a more efficient system for its production.Corynebacterium glutamicum is gram positive and is employed for the industrial production of amino acids, such as glutamate and lysine, that have been used in human food, animal feed, and pharmaceutical products for several decades (6). It is nonpathogenic and produces no hazardous toxins (6, 10). In a previous report we demonstrated that the pro-MTG was efficiently secreted by C. glutamicum when it carried a signal peptide derived from a cell surface protein of corynebacteria. Moreover, the proenzyme was processed to the active form of the enzyme by the subtilisin-like protease, SAM-P45, when the latter was cosecreted with the proenzyme (5). However, the N-terminal amino acid sequence of the processed transglutaminase differed from that of the native enzyme: four amino acid residues, Phe-Arg-Ala-Pro, at the C terminus of the prodomain were added. We have introduced a preferred SAM-P45 cleavage site at the C terminus of the prodomain in order to produce native-type MTG in C. glutamicum.Deletion analyses of the prodomain. DNA manipulations were carried out by the methods described by Sambrook et al. (13). PCR with Pyrobest DNA polymerase (Takara Shuzo, Kyoto, Japan) was performed in 50-l reaction mixtures for 5 min at 94°C, followed by 25 cycles of 10 s at 98°C, 30 s at 55°C, and 3 min at 72°C. Nucleotide sequences were determined by using a BigDye terminator cycle-sequencing FS ready reaction kit (Applied Biosystems) and a DNA sequencer (model 377; Applied Biosystems).Plasmids expressing pro-MTG with N-terminal, central, or C-terminal deletions (⌬1D, ⌬1D6E, ⌬25I31S, ⌬19A34A, ⌬42F45P, and ⌬44A45...

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