Secretion of Active-Form Streptoverticillium mobaraense Transglutaminase by Corynebacterium glutamicum : Processing of the Pro-Transglutaminase by a Cosecreted Subtilisin-Like Protease from Streptomyces albogriseolus

Abstract

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.

The Pro-peptide ofStreptomyces mobaraensisTransglutaminase Functions incisand intransto Mediate Efficient Secretion of Active Enzyme from Methylotrophic Yeasts

“…As a result, we derived a C. boidinii strain secreting a high amount of active TGase in the medium (about 90 mg/l), whose productivity was 20 times higher than that of the original strain and comparable to the level recently developed with a Corynebacterium expression system. 9) Since active TGase can be produced directly when the propeptide is coexpressed, neither simultaneous expression of exogenous protease nor refolding of the protein is necessary in the yeast expression system. Similar propeptide coexpression did not yield an active enzyme in the bacterial expression systems (data not shown), so the methylotrophic yeast expression system appears to be superior in this respect.…”

“…[7][8][9] The gene contains a 1,221-nucleotide open reading frame encoding a 407-amino acid protein, corresponding to the predicted pre-peptide of 31 amino acids, the pro-peptide of 45 amino acids, and the mature TGase of 331 amino acids. The pro-peptide inhibits enzyme activity and also increases enzyme thermostability, 8) but it is not necessary for catalytic activity.…”

Prediction and analysis of the secreteomic in Corynebacterium glutamicum ATCC 13032

“…However, there had been only few reports concerning heterologous protein secretion in C. glutamicum. Recently, in many studies it has being demonstrated that Streptomyces mobaraensis transglutaminase (Date et al, 2004;Kikuchi et al, 2003) another enzyme used in the food industry, and human epidermal growth factor can be efficiently secreted in active form by C. glutamicum; so this strain is a potential host for industrial-scale protein production. In addition, the Tat pathway in C. glutamicum has been demonstrated to specifically mediate the secretion of Arthrobacter globiformis isomaltodextranase and green fluorescent protein (GFP) carrying an E. coli TorA signal peptide.…”

MICROBIAL TRANSGLUTAMINASE OF INDONESIAN Streptomyces sp. : Screening and Production in several media

“…cinnamoneum (Duran et al, 1998), Streptoverticillium mobaraense (Kikuchi et al, 2003), Streptoverticillium ladakanum (Tellez-Luis et al, 2004), Streptomyces netropsis (Yu et al, 2008), and Streptoverticillium hygroscopicus (Aidaroos et al, 2011). It has also been reported to be found in Bacillus subtilis spores (Liu et al, 2014).…”