Dataset Information

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.

SUBMITTER: Kikuchi Y

PROVIDER: S-EPMC152470 | biostudies-literature | 2003 Jan

REPOSITORIES: biostudies-literature

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Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-Like protease from Streptomyces albogriseolus.

Kikuchi Yoshimi Y Date Masayo M Yokoyama Kei-ichi K Umezawa Yukiko Y Matsui Hiroshi H

Applied and environmental microbiology 20030101 1

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-do ...[more]

PMID: 12514016

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Project description:The industrial microbe Corynebacterium glutamicum is gaining substantial importance as a platform host for recombinant protein secretion. We recently developed a fluorescence-based (eYFP) C. glutamicum reporter strain for the quantification of Sec-dependent protein secretion by monitoring the secretion-related stress response and now demonstrate its applicability in optimizing the secretion of the heterologous enzyme cutinase from Fusarium solani pisi. To drive secretion, either the poor-performing PelSP or the potent NprESP Sec signal peptide from Bacillus subtilis was used. To enable easy detection and quantification of the secreted cutinase we implemented the split green fluorescent protein (GFP) assay, which relies on the GFP11-tag fused to the C-terminus of the cutinase, which can complement a truncated GFP thereby reconstituting its fluorescence. The reporter strain was transformed with different mutant libraries created by error-prone PCR, which covered the region of the signal peptide and the N-terminus of the cutinase. Fluorescence-activated cell sorting (FACS) was performed to isolate cells that show increased fluorescence in response to increased protein secretion stress. Five PelSP variants were identified that showed a 4- to 6-fold increase in the amount and activity of the secreted cutinase (up to 4,100 U/L), whereas two improved NprESP variants were identified that showed a ∼35% increase in secretion, achieving ∼5,500 U/L. Most of the isolated variants carried mutations in the h-region of the signal peptide that increased its overall hydrophobicity. Using site-directed mutagenesis it was shown that the combined mutations F11I and P16S within the hydrophobic core of the PelSP are sufficient to boost cutinase secretion in batch cultivations to the same level as achieved by the NprESP. Screening of a PelSP mutant library in addition resulted in the identification of a cutinase variant with an increased specific activity, which was attributed to the mutation A85V located within the substrate-binding region. Taken together the biosensor-based optimization approach resulted in a substantial improvement of cutinase secretion by C. glutamicum, and therefore represents a valuable tool that can be applied to any secretory protein of interest.

Project description:BACKGROUND:In recent years, the industrial workhorse Corynebacterium glutamicum has gained increasing interest as a host organism for the secretory production of heterologous proteins. Generally, the yield of a target protein in the culture supernatant depends on a multitude of interdependent biological and bioprocess parameters which have to be optimized. So far, the monitoring of such optimization processes depends on the availability of a direct assay for the respective target protein that can be handled also in high throughput approaches. Since simple assays, such as standard enzymatic activity assays, are not always at hand, the availability of a general protein secretion biosensor is highly desirable. RESULTS:High level secretion of proteins via the Sec protein export pathway leads to secretion stress, a phenomenon that is thought to be caused by the accumulation of incompletely or misfolded proteins at the membrane-cell envelope interface. We have analyzed the transcriptional responses of C. glutamicum to the secretory production of two different heterologous proteins and found that, in both cases, the expression of the gene encoding a homologue of the extracytosolic HtrA protease was highly upregulated. Based on this finding, a C. glutamicum Sec secretion biosensor strain was constructed in which the htrA gene on the chromosome was replaced by the eyfp gene. The fluorescence of the resulting reporter strain responded to the secretion of different heterologous proteins (cutinase from Fusarium solani pisi and alkaline phosphatase PhoA from Escherichia coli) in a dose-dependent manner. In addition, three differently efficient signal peptides for the secretory production of the cutinase could be differentiated by the biosensor signal. Furthermore, we have shown that an efficient signal peptide can be separated from a poor signal peptide by using the biosensor signal of the respective cells in fluorescence activated cell sorting experiments. CONCLUSIONS:We have succeeded in the construction of a C. glutamicum biosensor strain that allows for the monitoring of Sec-dependent secretion of heterologous proteins in a dose-dependent manner, independent of a direct assay for the desired target protein.

Project description:We previously isolated three extracellular endogenous enzymes from a Streptomyces albogriseolus mutant strain which were targets of Streptomyces subtilisin inhibitor (SSI) (S. Taguchi, A. Odaka, Y. Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In the present study, of the three enzymes the largest one, with a molecular mass of 45 kDa (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), termed SAM-P45, has been characterized in detail. The entire gene encoding SAM-P45 was cloned as an approximately 10-kb fragment from S. albogriseolus S-3253 genomic DNA into an Escherichia coli host by using a shuttle plasmid vector. The amino acid sequence corresponding to the internal region of SAM-P45, deduced from the nucleotide sequence of the gene, revealed high homology, particularly in three regions around the active-site residues (Asp, His, and Ser), with the amino acid sequences of the mature domain of subtilisin-like serine proteases. In order to investigate the enzymatic properties of this protease, recombinant SAM-P45 was overproduced in Streptomyces coelicolor by using a strong SSI gene promoter. Sequence analysis of the SAM-P45 gene and peptide mapping of the purified SAM-P45 suggested that it is synthesized as a large precursor protein containing a large C-terminal prodomain (494 residues) in addition to an N-terminal preprodomain (23 and 172 residues). A high proportion of basic amino acids in the C-terminal prodomain was considered to serve an element interactive with the phospholipid bilayer existing in the C-terminal prodomain, as found in other membrane-anchoring proteases of gram-positive bacteria. It is noteworthy that SAM-P45 was found to prefer basic amino acids to aromatic or aliphatic amino acids in contrast to subtilisin BPN', which has a broad substrate specificity. The hydrolysis by SAM-P45 of the synthetic substrate (N-succinyl-L-Gly-L-Pro-L-Lys-p-nitroanilide) most preferred by this enzyme was inhibited by SSI, chymostatin, and EDTA. The proteolytic activity of SAM-P45 was stimulated by the divalent cations Ca2+ and Mg2+. From these findings, we conclude that SAM-P45 interacts with SSI and can be categorized as a novel member of the subtilisin-like serine protease family.

Project description:BackgroundTransglutaminases (TGase), synthesized as a zymogen (pro-TGase) in Streptomyces sp., are important enzymes in food industry. Due to the important applications of TGase in food industry, obtaining robust and food-safe TGase-producing strains has attracted much attention during the past decade. In this study, Streptomyces hygroscopicus pro-TGase was efficiently expressed and secreted by a food-grade host, Yarrowia lipolytica, without antibiotic markers.ResultsThe pro-TGase gene was cloned into integrative vectors pINA1296 (monocopy) and pINA1297 (multicopy), and was used to transform the Y. lipolytica Po1g or Po1h strain, respectively. Expression was driven by a recombinant hp4d promoter and secretion obtained using a XPR2 pre-sequence as a signal peptide. The highest yield of extracellular pro-TGase produced by the recombinant Po1h strain corresponded to 5.3 U/mL of TGase, a level 8.8 fold higher than that obtained using the recombinant Po1g strain. Asparagines in two potential Asn-linked glycosylation sites (Asn160 and Asn355) from pro-TGase were mutated to glutamine individually or simultaneously, yielding the deglycosylated variants N160Q, N355Q, and N160Q/N355Q. The activities of N160Q, N355Q and N160Q/N355Q constructs were respectively 5.3 U/mL, 7.8 U/mL, and 3.0 U/mL, equivalent to 100 %, 147 %, and 57 % of that from wild-type pro-TGase. The TGase yield of N355Q variant was raised to 35.3 U/mL of by using a glycerol feeding strategy in a 3 L fermenter. The optimal pH and temperature of the activated pro-TGase, and of its deglycosylated variants, were in the range of 5.0-6.0 pH and 40-45 °C, respectively. The half-life of the recombinant wild-type pro-TGase at 37 °C reached 34.0 min, and those of the variants were from 24.2 min to 11.5 min. In contrast to the wild-type pro-TGase, all of the variants had decreased specific activities, and both the K m and k cat values of the variants decreased accordingly.ConclusionsThis study constitutes the first report of the heterologous expression of a pro-TGase in Y. lipolytica, and provides new possibilities for the efficient production of TGases used in food processing.

Dataset Information

Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-Like protease from Streptomyces albogriseolus.

Publications

Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-Like protease from Streptomyces albogriseolus.

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