Production of Native-Type Streptoverticillium mobaraense Transglutaminase in Corynebacterium glutamicum

We previously observed secretion of active-form transglutaminasein Corynebacterium glutamicum by coexpressing the subtilisin-likeprotease SAM-P45 from Streptomyces albogriseolus to processthe prodomain. However, the N-terminal amino acid sequence ofthe transglutaminase differed from that of the native Streptoverticilliummobaraense enzyme. In the present work we have used site-directedmutagenesis to generate an optimal SAM-P45 cleavage site inthe C-terminal region of the prodomain. As a result, native-typetransglutaminase was secreted.

INTRODUCTION

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Introduction

Transglutaminases (protein-glutamine ${gamma}$ -glutamyltransferase [EC2.3.2.13]) are a family of enzymes that catalyze an acyl transferreaction between a ${gamma}$ -carboxyamide group of a glutamine residuein a peptide chain and a ${gamma}$ -amino group of a lysine residue, resultingin the formation of an ${epsilon}$ -( ${gamma}$ -glutamyl) lysine cross-linkage (2).Transglutaminases are widely distributed, and their physiologicalproperties have been studied. Animal transglutaminases are calcium-dependentenzymes (2, 12, 17), while calcium-independent transglutaminaseshave been discovered in bacteria belonging to the actinomycetes(1, 16). Streptoverticillium mobaraense transglutaminase (mature-formtransglutaminase [MTG]) has been used in the food industry tomodify protein (3, 7, 11). Presently the enzyme is producedby conventional fermentation, but it would be desirable to developa more efficient system for its production.

Corynebacterium glutamicum is gram positive and is employedfor the industrial production of amino acids, such as glutamateand lysine, that have been used in human food, animal feed,and pharmaceutical products for several decades (6). It is nonpathogenicand produces no hazardous toxins (6, 10). In a previous reportwe demonstrated that the pro-MTG was efficiently secreted byC. glutamicum when it carried a signal peptide derived froma cell surface protein of corynebacteria. Moreover, the proenzymewas processed to the active form of the enzyme by the subtilisin-likeprotease, SAM-P45, when the latter was cosecreted with the proenzyme(5). However, the N-terminal amino acid sequence of the processedtransglutaminase differed from that of the native enzyme: fouramino acid residues, Phe-Arg-Ala-Pro, at the C terminus of theprodomain were added. We have introduced a preferred SAM-P45cleavage site at the C terminus of the prodomain in order toproduce native-type MTG in C. glutamicum.

Deletion analyses of the prodomain.

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Deletion analyses of the...

DNA manipulations were carried out by the methods describedby Sambrook et al. (13). PCR with Pyrobest DNA polymerase (TakaraShuzo, Kyoto, Japan) was performed in 50-µl reaction mixturesfor 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 sequenceswere determined by using a BigDye terminator cycle-sequencingFS ready reaction kit (Applied Biosystems) and a DNA sequencer(model 377; Applied Biosystems).

Plasmids expressing pro-MTG with N-terminal, central, or C-terminaldeletions ( ${Delta}$ 1D, ${Delta}$ 1D6E, ${Delta}$ 25I31S, ${Delta}$ 19A34A, ${Delta}$ 42F45P, and ${Delta}$ 44A45P) wereconstructed by crossover PCR (Fig. 1 and 2A). First PCRs wereperformed with CspB5 (as forward primer) and primer A (as reverseprimer) or with primer B (as forward primer) and Tg3 (as reverseprimer) with plasmid pPSPTG1 DNA (5) as a template (Fig. 1).Second PCRs were performed with CspB5 (as forward primer) andTg3 (as reverse primer) with the amplified fragments generatedby the first PCRs as templates (Fig. 1). Each amplified fragmentwas digested with ScaI and EcoO65I, and the digested fragmentswere inserted into the ScaI-EcoO65I site of pPSPTG1 to obtainpPTG1 ${Delta}$ 1D, pPTG1 ${Delta}$ 1D6E, pPTG1 ${Delta}$ 25I31S, pPTG1 ${Delta}$ 19A34A, pPTG1 ${Delta}$ 42F45P, andpPTG1 ${Delta}$ 44A45P. All cloned fragments made by PCR were sequencedto check for PCR-induced errors.

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FIG. 1. Construction of prepro-MTG genes with deleted or mutated prodomains by crossover PCR. (A) Schematic representation of crossover PCR with primer CspB5 and primer A, primer B and Tg3 for first PCRs, and primers CspB5 and Tg3 for second PCR. (B) Primer sequences.

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FIG. 2. Structures of the deleted or mutated prodomains. (A) Deleted prodomains. Each deleted region is indicated by a line above it ( ${Delta}$ 1D, ${Delta}$ 25I31S, or ${Delta}$ 44A45P) or is underlined ( ${Delta}$ 1D6E, ${Delta}$ 19A34A, or ${Delta}$ 42F45P). (B) Mutated prodomains. The mutated region (GPSFRAP) of the prodomain is underlined. The plasmids expressing prepro-MTG genes with mutated prodomains GPSFGPK, GPSFGPR, GPK, and GPR are pPSPTG11, pPSPTG12, pPSPTG13, and pPSPTG14, respectively.

C. glutamicum ATCC 13869 was transformed with pPSPTG1, pPTG1 ${Delta}$ 1D,pPTG1 ${Delta}$ 1D6E, pPTG1 ${Delta}$ 25I31S, pPTG1 ${Delta}$ 19A34A, pPTG1 ${Delta}$ 42F45P, and pPTG1 ${Delta}$ 44A45P(9). The transformants were cultured in MMTG medium (5) containing25 mg of kanamycin per liter at 30°C for 40 h, and secretionof the corresponding pro-MTG was assessed by high-performanceliquid chromatography as described previously (5). Accumulationof the pro-MTGs with central deletions ( ${Delta}$ 25I31S and ${Delta}$ 19A34A) wasgreatly decreased (about 1% of the intact pro-MTG) (Fig. 3)and removing six amino acid residues from the N terminus ofthe prodomain ( ${Delta}$ 1D6E) reduced the amount of secreted pro-MTGto approximately one-third. However, removal of one amino acidresidue from the N terminus ( ${Delta}$ 1D) or either two or four fromthe C terminus ( ${Delta}$ 44A45P or ${Delta}$ 42F45P) had hardly any effect on theamount of pro-MTG secreted. Evidently the central region ofthe prodomain, but not the C terminus, has an important rolein secretion. We therefore explored the possibility of mutatingthe C-terminal region of the proprotein in order to producenative-type MTG by SAM-P45 processing.

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FIG. 3. Secretion of prodomains harboring deletions. Values are averages of two independent experiments. The value for the amount of the pro-MTG accumulation with the native-type prodomain was set at 100% for each experiment.

Mutational analysis of C-terminal region.

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Mutational analysis of c...

Suzuki et al. (14) have shown that SAM-P45 exhibits strong hydrolyticactivity towards tripeptide substrates whose amino acid residuesC terminal of the cleavage site are Lys or Arg, preferably inthe sequence Gly-Pro-Lys or Gly-Pro-Arg. Plasmids expressingpro-MTGs with prodomains whose C termini had optimal SAM-P45cleavage sites were constructed by crossover PCR (Fig. 1 and2B). Each amplified fragment was digested with ScaI and EcoO65I,and the digested fragments with their prodomain deletions wereinserted into the ScaI-EcoO65I site of pPSPTG1 to yield pPTG11,pPTG12, pPTG13, and pPTG14. C. glutamicum ATCC 13869 was transformedwith these constructs, and the transformants were grown as describedabove. The accumulation of pro-MTG in these cultures is shownin Table 1, and as expected, none of the mutations in the Cterminus of the prodomain had much effect on the amount of secretedpro-MTG. The culture supernatants were incubated with purifiedSAM-P45 for 2 h at a 100:1 ratio of pro-MTG to SAM-P45. Afterthis, they were subjected to sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) (8), and the proteins were electroblottedonto polyvinylidene difluoride membranes (Bio-Rad). The segmentof gel containing active-form MTG was cut out, and N-terminalamino acid sequences were determined as described earlier (4).The N-terminal amino acid of the pPSPTG11 and pPSPTG12 MTGswas Asp, as in native S. mobaraense MTG (Fig. 4). However, Ser37of the prodomain was detected N terminally in the pPSPTG13 andpPSPTG14 MTGs in addition to Asp (Fig. 4). The ratio of SerN termini to Asp N termini was approximately 3 to 2 in both.

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TABLE 1. Relative amounts of pro-MTG accumulation in the culture supernatant of C. glutamicum

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FIG. 4. Sites of SAM-P45 cleavage of pro-MTGs with mutated prodomains. The amino acid sequence of the MTG is underlined, and the site of SAM-P45 cleavage is indicated by an arrow.

Production of native-type MTG in C. glutamicum.

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Production of native-type mtg...

In light of these results, we used pPSPTG11 to produce native-typeMTG in C. glutamicum. pPSPTG11 alone and pVSS1 expressing SAM-P45were introduced into C. glutamicum ATCC 13869, and a transformantwas cultured in MMTG at 30°C for 70 h. At various timessamples of the supernatant were subjected to SDS-PAGE. The pro-MTGderived from pPSPTG11 was processed by SAM-P45, and accumulationof native-type MTG was maximal at about 70 h (Fig. 5). The maximalaccumulation of native-type MTG achieved to date under thesecondition is 132 mg/liter. The N-terminal amino acid of thesecreted native-type MTG is Asp, indicating that the modifiedprodomain is processed in vitro. Using the methods describedby Yokoyama et al. (18), we purified the MTG from S. mobaraense,the active-form MTG with additional Phe-Arg-Ala-Pro residues(5), and the native-type MTG in this report. The specific activitiesof these enzymes were determined by the calorimetric hydroxamateprocedure as described by Yokoyama et al. (18), and the followingresults were obtained: specific activities of the MTG from S.mobaraense, the active-form MTG with additional Phe-Arg-Ala-Proresidues (5), and the native-type MTG used for this report were26, 30, and 26 U/mg, respectively.

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FIG. 5. SDS-PAGE analysis of the active-form MTG produced by C. glutamicum carrying plasmids expressing pro-MTG and SAM-P45. Ten microliters of supernatant and an equal volume of sample buffer were applied to each slot and were analyzed by SDS-PAGE. After electrophoresis, the gel was stained with Coomassie brilliant blue R-250. Lane 1, molecular weight markers; lanes 2 through 5, culture supernatants after 30, 45, 70, and 140 h of cultivation; and lane 6, purified MTG from S. mobaraense.

In this study we have obtained efficient secretion of native-typeMTG by C. glutamicum by using an altered prodomain and coexpressingSAM-P45. The accumulation of MTG (132 mg/liter) exceeded thatin other hosts, since no more than 0.1 and 5 mg of MTG per literwere secreted by Streptomyces lividans (16) and Escherichiacoli (15), respectively. This C. glutamicum protein expressionand cleavage system is therefore useful for producing a heterologousprotein with an N-terminal amino acid sequence identical tothat of the native form.

ACKNOWLEDGMENTS

We are grateful to S. Taguchi for his kind gift of purifiedSAM-P45 and to N. Onishi for helpful discussion.

FOOTNOTES

* Corresponding author. Mailing address: Institute of Life Sciences, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan. Phone: (81)-44-244-7123. Fax: (81)-44-222-0129. E-mail: yoshimi_kikuchi{at}ajinomoto.com.

REFERENCES

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